Simple Summary: T cells modified  with  a chimeric antigen receptor (CAR) that targets BCMA, a protein expressed on malignant  plasma cells, represent a novel treatment option for multiple myeloma.  Despite initially eliminating the disease, the function  of BCMA-directed  CAR-T cells diminishes within a year of administration, leading to disease relapse. The aim of this research was to alter the cytokines used in the ex vivo expansion of anti-BCMA  CAR-T cells, to avoid the development of an unfavorable phenotype that would impair in vivo function. We discovered that CAR-T cells expanded with IL-15 had reduced dysfunction and enhanced persistence compared to those grown with IL-2 or a combination of IL-15 and IL-7, which resulted in longer and improved anti-tumor responses in a mouse model. Therefore, the use of IL-15 alone in place of IL-2 or IL-15/IL-7 should be considered when designing CAR-T cell production protocols, to improve the duration of patient responses.

 

Abstract: Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction  are crucial features that enhance the durability  of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy.  We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with  clear in vitro activity.  Strikingly, however, IL-15- produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly  their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients.

 

1. Introduction

 

Adoptive immunotherapy using CD19-targeting chimeric antigen receptor (CAR)- modified T cells is now a proven treatment for hematological malignancies such as B-cell acute lymphoblastic  leukemia and non-Hodgkin’s lymphoma.   For multiple myeloma (MM),  BCMA has emerged as the most promising  target to which CAR-T cells can be directed [1,2]. Particularly for relapsed/refractory (R/R) MM, which is currently considered incurable [3], novel therapies are urgently required. Various studies have demonstrated impressive response rates for anti-BCMA CAR-T cells (BCMA-CARs) when used to treat R/R MM [4–8], culminating in the recent approval by the FDA of the first BCMA-CAR, idecabtagene vicleucel (ide-cel), for these patients [9].

Despite high rates of initial  response following BCMA-CAR treatment, relapses are frequently observed, resulting in average progression-free survival rates of approximately 8–10 months [2,8,10,11]. The most common parameter that has been described to negatively correlate with  BCMA-CAR  efficacy is a lack of in vivo expansion/persistence [5,6,8,12–14]. Several strategies to improve the survival and/or differentiation status, and thus the persistence, of CAR-T cells have been proposed [15]. For BCMA-CARs, some of these approaches for improving the final product have already been studied and implemented, including the use of a 1:1 CD4+:CD8+ ratio [16], the humanization of the CAR molecule [17], and the inclusion  of a phosphoinositide  3-kinase inhibitor in the ex vivo  culture [18]. However, the choice of cytokine(s) used to expand BCMA-CARs has not been previously studied in detail.

 

Currently, high-dose IL-2 is used to expand the BCMA-CAR products that are being investigated in most clinical studies [5,6,8,19]. Although high-dose IL-2 is a strong inducer of T-cell proliferation [20], it additionally promotes terminal  differentiation and T-cell exhaustion [21]. IL-15 is an attractive substitute for high-dose IL-2 because they have equal T-cell mitogenic properties [22], but IL-15 does not cause effector T cell exhaustion [21]. Moreover, IL-15 induces the formation of memory cells and enhances the fitness of T cells by delaying their senescence [23,24], which are desirable qualities for CAR-T cells [25]. IL-7 is frequently used in combination with IL-15 because it has been shown to preserve a naïve/stem cell memory phenotype [26]. For anti-CD19 CAR-T cells, replacing IL-2 with IL-15 or IL-15/IL-7 results in improved survival and persistence in pre-clinical models [27,28]. Critical to the observed superior in vivo efficacy was an improved  phenotype of the ex vivo expanded CAR-T cells prior to administration in patients, especially an increase in the proportion of memory stem cells and reduced dysfunction [27,28]. Interestingly, Alizadeh et al. showed that anti-CD19 CAR-T cells produced using both IL-15 and IL-7 were inferior to those generated using IL-15 alone [28].

