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When we took ventolin pill price the editorship of Evidence-Based Mental Health (EBMH) at the end of 2013, we set two main objectives. To promote and ventolin pill price embed an evidence-based medicine (EBM) approach into daily mental health clinical practice, and to get an impact factor (IF) for EBMH. Both aims have been big challenges and we ventolin pill price have learnt a lot.EBM has been around for about 30 years now, shaping and changing the way we practice medicine. When Guyatt and colleagues published their seminal paper in 1992,1 EBM was described as the combination of three intersecting domains. The best available evidence, the ventolin pill price clinical state and circumstances, and patient’s preferences and values.

EBM and EBMH have since continuously evolved to deepen our understanding of these three domains.The best available evidenceWe keep complaining about the poor ventolin pill price quality of studies in mental health. To properly assess the effects of interventions and devices before and after regulatory approval, we all know that randomised controlled trials are the best study design.2 3 However, real-world data are crucial to shed light on key clinical questions,4 especially when adverse events5 or prognostic factors6 are investigated. It necessarily …IntroductionQuality-adjusted life years (QALYs) have been increasingly used in ventolin pill price general medicine and in psychiatry to evaluate the impact of a disease on both the quantity and quality of life.1 One QALY is equal to 1 year in perfect health, can range down to zero (death) or may take negative values (worse than death). QALYs can be used to compare the burdens of various ventolin pill price diseases, to appreciate the impact of their interventions, to help set priorities in resource allocations across different diseases and interventions and to inform personal decisions.The representative method to evaluate QALYs is the generic, preference-based measure of health including the Euro-Qol five dimensions (EQ-5D)2 3 and the SF-6D based on Short Form Survey-36 (SF-36).4 5 Of these, the EQ-5D is the most frequently used and is the preferred instrument by the National Institute of Health and Care Excellence in the UK. While the responsiveness of such generic measures to various mental conditions, especially severe mental illnesses, has been questioned,6 its validity and responsiveness to common mental disorders including depression and anxiety have been generally established.7 8However, the traditional focus of measurements in mental health has ventolin pill price centred mainly on symptoms.

Many trials have, therefore, not administered the generic health-related quality of life measures. This has hindered comparison of impacts of mental disorders vis-à-vis other medical conditions on the one hand and also ventolin pill price evaluation of values of their interventions on the other.9 10We have been collecting individual participant-level data from randomised controlled trials of internet cognitive-behavioural therapies (iCBT) for depression,11 several of which administered both symptomatologic scales and generic health status scales simultaneously. This study, ventolin pill price therefore, attempts to link the depression-specific measure onto the generic measure of health in order to enable estimation of QALYs for depressive states and their changes. Such cross-walking should facilitate assessment of burden of depression at its various severity and of the impacts of its various treatments.MethodsDatabaseWe have been accumulating a data set of individual participant data of randomised controlled trials of iCBT among adults with depressive symptoms, as established by specified cut-offs on self-report scales or by diagnostic interviews.11 For this study, we have selected studies that have administered the EQ-5D and depression severity scales at baseline and at end of treatment. We excluded patients if they had missing data in either of the ventolin pill price two scales at baseline or at endpoint.

We excluded studies that focused on patients with general medical disorders (eg, ventolin pill price diabetes, glioma) and depressive symptoms.MeasuresEQ-5D-3LThe EQ-5D-3L comprises five dimensions of mobility, self-care, usual activities, pain/discomfort and anxiety/depression, each rated on three levels corresponding with 1=no problems, 2=some/moderate problems or 3=extreme problems/unable to do. This produces 3ˆ5=243 different health states, ranging from no problem at all in any dimension (11111) to severe ventolin pill price problems on all dimensions (33333). Each of these 243 states is provided with a preference-based score, as determined through the time trade-off (TTO) technique in a sample of the general population. In TTO, respondents are asked to give the relative length of time in full health that they would ventolin pill price be willing to sacrifice for the poor health states as represented by each of the 243 combinations above. The EQ-5D scores range between 1=full ventolin pill price health and 0=death to minus values=worse than death bounded by −1.

