"VSports手机版" IL15 enhances CAR-T-cell antitumor activity by reducing mTORC1 activity and preserving their stem cell memory phenotype

VSports在线直播 - Tumor cell lines

Raji-ffluc (CD19+; a kind gift from Dr. Michael Jensen in 2014), LCL (CD19+; generated by Epstein-Barr virus (EBV) transformation of human B cells as described previously (8), KG1A (CD19–;a kind gift from Dr. Ravi Bhatia in 2008) and human glioma cell line PBT030–2-ffLuc (IL13Rα2+; low passage tumor sphere line derived from human GBM tissue) were maintained as previously described (22). Cell banks of tumor lines were authenticated for the desired antigen/marker expression by flow cytometry and tested for mycoplasma VSports. Thawed cells from banks were maintained in culture for less than 1–3 months.

T cell isolation, lentiviral transduction and ex vivo CAR-T cell expansion

Blood products were obtained from healthy donors or discard kits with informed written consent (whenever necessary) after protocols were approved by the City of Hope (COH) Internal Review Board (IRB). All studies were conducted in accordance with U. S. Common Rule ethical guidelines. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation over Ficoll-Paque (GE Healthcare) and then underwent CD14 depletion (PBMC population). For the CD62L+ population, additional CD25 depletion (Miltenyi Biotec) followed by CD62L+ selection was performed. T cells were stimulated with Dynabeads Human T-Expander CD3/CD28 (Invitrogen) at a 1:3 ratio (T cell:bead) overnight in X-VIVO-15 (Lonza) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and different cytokine cocktails as follows: IL2/IL15 [50 U/mL IL2 (Novartis), 0. 5 ng/mL IL15 (CellGenix)]; IL15/IL7 [10 ng/mL IL15, 10 ng/mL IL7 (Miltenyi Biotec)]; IL15/IL7/IL21 [10 ng/mL IL15, 10 ng/mL IL7, 10 ng/mL IL21(Miltenyi Biotec)]; IL15 [10 ng/mL] or IL2 titrations [25, 20 or 10 U/mL IL2 (Novartis) 0 VSports app下载. 5 ng/mL IL15 (CellGenix)]. Stimulated T cells were then transduced or not with a lentivirus vector encoding the CD19-CAR or IL13Rα2-CAR as previously described (8,22,23). Mock and transduced cell cultures were given the indicated cytokines three times a week for 18–21 days of culture before subsequent analyses. For time point analyses comparing CAR-T/IL2 with CAR-T/IL15, exogenous cytokines were replaced three times per week for up to 32 days, and cells were cryopreserved at an early (days 14–16), mid (day 23) or late (day 32) time point post-bead stimulation for further detailed analyses. For rapamycin treated groups, CAR-T/IL2 cells were incubated with rapamycin (100nM) (LC Laboratories) beginning at 4 days post-bead stimulation and refreshed with media containing rapamycin three times per week.

Flow Cytometry

CAR-T cell lines were stained with fluorochrome-conjugated antibodies against the following markers: CD3, CD8, CD45, CD62L, CD27, CD45RA, CD45RO, CD127, CD95 (BD Biosciences); Lag3, PD-1 (eBioscience); Tim3 (R&D Systems); CCR7, or 2B4 (BioLegend). Staining with anti-EGFR (BioLegend) or anti-CD19 (BD Biosciences) was used as a surrogate for CAR expression in cells transduced to express CD19-CAR or IL13Rα2-CAR, respectively. For assessment of active caspase 3, CAR-T cell lines at the early, mid, and late time point were fixed and stained using the PE-Anti-Active Caspase3 Kit (BD Biosciences) according to the manufacturer’s instructions. For p-STAT5 staining, cells were fixed with 4% paraformaldehyde, followed by 10 min incubation in chilled methanol. Cells were stained with p-STAT5-Alexa Fluor 488 (BD biosciences).

For assessment of CAR-T cell proliferation and cytotoxic activity, 25,000 CD19-CAR-T cells and 25,000 tumor cells (Raji-ffluc, LCL, or KG1A) were incubated for seven days. The cell mixture was stained with anti-CD3, -CD8, and -EGFR. Absolute number of viable tumor cells and CAR-T cells was measured using flow cytometry.