 

For BCMA-CARs, the optimal  cytokine condition  for the expansion of CAR-T cell products is currently unknown. Moreover, whether IL-7 is beneficial or not to IL-15-driven CAR-T cell production is an unanswered question. In the present study, we compared how IL-2, IL-15 and a combination of IL-15/IL-7 shaped the phenotype and function of ARI2h cells [17], an in-house BCMA-CAR  that is currently  undergoing  assessment in a clinical trial for R/R MM (NCT04309981). We discovered that ARI2h cells grown in each of the three conditions were comparable in terms of expansion, CAR transduction  and in vitro activity, but that IL-15-cultured cells were better than those expanded in IL-15/IL-7 at ameliorating  disease progression in a MM xenograft model. Furthermore, we found that IL-15 drove a less-differentiated ARI2h phenotype than when IL-15/IL-7 was used in combination, and additionally, that IL-15-grown ARI2h cells had reduced dysfunction and were more memory stem cell-like than those cultured with IL-2. Finally, we showed that T cell senescence-linked pathways and the release of pathology-associated cytokines are alleviated by IL-15 compared to IL-2.

 

2. Results

 

2.1. IL-15-Grown BCMA-CARs (ARI2hIL-15) Express High Surface Levels of ARI2h  CAR and

 

Expand Well in Culture

 

Current protocols for the expansion of BCMA-CARs require the use of IL-2 or IL-15/IL-7  to amplify  the CAR-T cell number, following an initial  CD3-/CD28-mediated T-cell activation.   To compare IL-15 expansion with  IL-2 and IL-15/IL-7, T cells were cultured  with  a concentration of IL-2 that was used for preclinical  studies of ARI2h,100 IU/mL [17], and IL-15/IL-7 concentrations that were used in a previous study and that closely match those used in the clinical trial currently ongoing for ARI2h (NCT04309981)—10 ng/mL for both (Figure 1A) [28]. We found that in vitro expansion of ARI2h CAR-T cells with IL-15 (henceforth referred to as ARI2hIL−15) did not disrupt expression of the CAR molecule, alter the CD4+:CD8+ ratio of the CAR+  T cells, or impede CAR-T cell expansion, compared to ARI2h CAR-T cells cultured with IL-2 (ARI2hIL-2) or IL-15/IL-7 (ARI2hIL-15/IL-7) (Figure 1B–D). On the other hand, without adding cytokines to the in vitro culture, ARI2h CAR-T cells (ARI2hNone) had a lower CD4+:CD8+ ratio and did not expand to the same extent as ARI2hIL-15, ARI2hIL-2, or ARI2hIL-15/IL-7 (Figure 1C,D).

 

One of the major differences between IL-2- and IL-15-grown T cells is that IL-2-cultured cells have an increased cell size due to an augmented protein content [22]. In agreement with this, ARI2hIL-2  cells were larger and more complex than ARI2hIL-15  cells, according to the forward  scatter (FSC-A) and side scatter (SSC-A), respectively, of CD4+ and CD8+ cells, as determined by flow cytometry (Figure 1E,F and Figure S1). A similar DNA content in ARI2hIL-2, ARI2hIL-15 and ARI2hIL-15/IL-7 cells suggested that the difference in cell size was not explained by altered cell cycle progression (Figure 1G). Interestingly, the addition of IL-7 in the expansion of ARI2hIL-15/IL-7 made no difference to the cell size or cell cycle progression when compared to ARI2hIL-15 cells.