The scoring algorithm for the UK is based on TTO responses of a random sample (n=2997) of noninstitutionalised adults. Over the years, value sets for EQ-5D-3L have been produced for many countries/regions.2 3 7Depression severity scalesWe included any validated depression ventolin pill price severity measures. The scale scores were converted into the most frequently used scale, namely, the Patient Health Questionnaire-9 (PHQ-9),12 using the established conversion algorithms13 14 for the Beck Depression Inventory, second edition (BDI-II)15 or the Centre for Epidemiologic Studies Depression Scale (CES-D).16The PHQ-9 consists of the nine diagnostic criteria items of major depression from the DSM-IV, each rated on a scale between 0 and 3, making the total score range 0–27 ventolin pill price. The instrument has demonstrated excellent reliability, validity and responsiveness. The cut-offs have been proposed as 0–4, 5–9, 10–14, 15–19 and 20- for no, mild, moderate, moderately severe and severe depression, respectively.12Statistical analysesWe first calculated Spearman correlation coefficients between PHQ-9 and EQ-5D total ventolin pill price scores at baseline, at end of treatment and their changes, to establish if the linking is justified.

Correlations were ventolin pill price considered weak if scores were <0.3, moderate if scores were ≥0.3 and<0.7 and strong if scores were ≥0.7.17 Correlations ≥0.3 have been recommended to establish linking.18 We then applied the equipercentile linking procedure,19 which identified scores on PHQ-9 and EQ-5D or their changes with the same percentile ranks and allows for a nominal translation from PHQ-9 to EQ-5D by using their percentile values. This approach has been used successfully for scales in depression, schizophrenia or Alzheimer’s disease.14 20–22 We analysed all trials collectively rather than by trial to maximise the sample size, ensure variability in the included populations and attain robust estimates.We conducted a sensitivity analysis by excluding studies that require the conversion of various depression severity scores into PHQ-9.All the analyses were conducted in R V.4.0.2, with the package equate V.2.0.7.23Ethics statementThe authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the ventolin pill price Helsinki Declaration of 1975, as revised in 2008. Ethical approval was not required for this study as it used only deidentified patient data.FindingsIncluded studiesWe identified seven RCTs of iCBT (total n=2457), which administered validated depression scales and EQ-5D both at baseline and at endpoint (online supplemental eTable 1). Three studies included only patients with major depressive disorder (MDD), one only patients with subthreshold depression and the remaining ventolin pill price three included both. All the ventolin pill price studies administered EQ-5D-3L.

PHQ-9 scores were converted from the BDI-II in three studies24–26 and from the CES-D in one study.27 The mean age of the participants was 41.8 (SD=12.3) years, 66.0% (1622/2457) were women and they scored 14.0 (5.4) on PHQ-9 and 0.74 (0.20) on EQ-5D at baseline and 9.1 (6.0) and 0.79 (0.21), respectively, at endpoint. When using the standard cut-offs of the PHQ-9,12 2.4% (60/2449) suffered from no ventolin pill price depression (PHQ-9 scores <5), 20.2% (492/2449) from subthreshold depression (5≤PHQ-9 scores <10), 33.5% (820/2449) from mild depression (10≤PHQ-9 scores <15), 26.5% (649/2449) from moderate depression (15≤PHQ-9 scores <20) and 17.3% (424/2449) from severe depression (20≤PHQ-9 scores) at baseline.Supplemental materialEquipercentile linkingSpearman’s correlation coefficient between the PHQ-9 and the EQ-5D scores was r=−0.29 at baseline, increased to r=−0.50 after intervention and was r=−0.38 for change scores.Figure 1 shows the equipercentile linking between PHQ-9 and EQ-5D total scores at baseline and at endpoint. Figure 2 shows the ventolin pill price same between their change scores. Table 1 summarises the correspondences between the two scales.PHQ-9 and EQ-5D ventolin pill price total scores at baseline and endpoint. EQ-5D,Euro-Qol Five Dimensions.

PHQ-9, PatientHealth Questionnaire-9." data-icon-position data-hide-link-title="0">Figure 1 PHQ-9 and EQ-5D total scores at baseline and ventolin pill price endpoint. EQ-5D,Euro-Qol Five ventolin pill price Dimensions. PHQ-9, PatientHealth Questionnaire-9.PHQ-9 change scores and EQ-5D change scores. EQ-5D, Euro-Qol Five ventolin pill price Dimensions. PHQ-9, Patient ventolin pill price Health Questionnaire-9." data-icon-position data-hide-link-title="0">Figure 2 PHQ-9 change scores and EQ-5D change scores.