For the degranulation assay, 50,000 CD19-CAR-T cells and 50,000 tumor cells (Raji-ffluc, LCL, or KG1A) were co-cultured for 5 hours in the presence of GolgiStop Protein Transport Inhibitor (BD Biosciences). The cell mixture was stained with anti-CD3, -CD8, and –EGFR followed by intracellular staining with anti-IFNγ (BD Biosciences). Degranulation assay post adoptive T cell therapy was performed as previously described (5). Briefly, intratumoral CAR-T cells were isolated post therapy and co-cultured ex vivo with tumor at a 1:1 ratio for 5 hours in the presence of GolgiStop Protein Transport Inhibitor (BD Biosciences). The cell mixture was stained with anti-CD45, -CD8, -CD107a followed by intracellular staining with anti-IFNγ and -TNFα (BD Biosciences).

Recursive killing assay was performed as previously described (5). Briefly, CAR-T/IL2 or CAR-T/IL15 cells were co-cultured with tumor (1:4 for IL13Rα2-CAR:tumor; 1:3 for CD19-CAR:tumor). CAR-T cells were rechallenged with additional tumor cells as described in the results. At the end of repetitive tumor challenge (5–7 days), viable tumor cells and CAR-T cells were counted using flow cytometry.

All samples were acquired on MACSQuant Analyzer 10 (Miltenyi Biotec) and analyzed with FlowJo software (v10.1, TreeStar) and GraphPad Prism Software (v5).

Metabolism Assays:

Mitochondrial oxygen consumption rate (OCR) was measured using the Seahorse Bioscience XF96 Extracellular Flux Analyzer (Agilent). Briefly, 0.2 or 0.4 million cells were seeded in cell culture microplates on the day of the experiment. Cells were suspended in OCR XF Assay medium (Agilent, 102365–100) supplemented with glucose (25 mM, Sigma) and sodium pyruvate (1 mM, Gibco). The pH value of the assay medium was adjusted to 7.4. OCR was measured following sequential injections of oligomycin (1 mM, Sigma), FCCP (0.5 mM, Sigma), and rotenone (2.5 mM, Sigma) according to the manufacturer’s instructions. The OCR measurements were normalized to cell numbers.

To measure mitochondria membrane potential, T cells were incubated with tetramethyl rhodamine methyl ester (TMRM; Invitrogen) at a final concentration of 25 nm for 15 min at 37°C. To quantify glucose uptake, T cells were incubated with 2-NBDG (fluorescent glucose analog; Invitrogen) at a final concentration of 50 μM for 30 min at 37°C. Mean fluorescent intensity (MFI) of cells was measured using flow cytometry.

"V体育官网入口" RNA Sequencing analysis:

Thawed CD19-CAR-T cells were labelled with fluorescently-labelled anti-CD4, -CD8 (BD Biosciences), and -EGFR (BioLegend). CAR-expressing (EGFR⁺) CD4⁺ or CD8⁺ cell populations were separated using a BD FACSAriaII (BD Biosciences). RNA was isolated from pure CD4⁺ or CD8⁺ populations using the RNeasy Mini kit (Qiagen), and cDNA was reverse transcribed using the SuperScript VILO Mastermix (Life Technologies) according to the manufacturer’s instructions. RNA sequencing library preparation was conducted as previously described (5). The 51-bp single-ended sequence reads were mapped to human genome (hg19) using TopHat (Version 2.0.8), and the expression of Refseq genes was counted with customized R scripts. A gene was considered expressed with an RPKM (reads per kilobase of gene per million reads mapped to exons) value more than one. Only genes expressed in at least one sample were kept for differential expression analysis. The raw counts were then normalized using the TMM (weighted trimmed mean of M-values) method, and differentially expressed genes were identified by two-fold change (≥ 2 and ≤ 0.5 for up- and down-regulated genes respectively) via exactTest using the Bioconductor package “edgeR” (24).

Multidimensional scaling (MDS) analysis

Classical multidimensional analysis (MDS) plot was generated using the plotMDS function in edgeR Bioconductor package (24). Briefly, the distance matrix was computed from RNA-seq data in units of RPKM and used as input for MDS analysis. The first two coordinates of the configuration matrix are plotted with numbers corresponding to the differentially expressed genes (including both up- and down-regulated genes) with a log2 (fold change) ≥ 1 or ≤ −1 in gene expression between indicated samples. Graph corresponds to four samples: CAR-T/IL2 – Day 14, CAR-T/IL2 – Day 32, CAR-T/IL15 – Day 14 and CAR-T/IL15 – Day 32.