2.2. ARI2hIL-15 Functional Responses Are Indistinguishable from Those Exhibited  by ARI2hIL-2 or ARI2hIL-15/IL-7 To test their cytotoxic function,  ARI2h CAR-T cells expanded with  IL-2, IL-15, IL-15/IL-7,  or no cytokine were co-cultured with  two luciferase-expressing MM cell lines, ARP-1-GFP and U266-GFP. The cytotoxicity exhibited by ARI2hIL−15 against these tumor cells was similar to ARI2hIL-2 and ARI2hIL-15/IL-7 at effector:target (E:T) ratios ranging from

1:1 to 0.125:1 (Figure 2A). Furthermore, ARI2hIL-15, ARI2hIL-2  and ARI2hIL-15/IL-7 fully eliminated ARP-1-GFP cells within 96 h, even at a low E:T ratio, but, in contrast, ARI2hNone cells exhibited little  to no function  (Figure 2B—left).  When ARI2hIL-15, ARI2hIL-2  and ARI2hIL-15/IL-7 were rechallenged with  tumor cells (Figure S2A), they exhibited a more efficient cytotoxic functionality than after the first challenge (Figure 2B) but expanded slightly  less (Figure S2B). For ARI2hIL-15, ARI2hIL-2  and ARI2hIL-15/IL-7, the proportion of CAR+  cells increased after each challenge (Figure S2C), as previously  reported for ARI2hIL-2 [17], and the CD4+:CD8+ ratio of CAR+ T cells decreased (Figure S2D).

 

Critical to the function of CAR-T cells is their ability to produce cytokines and effector molecules in response to target tumor cells. When exposed to MM cells for 6 h, ARI2hIL−15 cells produced a high quantity of IFNγ, similar to that made by ARI2hIL−2 cells, but less than that released by ARI2hIL-15/IL-7 cells (Figure 2C and Figure S2E). IL-2, which is critical for effector T cell proliferation and function [29], was also produced at high concentrations by ARI2h cells expanded in each of the cytokine conditions (Figure 2D and Figure S2F). The cytokine(s) used to expand the ARI2h cells also had little effect on the ARP-1-mediated degranulation, as measured by surface levels of CD107a (Figure 2E), or the production of cytotoxic molecule granzyme B (Figure 2F). Basal expression of granzyme B was similar across all ARI2h CARs, suggesting a similar ability  to make granzyme B in response to cytokine signaling (Figure S2G).

Put together, these results show that, in vitro,  ARI2hIL-15  cells are not inferior  to ARI2hIL-2  or ARI2hIL-15/IL-7 in terms of the production of functional CAR-T cells. These findings also confirm that the presence of a γ-chain cytokine in the expansion of ARI2h is critical.

 

2.3. ARI2hIL-15 Has a Superior In Vivo Function Than ARI2hIL-15/IL-7

 

The relative efficacies of ARI2hIL−15, ARI2hIL−2 and ARI2hIL-15/IL-7 were further ex- amined in an in vivo murine model of MM. For this experiment, NOD-SCIDIL2gc− /− mice that were previously engrafted with ARP-1-GFP cells until developing a high tumor bur- den were injected with untransduced (UT) T cells, ARI2hIL-15, ARI2hIL-2, or ARI2hIL-15/IL-7 (Figure 3A,B). All  of the mice that received ARI2h exhibited an initial  decline in their tumor levels, whereas the disease continued to progress in animals that received UT T cells (Figure 3B,C). However, many of the mice treated with ARI2h eventually experienced tumor regrowth, with those treated with ARI2hIL-15/IL-7 relapsing more quickly than those injected with ARI2hIL-15 or ARI2hIL-2 (Figure 3B,C). Correspondingly, the overall survival time of mice from the ARI2hIL-15 group was significantly longer than that of mice from the ARI2hIL-15/IL-7 set (Figure 3D). Interestingly, the two surviving mice from the ARI2hIL-15 group did begin to develop new tumors four weeks after the CAR-T cell infusion, but these were cleared without any reinfusion of CAR-T cells (Figure 3B—IL-15 mice 1 and 2 from day 54 onward). Strikingly, these surviving ARI2hIL-15-treated animals were the only mice in which CAR-T cells were found in substantial numbers in the spleen and bone marrow following sacrifice (Figure 3E and Figure S3A).