EQ-5D,Euro-Qol Five ventolin pill price Dimensions. PHQ-9, PatientHealth Questionnaire-9.View this table:Table 1 Conversion table from PHQ-9 to EQ-5D total and change scoresSensitivity analysisWhen we limited the samples to the three studies28–30 that administered PHQ-9 (total n=1375), the linking results were replicated (online supplemental eFigure 1).DiscussionThis is the first study to link a depression severity measure with the EQ-5D-3L both for total and change scores. To summarise, subthreshold depression corresponded with EQ-5D-3L ventolin pill price index values of 0.9–0.8, mild major depression with 0.8–0.7, moderate depression with 0.7–0.5 and severe depression with 0.6–0.0. A five-point improvement in PHQ-9 corresponded approximately with an increase in EQ-5D-3L index values by ventolin pill price 0.03, and a ten-point improvement can lead to an increase by approximately 0.25.A systematic review of utility values for depression31 found that the pooled mean (SD) utilities based on studies using the standard gamble as a direct valuation method were 0.69 (0.14) for mild, 0.52 (0.28) for moderate and 0.27 (0.26) for severe major depression. The estimates based on studies using EQ-5D as an indirect valuation method were 0.56 (0.16) for mild, 0.52 (0.28) for moderate and 0.25 (0.15) for severe depression.

One recent study regressed PHQ-9 on SF-6D ventolin pill price scores among 394 patients in theimproving Access to Psychological Therapies (IAPT) cohort7 32 and estimated none/mild depression on PHQ-9 to be worth 0.73 SF-6D scores, moderate depression 0.65 and severe depression 0.56. Our results are largely in line with these aforementioned ventolin pill price studies.There was a consistent difference of about 0.07 EQ-5D scores for the same PHQ-9 score if it represented the baseline or endpoint measurements (figure 1). This is understandable because a patient would rate their health status less satisfactory if they stayed equally symptomatic as before after the treatment and also because it means that they continued to suffer from depression for longer. It is, therefore, reasonable to use the conversion table at baseline for relatively new cases of depression and that at end of treatment for more chronic cases (table 1).An effect size to be ventolin pill price typically expected after 2 months of antidepressant pharmacotherapy33 or psychotherapy27 34 over the pill placebo condition is 0.3. Given that the average SD of PHQ-9 in the studies was about 6, ventolin pill price an effect size of 0.3 corresponds to a difference by two points on PHQ-9.

The differences in EQ-5D scores corresponding with the end-of-treatment PHQ-9 scores of ventolin pill price x versus x+2, where x is between 5 and 15 (table 1), ranges between 0.08 and 0.13, producing an approximate average of 0.1 EQ-5D scores. If we assume that the same difference would continue for the ensuing 10 months, the gain in QALY per year would be equal to 0.09 QALY. If we assume that the difference would eventually wear out over the course of the year due to naturalistic improvements to be expected in the control group, the gain in QALY per year would be equal to ventolin pill price 0.05 QALY. (See figure 3 for a schematic drawing to help understand the calculation of QALYs based on ventolin pill price changing EQ-5D scores. In reality, the changes will be more smoothly curvilinear but the calculation will be similar.) Since one QALY is typically valuated at US$50 000 or 3000 Stirling pounds,35 such therapies would be cost-effective if they cost US$2500 to US$4500 (150 to 270 pounds) or less.

If a 1 day fill of generic selective serotonergic reuptake inhibitor antidepressants costs 1–3 dollars and a 1-year prescription costs US$400–1200 dollars, or if 8–16 sessions of psychotherapy cost US$1600–3200 dollars, both therapies would be ventolin pill price deemed largely cost-effective. An individual’s decision, by contrast, will and should ventolin pill price be more variable and no one can categorically reject nor require such treatments for all patients.A schematic graph showing gains in QALY due to typical pharmacotherapies or psychotherapies. A patient may start with ventolin pill price PHQ-9 of 20, corresponding with EQ-5D index value of 0.5. Then they may improve after 2 months of antidepressant therapy to EQ-5D score of 0.9 (solid line), while they may improve to EQ-5D score of 0.8 even if on placebo (dashed line). If we ventolin pill price assume that the same difference would continue for the ensuing 10 months while showing slow gradual improvement in both cases, the gain in QALY per year would be equal to 0.09 QALY.