Immunoblot analysis

CD19-CAR-T cells were lysed in ice-cold RIPA buffer (Thermo Scientific) containing protease inhibitor cocktail (Roche). Supernatants from centrifuged whole cell lysates were prepared in 4x LDS Sample Buffer (Life Technologies) and run on a 4–12% Bis-Tris gel (Life Technologies). Gels were transferred to a 0.45μm PVDF membrane (Life Technologies) and incubated overnight at 4°C using primary antibody to Bcl2, pAkt-S473, Akt, prpS6-S235/236, rpS6, Glut1, and CPT1a (Cell Signaling); p-STAT5 (BD biosciences); actin (Abcam); or GAPDH (Millipore), then incubated with HRP-conjugated secondary antibody (Millipore) and imaged using ECL detection reagent (GE Healthcare Amersham) and TI-BA Series 2000A film processor on autoradiography film (Denville Scientific).

Q-RT-PCR analysis

At the early and late time-points, CAR-T cells were separated into CD4⁺ or CD8⁺ populations using the EasySep CD8 or CD4 Positive Selection Kits (Stem Cell Technologies), respectively. RNA was isolated using the RNeasy Mini Kit (Qiagen). cDNA was reverse transcribed using the SuperScript VILO Mastermix (Life Technologies) according to the manufacturer’s instructions. qPCR reactions were performed as previously described (5). Primers are described in Supplementary Table S1.

In vivo xenograft models

All mouse experiments were approved by the City of Hope Institutional Animal Care and Use Committee (IACUC). For CD19-CAR-T studies, NOD/Scid IL2RγC^null (NSG) mice (6–10-week-old) were injected with 0.5×10⁶ Raji-ffluc cells (CD19⁺) intravenously (i.v.) on day 0. CD19-CAR-T cells or mock-transduced T cells (1×10⁶) that were expanded in vitro under different cytokine conditions were injected i.v. into mice 3 days after tumor inoculation. For IL13Rα2-CAR-T studies, NSG mice (6–10 week-old) were injected with 0.1×10⁶ human glioma cell line (IL13 Rα2⁺) intracranially (i.c.) as described before (22). On day 8, IL13Rα2-CAR-T cells or mock (5×10⁴ cells), expanded in different cytokine conditions, were administered intratumorally. Tumor burden was monitored with Xenogen IVIS (Xenogen) or SPECTRAL Ami X (Spectral Instruments Imaging) and analyzed using Living Image 2.50 (Perkin Elmer) or AMIView software (v1.7.061, Spectral Instruments Imaging). Survival curves were generated by GraphPad Prism Software (v5). For some experiments, post-therapy, retro-orbital blood samples were RBC-lysed using PharmLyse buffer (BD Biosciences), then assessed using anti-human CD45 and anti-human CD3 antibodies (BD Biosciences) to measure the frequency of adoptively transferred T cells by flow cytometry. Assessment of CAR-T function post-therapy was conducted as previously described (5). Briefly, tumors were injected (1×10⁶ cells) after 7 days, CAR-T/IL15 or CAR-T/IL2 was injected intratumorally. T cells were isolated and re-cultured with tumor at 1:1 ratio and degranulation assay was conducted as described above.

Statistical analysis

Statistical significance was determined using Student t test (two groups) or one-way ANOVA analysis with a Bonferroni (three or more groups). Survival was plotted using a Kaplan-Meier survival curve and statistical significance was determined by the Log-rank (Mantel-Cox) test. All analyses were carried out using GraphPad Prism software (v5). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 as significant; ns, as not significant.

CAR-T cells expanded ex vivo in IL15 maintain a less-differentiated phenotype (V体育官网)