 

Further analysis of the ARI2hIL-15  cells from these mice showed that the CD8+ cells expressed intermediate levels of the exhaustion markers PD-1, TIM-3 and TIGIT, but low levels of LAG-3 (Figure 3F). The CD4+ ARI2hIL-15  cells displayed a similar  exhaustion phenotype (Figure S3B). To characterize the dysfunction  of the ARI2hIL-15  cells in more detail, the expression of cell surface markers of T cell senescence, namely, CD28, CD27, KLRG-1 and CD45RA [30], were analyzed. Although over 70% of the CD8+ cells expressed KLRG-1, they were predominantly CD28+CD27+CD45RA-, suggesting that they had a central memory phenotype and were not late-differentiated senescent cells (Figure 3G). Similarly, the CD4+ ARI2hIL-15 cells were mostly KLRG-1+ and CD28+CD45RA-, but they did not express CD27 (Figure  S3C). Further analysis of the memory phenotype revealed that the majority of ARI2hIL-15  cells, especially CD8+, were CCR7-CD45RA- effector memory cells (Figure S3D).

 

Overall, these data show that ARI2hIL-15 CAR-T cells have enhanced persistence and are superior to ARI2hIL-15/IL-7 CAR-T cells at preventing or reducing tumor growth in a MM  murine model.  In addition,  ARI2hIL-15  cells did not express LAG-3, an important marker of exhaustion, and did not exhibit a senescent phenotype.

 

2.4. IL-15-Cultured CAR-T  Cells Have a More Differentiated  Phenotype When Additionally Expanded with IL-7 To understand the improved in vivo efficacy of ARI2hIL-15 compared to ARI2hIL-15/IL-7, we explored the phenotype of the cells following nine days of in vitro culture. The cytokine used for the in vitro proliferation  of T cells influences their memory phenotype, which in turn, alters their longevity and functional responses. Indeed, for CD19-directed CAR-T cells, an increase in memory-like cells has been linked with improved anti-tumor function and the ability to maintain proliferation in a host [31]. Interestingly, CD8+ ARI2h CAR-T cells were less differentiated  than UT CD8+ T cells, regardless of the cytokine that was added to the culture, displaying  a lower percentage of cells with  an effector memory (CD45RA-CCR7-) or effector (CD45RA+CCR7− ) phenotype (Figure 4A and Figure S4A). CD8+ ARI2hIL-15 and ARI2hIL-2 T cells had a remarkably similar overall percentage of mem- ory/effector phenotype cells, but the makeup of CD8+ ARI2hIL-15/IL-7 cells was different (Figure 4A). When explored in more detail, both CD4+ and CD8+ ARI2hIL-15/IL-7 cells had a lower proportion of central memory (CD45RA-CCR7+) phenotype cells than ARI2hIL-15 (Figure 4B), while exhibiting a higher fraction of effector phenotype cells (Figure 4C). CD4+ ARI2hIL-15/IL-7 cultures had more cells with  a naïve (CD45RA+CCR7+) phenotype, but within the CD8+ population,  ARI2hIL-2, ARI2hIL-15  and ARI2hIL-15/IL-7 naïve phenotype cells were found at similar percentages (Figure S4B).

 

Another  indicator  of low  differentiation and high self-renewal capacity within a T cell population  is the presence of memory  stem cells, which  can be identified  as a subset of the CD45RA+CCR7+ population that additionally express the chemokine receptor CXCR3 [32]. The expression of CXCR3 was higher within CD45RA+CCR7+ ARI2hIL-15 cells than ARI2hIL-2 cells (Figure 4D), both for CD4+ and CD8+ T cells, suggesting an increased proportion of stem-like cells.