If we assume that the difference would eventually wear out over the course of the year due to naturalistic improvements to be expected in the control group, the gain in QALY per year would be equal to 0.05 ventolin pill price QALY. Please note that this is a schematic drawing for illustrative purposes. In reality, the changes will be more smoothly curvilinear but the calculation ventolin pill price will be similar. EQ-5D, Euro-Qol ventolin pill price Five Dimensions. PHQ-9, Patient Health Questionnaire-9 ventolin pill price.

QALY, quality-adjusted life years." data-icon-position data-hide-link-title="0">Figure 3 A schematic graph showing gains in QALY due to typical pharmacotherapies or psychotherapies. A patient may start with PHQ-9 of 20, corresponding with EQ-5D index ventolin pill price value of 0.5. Then they ventolin pill price may improve after 2 months of antidepressant therapy to EQ-5D score of 0.9 (solid line), while they may improve to EQ-5D score of 0.8 even if on placebo (dashed line). If we assume that the same difference would continue for the ensuing 10 months while showing slow gradual improvement in both cases, the gain in QALY per year would be equal to 0.09 QALY. If we assume that the difference would eventually wear out over the course of the year due to naturalistic improvements to be expected in the control group, the gain in QALY per year would be ventolin pill price equal to 0.05 QALY.

Please note that this is a schematic drawing ventolin pill price for illustrative purposes. In reality, the changes will be more smoothly curvilinear but the calculation will be similar. EQ-5D,Euro-Qol Five Dimensions ventolin pill price. PHQ-9, PatientHealth ventolin pill price Questionnaire-9. QALY, quality-adjustedlife years.Several caveats should be considered when interpreting ventolin pill price the results.

First, our sample was limited to participants of trials of iCBT. It may be argued that the ventolin pill price results, therefore, would not apply to patients with depression undergoing other therapies or in other settings. Second, the correlations between PHQ-9 and EQ-5D were strong enough for total scores at endpoint and for change scores to justify linking but were somewhat weaker at baseline, probably due to limited variability in ventolin pill price PHQ-9 scores at baseline because some studies required minimum depression scores. However, the overall correspondence between PHQ-9 scores and EQ-5D had the same shape between baseline and endpoint, which will increase credibility of the linking at baseline as well. Third, we were able to compare PHQ-9 to ventolin pill price EQ-5D-3L only.

The EQ-5D-5L, which measures health in five levels instead of three, has been developed to be ventolin pill price more sensitive to change and to milder conditions.36 When data become available, we will need to link PHQ-9 and EQ-5D-5L to examine if we can obtain similar conversion values.Our study also has several important strengths. First, our sample included patients with subthreshold depression and major ventolin pill price depression and from the community or workplace and the primary care. Furthermore, they encompassed mild through severe major depression in approximately equal proportions. Second, all the patients in our sample received iCBT or control interventions including care ventolin pill price as usual. Potential side effects of different antidepressants, repetitive brain stimulation, electroconvulsive therapy and other more aggressive therapies must of course be taken into consideration when ventolin pill price evaluating their impacts, but our estimates, arguably independent of major side effects, can better inform such considerations.

Finaly, unlike any prior studies, we were able to link specific PHQ-9 scores and their changes scores to EQ-5D-3L index values.Conclusion and clinical implicationsIn conclusion, we constructed a conversion table linking the EQ-5D, the representative generic preference-based measure of health status, and the PHQ-9, one of the most popular depression severity rating scale, for both its total scores and change scores. The table will enable fine-grained assessment of burden of depression at its various levels of severity and of impacts of its various treatments which may bring various degrees of improvement at ventolin pill price the expense of some potential side effects.Data availability statementData are available upon reasonable request. The overall ventolin pill price database used for this IPD is restricted due to data sharing agreements with the research institutes where the studies were conducted. IPD from individual studies are available from the individual study authors.Ethics statementsPatient consent for publicationNot required..