Previous studies have indicated that IL2 promotes the generation of highly differentiated T cell subsets such as Tem and Teff (10,25). By comparison, IL15 is reported to promote the generation of memory T cells (11,26,27), and promote T cell survival and antitumor activity in mice (28,29). To assess whether the replacement of IL2 by IL15 may overcome this pitfall for human CAR-T cells expanded under clinically relevant manufacturing processes, we compared ex vivo expanded CD19-CAR-T cells cultured with either IL2 (CAR-T/IL2) or IL15 (CAR-T/IL15). The CD19-CAR is a second-generation CD28 CAR (30), which is currently being evaluated clinically (; ; ). For these studies, CAR-T cells were cultured in 10ng/ml IL15 or our current clinical cGMP manufacturing cytokine condition, which uses 50U/ml IL2 with low levels of IL15 (0.5ng/ml) (IL2/IL15low) (23,31). CAR-T cells cultured with IL2/IL15low (manufacturing platform) show no phenotypic differences when compared with 50 U/mL IL2 alone; therefore CAR-T cells generated from IL2/IL15low were labeled as CAR-T/IL2 (Supplementary Fig. S1). For these studies, unless otherwise stated, we also utilized our current clinical manufacturing platform, which enriches CD62L⁺ T cells, primarily consisting of naïve (Tn), stem cell memory (Tscm) and central memory (Tcm) subsets, for CAR-engineering. Flow cytometry analysis of selected T cells post-enrichment confirms that >85% of the T cells are CD62L⁺ and 55±10% are CD3⁺CD45RA⁺ CD62L⁺ Tn or Tscm (3 representative healthy donors; Supplementary Fig. S2).

To compare IL2 to IL15 ex vivo expansion conditions, we expanded cells over an extended period of time (up to 32 days) as this process allows the two populations to be compared for differentiation, exhaustion and apoptosis status (32). Our data indicate that throughout extended culture, CAR-T/IL15 maintains a higher proportion of T cells (both CD4⁺ and CD8⁺) that exhibit a Tscm phenotype (defined as CD95⁺, CD45RA⁺ CCR7⁺, CD62L⁺ CD27⁺ or CD62L⁺ CD127⁺) as compared to CAR-T/IL2 (Fig. 1A and B; Supplementary Fig. S3A), which corresponded to a higher CD45RA:CD45RO ratio for CAR-T/IL15 (Supplementary Fig. S3B). Further qPCR analysis demonstrated an up-regulation of key memory stem-like-associated transcription factors (i.e. LEF1 and TCF7) in CAR-T/IL15 as compared to CAR-T/IL2, which was confirmed with the RNA sequencing analysis conducted on purified CD4⁺ and CD8⁺subsets (Fig. 1C and D). In line with this finding, CAR-T/IL15 displayed reduced expression of genes regulating effector differentiation such as eomesodermin (EOMES), T-box 21 (TBX21), as well as cytotoxic molecules (for example, granzyme B and perforin) as compared to CAR-T/IL2 (Fig. 1E and F). Furthermore, CAR-T/IL15 cells expressed less IFNγ (a marker of Teff cells) following antigen stimulation with CD19⁺ Raji cells compared to CAR-T/IL2 cells (Fig. 1G and Supplementary Fig. S3C). With extended culture, we observed a decrease in the frequency of CD4⁺ T cells in both culture conditions, which resulted in insufficient coverage by RNA-seq for analysis on day 32 (Fig. 1D, F).

Next, to further assess the influence of IL15 and IL2 on CAR-T cells during ex vivo expansion, CD8⁺CAR⁺ cells from each culture condition at early and late time points (days 14 and 32) were compared for global gene expression changes. Hierarchical clustering highlighted extensive differences between the two culture conditions (Fig. 2A). Multidimensional scaling (MDS) analysis showed that by day 14 in culture, CAR-T/IL2 and CAR-T/IL15 exhibited different expression profiles (721 differentially expressed genes) and even greater differences by day 32 (1687 differentially expressed genes, P < 0.01 and greater than twofold change in expression, Fig. 2B). Extended culture over time altered the gene expression profiles more for CAR-T/IL2 cells (1674 differentially expressed genes) than CAR-T/IL15 cells (782 differentially expressed genes). Furthermore, as early as day 14, 123 genes were differentially expressed in CD4⁺ T cell population cultured in IL2 as compared to IL15 (Supplementary Fig. S4). These data thus confirm that CAR-T/IL15 cells retain their phenotypic characteristics in both CD4⁺ and CD8⁺ T cell populations compared to their CAR-T/IL2 counterparts.