 

When CAR-T cells are administered to cancer patients and they encounter tumor cells, they differentiate further along either an effector or memory pathway. mTORC1 activity is critical to promoting the effector differentiation  lineage of T cells, and as such, reduced mTORC1 activation is thought to be favorable for durable responses. We found that MM cell-induced mTORC1 activity, as measured  by phosphorylation of the downstream riboso- mal S6 protein, was lower in ARI2hIL-15 cells than in either ARI2hIL-2 or ARI2hIL-15/IL-7 cells, which exhibited similar levels of S6 protein phosphorylation (Figure 4E). This difference was dependent on mTORC1 activity as it was blocked by the mTORC1-specific inhibitor rapamycin (Figure S4C), and moreover, it was not caused by an overall decrease in cell activation, as the induction of CD69 expression was the same in ARI2h CAR-T cells from all groups (Figure S4D). Interestingly, basal mTORC1 activity, in the absence of MM cells, was higher in ARI2hIL-2  cells compared to ARI2hIL-15  or ARI2hIL-15/IL-7, although many cells demonstrated low mTORC1 signaling (Figure S4E).

 

Thus, these data provide evidence that the addition of IL-15 alone is the best in vitro condition  in which  to produce ARI2h cells that are less differentiated  and have more memory stem-like cells.

 

2.5. ARI2hIL-15 Cells Have Reduced Expression of LAG-3  and Augmented  Levels of CD27 Compared to ARI2hIL-2

 

An important indicator of CAR-T cell efficacy is the expression of exhaustion markers that contribute to T cell dysfunction.  In particular, PD-1, LAG-3 and TIM-3 have been proposed as the most significant markers. ARI2hIL-15 and ARI2hIL-15/IL-7 expressed reduced levels of LAG-3 compared to ARI2hIL-2 in both CD4+ and CD8+ cells (Figure 5A). Although LAG-3 expression was similar on ARI2hIL-15 and ARI2hIL-15/IL-7 cells, levels of TIM-3 were lower on ARI2hIL-15  CD4+ cells (Figure 5B). However,  PD-1 expression was marginally but significantly lower on ARI2hIL-15/IL-7  cells (Figure 5C), suggesting an overall similar exhaustion phenotype between ARI2h cells of the IL-15 and IL-15/IL-7 groups. Another immune checkpoint, TIGIT, inhibits endogenous T cell anti-MM activity, due to the high levels of TIGIT ligands expressed on malignant plasma cells in the BM [33], but expression of this receptor was not different on ARI2hIL-15, ARI2hIL-2 or ARI2hIL-15/IL-7 cells (Figure S5A).

 

A further  source of T cell dysfunction  is the loss of expression of costimulatory molecules, particularly CD27 and CD28. CD27 expression was elevated on ARI2hIL-15 cells compared to ARI2hIL-2 cells, particularly in CD4+ cells, where ARI2hIL-15 cell CD27 levels were also significantly greater than those on ARI2hIL-15/IL-7 cells (Figure 5D). However, on CD8+ cells, CD27 expression was similar on ARI2hIL-15 and ARI2hIL-15/IL-7 cells. Inter- estingly, CD4+CAR+ ARI2hIL-2  CD27 levels were less than half that seen on IL-2-cultured UT CD4+ T cells (Figure 5D). Likewise, CD28 expression was lower on ARI2hIL-2  cells compared to UT T cells, but there was no significant difference in CD28 levels between ARI2hIL−2 and ARI2hIL-15 or ARI2hIL-15/IL-7 (Figure S5B).

Altogether, ARI2hIL-15 and ARI2hIL-15/IL-7 cells exhibited a less dysfunctional pheno- type than ARI2hIL-2 cells.

 

2.6. ARI2hIL-15 Cells Have Reduced DNA Damage Compared to ARI2hIL-2

 

As the loss of CD27 expression is a marker of senescence in T cells [34], this was explored further.  T cell senescence is a phenomenon that is associated with  aging, and naturally occurs due to multiple rounds of replication and telomere loss, but it can also be induced or accelerated by external factors such as nutrient deprivation or the presence of certain metabolites [30]. To investigate whether ARI2h CAR-T cells expanded in IL-2, IL-15 or IL-15/IL-7 exhibited signs of altered senescence, the widely used senescence biomarker, senescence-associated β-galactosidase  (SA-β-gal) activity, was analyzed. ARI2hIL-15 cells showed reduced SA-β-gal activity  compared to ARI2hIL-2  cells (Figure 6A), suggesting that ARI2hIL-2  cells may be more senescent. Interestingly, this difference was not due to altered p38 MAPK signaling (Figure 6B and Figure S6A), a pathway that is often perturbed in senescent T cells.