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Michigan on Tuesday finalized a requirement that all 440,000 licensed or registered health Kamagra oral jelly thailand price workers in the state undergo annual hidden bias training to help address disparities best online ventolin in how patients are treated. The rule, which was initially ordered by Gov. Gretchen Whitmer last July, best online ventolin will take effect on June 1, 2022. Health workers renewing their license or registration will have to complete at least one hour of training each year.

New applicants will be required to receive at least two hours initially. Only those in veterinary medicine will be best online ventolin exempt. "We all have some form of implicit bias. We've got to acknowledge that and use proven methods to lessen the impact of that bias that we all bring to the table," the Democratic governor said at the Forest Community Health Center in Lansing.

The asthma ventolin, she said, has exposed best online ventolin and exacerbated underlying inequities such as the disparate impact of health outcomes by race. Whitmer last year ordered state employees to complete implicit bias training. Implicit bias is defined as the attitudes or stereotypes that affect people's understanding, actions and decisions in an unconscious manner, according to the Kirwin Institute for the Study of Race and Ethnicity at Ohio State University..

Michigan on Tuesday finalized a requirement that all 440,000 licensed or registered health workers in the state undergo annual hidden bias training to help address ventolin pill price disparities in Kamagra oral jelly thailand price how patients are treated. The rule, which was initially ordered by Gov. Gretchen Whitmer last July, will ventolin pill price take effect on June 1, 2022. Health workers renewing their license or registration will have to complete at least one hour of training each year.

New applicants will be required to receive at least two hours initially. Only those ventolin pill price in veterinary medicine will be exempt. "We all have some form of implicit bias. We've got to acknowledge that and use proven methods to lessen the impact of that bias that we all bring to the table," the Democratic governor said at the Forest Community Health Center in Lansing.

The asthma ventolin, she said, has exposed and exacerbated underlying ventolin pill price inequities such as the disparate impact of health outcomes by race. Whitmer last year ordered state employees to complete implicit bias training. Implicit bias is defined as the attitudes or stereotypes that affect people's understanding, actions and decisions in an unconscious manner, according to the Kirwin Institute for the Study of Race and Ethnicity at Ohio State University..

What should I tell my health care providers before I take Ventolin?

They need to know if you have any of the following conditions:

• diabetes
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• pheochromocytoma
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• an unusual or allergic reaction to albuterol, levalbuterol, sulfites, other medicines, foods, dyes, or preservatives
• pregnant or trying to get pregnant
• breast-feeding

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Each year, during the winter days as how much does ventolin hfa cost without insurance the daylight decreases, some individuals dangers of ventolin may experience a mood disorder known as Seasonal Affective Disorder (SAD). Also known as Seasonal Depression, SAD is considered a depressive episode which reoccurs the same time each year, usually in the fall, worsens during the winter months and ends as daylight increases in the spring.“Researchers have discovered that 75 percent of SAD sufferers are women with the disorder, typically beginning in early adulthood,” said Michelle Lucchesi, M.A., L.L.P., therapist of the Psychiatric Partial Hospitalization Program at MidMichigan Medical Center – Gratiot. €œHowever, SAD can also occur in men, children and adolescents.”The National Institute of Mental Health has found that SAD occurs as a response to the decrease daylight during dangers of ventolin the winter months. Symptoms of SAD are similar to depression and may include.

Sadness. Loss of interest in usual activities. Difficulty concentrating. Irritability.

Feeling tired. Lack energy. Weight gain, craving sweets and starchy food, and difficulty with sleep. While it is important to talk to a healthcare professional for a proper diagnosis and treatment of SAD, light therapy is found to be the most effective treatment.“Light therapy is an alternative to using antidepressant medications for those who have mild SAD and do not want to take medications,” explained Lucchesi.

€œLight therapy is used daily with individuals sitting for 30 minutes in front of a light box after waking up in the morning.”It is recommended that individuals begin light therapy each fall when symptoms of SAD often set in and continue every day throughout the winter months. Lamps for light therapy are widely available and much more affordable than when they first were introduced years ago.In addition to light therapy, additional options to help reduce symptoms of SAD include. Spending time outside every day. Eating a well-balanced diet.

Establishing a good sleep routine. Getting at least 30-minutes of exercise a day, as well as staying socially connected with loved ones and community (as safely as possible during asthma treatment).Those needing additional help to overcome mood disorders such as SAD are encouraged to seek help from their health care provider. In addition, the Psychiatric Partial Hospitalization Program (PHP) mental health day program at MidMichigan Medical Center – Gratiot is available for those who need additional support. Those with questions may call (989)466-3253.