IL15 promotes T cell survival and inhibits T cell exhaustion

The role of IL2 in proliferation and activation induced cell death (AICD) is well established (33), and IL15 is a known anti-apoptotic factor in several systems (34). Inhibition of caspase-3 activity through IL15-mediated posttranslational modifications improves T cell survival (28). Further, IL15 enhances antioxidant capacity of T cells thus leading to increased T cell persistence (35). To extend these studies to the ex vivo expansion of human CAR-T cells, we evaluated the CD19-CAR-T cell apoptotic phenotype under these conditions. Our data indicate that CD19-CAR-T/IL15 cells expressed significantly less active caspase-3 compared to CAR-T/IL2 (Fig. 3A). Up-regulation of the anti-apoptotic molecule Bcl2 in CAR-T/IL15 compared with CAR-T/IL2 further confirms that IL15 exerts an anti-apoptotic effect on T cells (Fig. 3B).

T cell exhaustion is a state of dysfunction that at early and intermediate stages involves up-regulation of inhibitory receptors such as PD-1, Lag3 and 2B4 and is accompanied by reduced antitumor function (36). Exhaustion impacts CAR-T cell function and persistence, which is a barrier for effective CAR-T cell responses (37). Methods that overcome or prevent T cell exhaustion could improve the effectiveness of CAR-T therapy. To determine whether IL15 impacts T cell exhaustion, T cells were cultured for up to 32 days to allow for induction of inhibitory molecules. Although no difference in frequency of PD-1⁺ T cells was observed (Supplementary Fig. S5A), a significant increase in frequency of Lag3⁺ and 2B4⁺ T cells in CAR-T/IL2 over the extended culture period was detected. Up-regulation of Lag3 and 2B4 also corresponded to increase in LAG3 and CD244 (2B4) gene expression in the CD8⁺ population (Fig. 3C and D). As early as 14 days post ex vivo culture, CD4⁺ T cells cultured in IL2 showed an overall higher expression of inhibitory molecules such as CTLA4 (CTLA4), PD-1 (PDCD1), Lag3 (LAG3), PD-L1 (CD274) and suppressive cytokines and transcription factors such as IL13 and FOXP3, respectively (Fig. 3D). Taken together, these studies demonstrate that human CAR-T cells expanded in IL15 are programmed for survival and sustain a less-exhausted phenotype.

IL15-cultured CAR-T cells exhibit less mTORC1 activity and reduced expression of glycolytic enzymes

We next sought to identify alterations in signaling pathways that may explain the phenotypic and functional differences observed in cells generated in IL15 versus IL2 cytokine conditions. Multiple signal transduction pathways have been implicated in regulating cell differentiation and preserving Tscm phenotype. Although Akt plays a role in T cell effector differentiation (38), our results indicated that phosphorylation of Akt (pAkt) (Fig. 4A) in CAR-T/IL2 cells did not differ from that in CAR-T/IL15 cells. Similarly, STAT5 activation (p-STAT5), which functions downstream of cytokine signaling was also not different between the IL2 and IL15 products (Supplementary Fig. S5B). However, mTORC1 activity was decreased in CAR-T/IL15 by more than 90%, as measured by reduced phosphorylation of ribosomal protein S6 (rpS6) (Fig. 4A). mTORC1 signaling pathway is involved in metabolic changes and regulation of glucose transport (39). Specifically, mTOR signaling increases glycolysis by increasing glucose transporter GLUT1 (SLC2A1) expression and stimulating glycolytic activity (40). Consistent with this observation, IL15 reduced GLUT1expression in CAR T cells and increased expression of CPT1a (CPT1A), an enzyme that regulates fatty acid oxidation (Fig. 4A). To determine if decreased mTORC1 activity in CAR-T/IL15 cells leads to commensurate changes in expression of genes in metabolism of T cells, we evaluated expression of other enzymes involved in glycolysis and fatty acid oxidation pathways. CD19-CAR-T/IL15 had reduced expression of glycolytic enzymes and conversely increased expression of enzymes involved in fatty acid oxidation pathway (Fig. 4B and C) and exhibited overall low mitochondrial potential (Δψm) (Fig. 4D), suggesting that IL15-mediated decrease of mTORC1 activity is associated with metabolic changes in CAR-T cells. Additionally, decrease in GLUT1 expression correlated with reduced glucose uptake in CAR-T/IL15 (Fig. 4D). To determine if these gene expression and metabolic measurements were associated with functional changes in cellular metabolism, we evaluated the mitochondria oxygen consumption rate (OCR), a measure of overall mitochondrial respiration and also an indicator of oxidative phosphorylation (OXPHOS). Consistent with the observed phenotypic changes, CAR-T/IL15 cells exhibited greater OCR and spare respiratory capacity, a feature of long-lived memory T cells (Fig. 4E). These findings are in line with previous studies indicating that Tscm and Tcm subsets are characterized with higher OCR and exhibit low-Δψm, which correlate with superior antitumor activity and in vivo persistence (41).