 

We hypothesized that the observed divergences in T cell dysfunctional phenotype between ARI2hIL-15  and ARI2hIL-2  could be derived from a difference in DNA  damage accumulation. Indeed, we found that the proportion of ARI2hIL-15 cells that were positive for H2AX phosphorylation (γH2AX)  was lower than for ARI2hIL-2  cells (Figure 6C and Figure S6B), thus showing that IL-15 causes fewer CAR-T cells to engage in a DNA damage response (DDR). Interestingly,  there was a large difference between the proportion of γH2AX+ cells in the ARI2hIL-2 group compared to UT T cells, indicating that the presence of the CAR induces a strong DDR.

 

Another hallmark of senescence in T cells is mitochondrial dysfunction and reduced mitochondrial content.  Based on the incorporation of tetramethyl  rhodamine  methyl ester (TMRM), which demonstrates mitochondrial transmembrane potential, ARI2hIL-2, ARI2hIL-15 and ARI2hIL-15/IL-7 all had a high proportion of hyperpolarized cells, suggesting a similar  mitochondrial fitness (Figure 6D and Figure S6C). In addition,  the uptake of MitoTracker Far Red dye was similar for ARI2h CAR-T cells grown in all three cytokine conditions, suggesting that there was no difference in mitochondrial mass (Figure 6E and Figure S6D,E).

 

Taken together, these data confirm that ARI2hIL-2 exhibits a more senescent phenotype compared to ARI2hIL-15, which is associated with increased DNA damage, but not changes in p38 MAPK signaling or mitochondrial function.

 

2.7. ARI2hIL Cells Secrete Loωer Levels of Cytokines  That Aγe Related  to CAR-T Cell Toxicities or MM Progression

 

Amor unresolved issue with CAR-T cell erapy is the triggering of cytokine-release syndrome (CRS)which is the most  frequent toxicity  associated with this treatment.CRS is caused by cytokines and chemokines that are secreted by acti rated CAR-T cellsas well as In summary, ARI2hIL-15  cells produce high levels of effector cytokines/chemokines when activated by tumor cells, but they release lower levels of secreted molecules that are associated with deleterious effects, such as CRS.

 

3. Discussion

 

BCMA-directed CAR-T cells are emerging as a promising therapy for incurable R/R MM. Although  anti-BCMA CAR-T cells have demonstrated excellent anti-cancer efficacy, a lack of CAR-T cell persistence, resulting in further relapse, is a major shortcoming [8]. The development of a suboptimal  phenotype following in vitro  CAR-T cell culture is impacted by the cytokine used for their expansion [27,28]. In this report, we show that IL-15 did not adversely affect cell expansion, CAR transduction, or in vitro anti-MM  cell activity, compared to IL-2 or a combination of IL-15 and IL-7. Indeed, we conclude that IL-15 alone is the optimal  cytokine condition  in which  to expand ARI2h cells.  IL-15 demonstrated superior in vivo function and generated less-differentiated CAR-T cells than IL-15/IL-7 combined, and moreover, IL-15 promoted a more stem cell-like phenotype, reduced dysfunction  and caused lower production of cytokines associated with  CRS or MM progression compared to IL-2.