Those interested in more information on MidMichigan’s comprehensive behavioral health programs may visit www.midmichigan.org/mentalhealth.Adapted by Michelle Lucchesi MA L.L.P. From an article by Callie Neyer, M.A./L.P.C..

Each year, during the winter days as the daylight decreases, ventolin price without insurance some individuals may experience a mood disorder ventolin pill price known as Seasonal Affective Disorder (SAD). Also known as Seasonal Depression, SAD is considered a depressive episode which reoccurs the same time each year, usually in the fall, worsens during the winter months and ends as daylight increases in the spring.“Researchers have discovered that 75 percent of SAD sufferers are women with the disorder, typically beginning in early adulthood,” said Michelle Lucchesi, M.A., L.L.P., therapist of the Psychiatric Partial Hospitalization Program at MidMichigan Medical Center – Gratiot. €œHowever, SAD can also occur in men, children and adolescents.”The National Institute of Mental Health has found that SAD occurs as ventolin pill price a response to the decrease daylight during the winter months. Symptoms of SAD are similar to depression and may include.

Sadness. Loss of interest in usual activities. Difficulty concentrating. Irritability.

Feeling tired. Lack energy. Weight gain, craving sweets and starchy food, and difficulty with sleep. While it is important to talk to a healthcare professional for a proper diagnosis and treatment of SAD, light therapy is found to be the most effective treatment.“Light therapy is an alternative to using antidepressant medications for those who have mild SAD and do not want to take medications,” explained Lucchesi.

€œLight therapy is used daily with individuals sitting for 30 minutes in front of a light box after waking up in the morning.”It is recommended that individuals begin light therapy each fall when symptoms of SAD often set in and continue every day throughout the winter months. Lamps for light therapy are widely available and much more affordable than when they first were introduced years ago.In addition to light therapy, additional options to help reduce symptoms of SAD include. Spending time outside every day. Eating a well-balanced diet.

Establishing a good sleep routine. Getting at least 30-minutes of exercise a day, as well as staying socially connected with loved ones and community (as safely as possible during asthma treatment).Those needing additional help to overcome mood disorders such as SAD are encouraged to seek help from their health care provider. In addition, the Psychiatric Partial Hospitalization Program (PHP) mental health day program at MidMichigan Medical Center – Gratiot is available for those who need additional support. Those with questions may call (989)466-3253.

Those interested in more information on MidMichigan’s comprehensive behavioral health programs may visit www.midmichigan.org/mentalhealth.Adapted by Michelle Lucchesi MA L.L.P. From an article by Callie Neyer, M.A./L.P.C..

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Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, buy ventolin online australia but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing Buy real levitra online rugstore toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM buy ventolin online australia cell) phenotype.

We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells buy ventolin online australia in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach to ACT involves the redirection of peripheral blood T buy ventolin online australia cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding. CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016.

Irving et al., 2017). A range of physical (Lanitis et al., 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and buy ventolin online australia effector function limit the potency of CAR-T cell therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of buy ventolin online australia CAR-T cells may help to overcome some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010).

In addition, CAR-T cells can buy ventolin online australia be armed with secretory molecules or additional receptors to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al., 2018. Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011.

Mardiros et al., 2013 buy ventolin online australia. Lanitis et al., 2012). Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012.

Avanzi et al., buy ventolin online australia 2018). As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of CAR evaluation in syngeneic models stems from inadequate methodologies buy ventolin online australia for efficient murine T cell transduction and expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et al., 2013 buy ventolin online australia.

Wang et al., 2014. Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, this was buy ventolin online australia assessed at 2 to 3 d after transduction (Tran et al., 2013.

Kuhn et al., 2019. Kusabuka et al., 2016) and thus may buy ventolin online australia include false positives due to transient expression from nonintegrated vector DNA (i.e., pseudo-transduction. Case et al., 1999, Costello et al., 2000).

Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et buy ventolin online australia al., 2019. Davila et al., 2013).

Finally, while stable retroviral packaging and producer cell lines may buy ventolin online australia enable transduction efficiencies for 2G and third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming.