Lastly, to determine whether IL15-mediated reduction in mTORC1 activity is the factor that preserves the Tscm phenotype, CAR-T/IL2 cells were cultured in the presence or absence of rapamycin (mTORC1 inhibitor) and compared to CAR-T/IL15. Expanding CAR-T/IL2 in the presence of rapamycin promoted a Tscm phenotype, similar to CAR-T/IL15 (Fig. 5A and 5B). Western blot analysis confirms down-regulation of p-rpS6 and Glut1 and up-regulation of CPT1a and Bcl2 in CAR-T/IL15 and IL2 plus rapamycin-cultured cells (Fig. 5C). Collectively, these data demonstrate that IL15-mediated reduction in mTORC1 activity leads to decreased glycolysis and prevents T cell differentiation.

Enhanced proliferative capacity of CAR-T/IL15 cells correlates with antitumor activity and persistence

The phenotypic differences observed in CAR-T/IL15 compared to CAR-T/IL2 indicated that IL15 was superior at preserving the stem-like properties of T cells, which has been previously shown to correlate with improved antitumor potency (11). We therefore compared the in vitro antitumor effects of CD19-CAR-T/IL2 and CAR-T/IL15 cells against CD19⁺ Raji tumor cells in a 7-day co-culture assay. Upon tumor antigen stimulation, CAR-T/IL15 exhibited 1.5-fold higher proliferative capacity and persistence and maintained superior overall in vitro killing capacity as compared to CAR-T/IL2 (Fig. 6A and B). To further evaluate recursive killing capacity of each population, CAR-T cells were rechallenged with additional tumor cells. At the end of repetitive tumor challenge, CAR-T/IL15 cells outperformed CAR-T/IL2 cells in tumor killing capacity (Supplementary Fig. S5C).

Lastly, we compared how extended culture conditions in either IL15 or IL2 can impact CAR-T cell antitumor activity in vivo. CD19-CAR-T/IL2 or CAR-T/IL15 cells expanded for 14 or 32 days were administered to mice bearing Raji-ffluc tumors, an aggressive CD19⁺ lymphoma mouse model. The adoptive transfer of CAR-T/IL15 cells mediated superior antitumor activity as measured by ffluc flux and promoted significantly greater survival advantage than CAR-T/IL2 at both culture time-points (Fig. 6C–E). CAR-T/IL15 cells expanded for 32 days showed similar efficacy as CAR-T/IL2 cells expanded for only 14 days. In line with these findings, CAR-T/IL15 cells exhibited significantly longer persistence than CAR-T/IL2 cells at both early and late expansion period (Fig. 6F and G). CAR-T/IL2 cells taken from extended culture condition (Day 32) exhibited the lowest persistence 7 days after infusion, which correlated with the least efficacious survival outcome (Fig. 6E). Together these data indicate that CAR-T/IL15 cells outperform CAR-T/IL2 cells in antitumor potency both in vitro and in vivo and furthermore, the negative impact of extended culture is partially rescued by culture in IL15.

IL15-mediated effects are observed across various manufacturing processes and CAR designs

To evaluate whether the phenotypic profile of CAR-T/IL15 cells was also observed in PBMC-derived CAR-T cell products, we also assessed the phenotypic changes for PBMC-expanded CAR-T cells cultured in IL2 compared to IL15. As anticipated, PBMC-derived CD19-CAR-T cells, which contain Tn, Tscm, and Tcm, also exhibited IL15-mediated phenotypic changes, which include maintenance of Tscm phenotype (CCR7⁺CD45RA⁺; CD62L⁺CD27⁺; CD62L⁺CD127⁺) and enhanced proliferative capacity upon antigen stimulation (Supplementary Fig. S6A and B). Furthermore, PBMC-derived CAR-T cells cultured in IL15 also exhibited increased expression of Bcl2 and reduced mTORC1 activity as indicated by reduced expression of p-rpS6 (Supplementary Fig. S6C). In vivo, both PBMC- and CD62L⁺-derived CAR-T cells cultured in IL15 showed superior survival benefit compared to IL2-cultured cells (Supplementary Fig. S6D). Together these data suggest that IL15 has similar biological impact on CAR-T cells derived from unselected T cell populations.

To determine whether the phenotypic and functional characteristics observed for CAR-T/IL15 cells were generalizable to other CAR designs, similar studies were conducted using our glioma-targeted IL13Rα2-CAR-T cells, which utilize a 4–1BB costimulatory domain (22). IL13Rα2-CAR-T cells cultured with IL15 also sustained their stem-like phenotype and remained less exhausted when compared with IL2 cultured cells (Supplementary Fig. S7A and B). Similar to CD19-CAR-T cells, IL13Rα2-CAR-T cells cultured in IL15 also exhibited reduced mTORC1 activity and GLUT1 expression, which correlated with greater OCR, low Δψm, and decreased glucose uptake (Supplementary Fig. S7C and D). Furthermore, intratumoral CAR-T/IL15 cells exhibit a greater polyfunctional phenotype (TNFα⁺ CD107a⁺ and IFNγ⁺TNFα⁺) as compared to CAR-T/IL2 cells (Supplementary Fig. S7E). IL13Rα2-CAR-T cells cultured with IL15 exhibited greater antitumor activity upon multiple tumor rechallenge in vitro as well as in vivo against orthotopic GBM tumors (Supplementary Fig. S7F and G). Together these studies suggest that the effect of IL15 is independent of CAR design (CD28 or 4–1BB costim) as both CD19⁻ and IL13Rα2-CAR-T products exhibited similar phenotypic and metabolic advantages when cultured in IL15 alone as compared to expansion in IL2.

"V体育安卓版" Inclusion of other γc-cytokines with IL15 does not improve antitumor activity

Our studies highlight the benefit of culturing in IL15 as compared to IL2. Previous reports demonstrate a benefit in combining IL15 with other cytokine combinations for the ex vivo expansion of human T cells, particularly IL21(42) or IL7 (2). With the goal of defining the optimal condition for the production of improved CAR-T cells, additional cytokine combinations (IL7/IL15 and IL7/IL15/IL21) (2,13–15) were assessed. CAR-T/IL15 cells expressed a more prominent CD45RA⁺CCR7⁺ and CD62L⁺CD27⁺ less-differentiated memory phenotype compared to cells in other culture conditions (Fig. 7A). Further, addition of IL7 and/or IL21 with IL15 increased expression of inhibitory receptors such as Lag3 and 2B4 as compared to IL15 alone (Fig. 7B). The observed changes were detected in both CD4⁺ and CD8⁺ T cell subsets, with CD8⁺ T cells showing a larger difference in phenotypes between the cytokine culture conditions (Fig. 7A and 7B). T cells cultured in IL7/IL15/IL21 exhibited the least favorable phenotype, defined as increased expression of inhibitory molecules and very low frequency of CD45RA⁺CCR7⁺ T cells (7±5 %) (Fig. 7A and B). These studies indicate that addition of other γc-cytokines to the culture condition does not result in a superior CAR-T product.

To compare how CAR-T cells cultured in various cytokine conditions perform in vivo, CD19-CAR-T cells cultured in IL2 (i.e., clinical cGMP manufacturing condition IL2/IL15low), IL15, IL7/IL15, IL7/IL2/IL15 were adoptively transferred at a suboptimal dose against mice bearing CD19⁺ Raji-ffluc tumors. In vivo assessment of CAR-T cell products showed superior antitumor activity of CD19-CAR-T cells cultured in IL15, as compared to other cytokine conditions (Fig. 7C and D). Additional γ_c-cytokines in culture condition resulted in a less efficacious CAR product even with relatively comparable CAR and CD4/CD8 T-cell composition (Supplementary Fig. S8A–D). Furthermore, assessment of T cells isolated from blood post CAR-T therapy showed reduced expression of inhibitory molecules (PD-1, Lag3, 2B4) in CAR-T/IL15 group (Fig. 7E), as compared to other cytokine combinations. Reducing the IL2 concentration did not enhance antitumor activity of CAR-T cells. The phenotypic characteristics of low dose IL2 was similar to standard IL2 (Supplementary Fig. S9A–C). Together this data highlights the impact of IL15 on T cells as compared with other cytokine culture conditions and suggests that inclusion of other γc-cytokines with IL15 can reduce the beneficial properties of IL15 alone.