Overall, our work provides important tools for enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing mice, which we believe is critical for buy ventolin online australia effective therapies reaching the clinic. We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig.

1 and is described buy ventolin online australia in detail in Materials and methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ. The anti-VEGFR-2 CAR retroviral vector is buy ventolin online australia abbreviated as DC101-28z (Fig.

2 A). Because retroventolines infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et al., buy ventolin online australia 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 beads consistently resulted in the highest frequency of buy ventolin online australia CD44+ CD62L− (recently activated, memory), CD25+ or CD69+ (activated), and Ki67+ (proliferating) CD3+ T cells (Fig.

2 B and Fig. S1 A) buy ventolin online australia. We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml.

Fig. 2 C) and enabled significantly higher transduction of primary buy ventolin online australia activated primary murine T cells as compared with unconcentrated retroventolin (Fig. 2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression.

Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig.

2, E and F). A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G.

Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I). Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig.

S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A).

Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002. Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig.

2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B). Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig.

3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E).

We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig. 3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig.

3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen. On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig.

3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2).

Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig. 3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig.

3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells. The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance.

Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retroventolin (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig.

4, B and C), as well as high intracellular expression of mIL-15 (Fig. 4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig.

4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retroventolin producer cell line. Fig.

4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig.

4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017). We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig.

4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig. 4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site.

Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM.

CD62Lhigh CD44high) and effector memory (EM. CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig.

5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1. Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig.

5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig.

5 H). Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retroventolin coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15.

We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A). Viability was similarly high for both over a 10-d period (Fig.

6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig.

S3 D). Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig.

6 E and Fig. S3 D). We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells.

Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I).

The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig. 6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L).

The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017). We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors.

Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B).

Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig. 7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig.

7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig. 7, D and F).

Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted.

Following the same ACT strategy as demonstrated above (Fig. 8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig.

S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig. 8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig.

8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer.

Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig. S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F).

Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig. S5, G and H).

The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005.

Gattinoni et al., 2005. Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo.

Retroviral vectors, most commonly derived from the murine stem cell ventolin (MSCV), a derivative of the Moloney murine leukemia ventolin, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentiventolin, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004. Tsurutani et al., 2007).

Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retroventolin-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016.

Kochenderfer et al., 2010). Despite that it is common procedure to concentrate lentiventolin via ultracentrifugation, this is usually not performed for CAR-encoding retroventolines. In this study, we demonstrated that retroventolin can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies.

We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d. Tran et al., 2013.

Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function.

Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998). In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015.

Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009).

Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014. Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells.

Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement.

The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002. Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge.

The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors. We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene.

Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018).

In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014). In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control.

As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate.

Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell ventolin [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010).

The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation. To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized.

The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retroventolin was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retroventolin Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific.

Fig. 1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific).

For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig. 1 A, 2).

Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium. The retroventolin was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig.

1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies. Fig.

1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2).

1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig. 1 B, 2.1). Subsequently, plates were washed once, retroventolin was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig.

1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates. Fig.

1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3).

At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig. 1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml.

For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform. DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017.

Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend).

For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C. Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322.

Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3. EBioscience), Bcl-2 (clone 10C4.

EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star).

Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer.

Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows. DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′.

DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′.

GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′. Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method.

We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions.

Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E. Lanitis, G.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies.

Romero and D. Dangaj reviewed the data and manuscript and provided suggestions. All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &.

Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.

Limited clinical ventolin pill price benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses http://steveplattner.com/buy-real-levitra-online-rugstore/. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of ventolin pill price murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages.

The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, ventolin pill price an important step toward the acceleration of effective therapies reaching the clinic. The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach ventolin pill price to ACT involves the redirection of peripheral blood T cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding.

CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016. Irving et al., 2017). A range of physical (Lanitis et al., 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency of CAR-T cell ventolin pill price therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of ventolin pill price CAR-T cells may help to overcome some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010). In addition, CAR-T cells can be ventolin pill price armed with secretory molecules or additional receptors to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al., 2018.

Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011. Mardiros et ventolin pill price al., 2013. Lanitis et al., 2012).

Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012. Avanzi et ventolin pill price al., 2018). As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of ventolin pill price CAR evaluation in syngeneic models stems from inadequate methodologies for efficient murine T cell transduction and expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et ventolin pill price al., 2013. Wang et al., 2014.

Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, this was assessed at 2 to 3 d ventolin pill price after transduction (Tran et al., 2013. Kuhn et al., 2019. Kusabuka et al., 2016) and thus ventolin pill price may include false positives due to transient expression from nonintegrated vector DNA (i.e., pseudo-transduction.

Case et al., 1999, Costello et al., 2000). Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et al., ventolin pill price 2019. Davila et al., 2013).

Finally, while stable retroviral packaging and producer cell lines may enable transduction efficiencies for 2G and ventolin pill price third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming. Overall, our work provides important tools for enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing mice, which ventolin pill price we believe is critical for effective therapies reaching the clinic.

We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig. 1 and is described in detail in Materials and ventolin pill price methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ.

The anti-VEGFR-2 CAR retroviral vector is abbreviated ventolin pill price as DC101-28z (Fig. 2 A). Because retroventolines infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et al., ventolin pill price 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 beads consistently resulted in the highest frequency of CD44+ CD62L− (recently ventolin pill price activated, memory), CD25+ or CD69+ (activated), and Ki67+ (proliferating) CD3+ T cells (Fig. 2 B and Fig.

S1 A) ventolin pill price. We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml. Fig. 2 C) ventolin pill price and enabled significantly higher transduction of primary activated primary murine T cells as compared with unconcentrated retroventolin (Fig.

2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression. Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig. 2, E and F).

A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G. Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I).

Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig. S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A). Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002.

Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig. 2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B).

Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig. 3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E). We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig.

3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig. 3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen.

On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig. 3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2). Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig.

3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig. 3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells.

The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance. Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retroventolin (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig. 4, B and C), as well as high intracellular expression of mIL-15 (Fig.

4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig. 4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retroventolin producer cell line.

Fig. 4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig. 4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017).

We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig. 4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig.

4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site. Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM. CD62Lhigh CD44high) and effector memory (EM.

CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig. 5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1.

Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig. 5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig. 5 H).

Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retroventolin coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15. We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A).

Viability was similarly high for both over a 10-d period (Fig. 6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig. S3 D).

Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig. 6 E and Fig. S3 D).

We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells. Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I). The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig.

6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L). The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017).

We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors. Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B). Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig.

7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig. 7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig.

7, D and F). Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted. Following the same ACT strategy as demonstrated above (Fig.

8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig. S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig.

8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig. 8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer. Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig.

S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F). Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig.

S5, G and H). The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo. Retroviral vectors, most commonly derived from the murine stem cell ventolin (MSCV), a derivative of the Moloney murine leukemia ventolin, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentiventolin, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004.

Tsurutani et al., 2007). Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retroventolin-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016. Kochenderfer et al., 2010).

Despite that it is common procedure to concentrate lentiventolin via ultracentrifugation, this is usually not performed for CAR-encoding retroventolines. In this study, we demonstrated that retroventolin can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies. We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d.

Tran et al., 2013. Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function. Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998).

In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015. Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009). Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014.

Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells. Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement. The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002.

Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge. The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors.

We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene. Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018). In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014).

In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control. As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.

The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate. Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell ventolin [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010). The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation.

To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized. The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retroventolin was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retroventolin Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific. Fig.

1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific). For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig.

1 A, 2). Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 3). At 48 (Fig. 1 A, 4) and 72 h (Fig. 1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium.

The retroventolin was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig. 1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies.

Fig. 1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2). 1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig.

1 B, 2.1). Subsequently, plates were washed once, retroventolin was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig. 1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates.

Fig. 1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3). At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig.

1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml. For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform.

DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017. Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend). For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C.

Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322. Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3.

EBioscience), Bcl-2 (clone 10C4. EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star). Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific).

Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer. Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows.

DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′. DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′. GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′.

Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method. We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions. M.

Irving, G. Coukos, and E. Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E.

Lanitis, G. Rota, P. Kosti, C. Ronet, and A.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies. B.

Seijo built essential constructs. P. Romero and D. Dangaj reviewed the data and manuscript and provided suggestions.

All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &. Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.