 

It is well established that less-differentiated cells with a more stem cell-like phenotype make for better CAR-T cell products.  Many studies have shown that IL-2 expansion induces fewer cells with  a naïve or stem cell memory-like  phenotype than IL-15 or IL-15/IL-7  [27,28,38]. Our data confirm that IL-2-grown  CARs had fewer naïve CD4+ cells than IL-15/IL-7-grown CARs, but in contrast, no difference was observed with CD8+ cells. This could be explained by the difference in length of ex vivo culture between our study and others. We cultured the CAR-T cells for 9 days to correspond with the protocol used for the clinical-grade production of ARI2h cells [17], whereas in many other studies, CAR-T cells are expanded for two weeks or more. In terms of the stem cell memory phenotype, ARI2hIL-15 naïve cells presented the highest expression of the chemokine receptor CXCR3. Furthermore, the addition of IL-7 to IL-15 cultures reduced the proportion  of ARI2h cells with a central memory phenotype and increased that of effector cells. Put together with the in vivo results, which showed that ARI2hIL-15/IL-7 was an inferior murine therapeutic compared to ARI2hIL-15, our data suggest that IL-7 is detrimental  to the production of ARI2h cells.  Correspondingly, a recent publication  found  that IL-15-expanded CD19- CAR-T cells demonstrated enhanced anti-tumor efficacy compared to those cultured with IL-15/IL-7 [28]. We believe that the adverse effects caused by IL-7 on CAR-T cell activity is an interesting observation that warrants further investigation.

 

Another  cell surface protein that is a marker of memory cells and self-renewal is CD27. We observed that CD27 was dramatically higher on the surface of ARI2hIL-15 cells compared to ARI2hIL-2. This is of particular interest because CD27 expression, in the form of CD27+CD45RO- cells, was one of the few parameters associated with  CART-BCMA efficacy in an R/R MM clinical study [6]. CD27 expression is also associated with T cell dysfunction—lack  of CD27 and CD28 expression on T cells is a sign of senescence [30]. We found that, despite CD28 levels being similar, SA β-gal activity  and γH2AX  levels were lower in ARI2hIL-15 cells compared to ARI2hIL-2, suggesting reduced senescence and DDR engagement. Based on these results, and previous work identifying that delayed replicative senescence improves anti-CD19 CAR-T cell persistence and function [39], the role of senescence and the DDR merits further exploration in BCMA-targeting and other types of CAR-T cell.

 

Another  observed difference was the expression of the inhibitory receptor LAG-3. In vivo results demonstrated that IL-15 prolonged the persistence of CD8+ ARI2h cells that were characterized by low expression of LAG-3 and a CD45RA-CD27+ central memory phenotype, suggestive of a higher fitness for ARI2hIL-15  CARs. Overall, the expression of inhibitory receptors in the ARI2h cells isolated from mice was high, indicating  that a combination therapy of ARI2h and checkpoint blockers could act synergistically in the treatment of MM. The loss of CD27 is also associated with CAR-T cell tonic signaling [40],

which is another well-known phenomenon that contributes to CAR-T cell dysfunction and the loss of in vivo persistence and tumor-clearing ability [41,42]. Here, we observed that IL-2-grown CAR-T cells exhibited other features of tonic signaling, such as increased cellular size, complexity and DNA content.

In addition  to enhancing CAR-T cell tumor clearance and persistence, eliminating or dampening  the effect of CRS would  be another major improvement  to CAR-T cell therapy. In this regard, limiting the production of IFNγ and TNFα by CAR-T cells, which are important  CRS-initiating molecules, could be beneficial. Here, we demonstrate that ARI2hIL-15  cells, which achieve superior in vivo activity,  secrete less IFNγ compared to ARI2hIL-15/IL-7 cells. Furthermore, ARI2hIL-15  cells produced a lower amount of TNFα, IL-9, SDF-1α and IL-22, all of which are cytokines associated with MM disease progression and/or CAR-T cell-induced toxicity [5,13,35–37].

 

4. Materials and Methods

 

4.1. Samples

 

All  donors provided  informed  written  consent in accordance with  the Declaration of Helsinki,  and all research involving human-derived  material was approved by the Ethical Committee of Clinical Research at Hospital  Clínic, Barcelona. Buffy coats from healthy donors were obtained from the local blood and tissue bank (Banc de Sang i Teixits, Catalonia). Human T cells were isolated from buffy coats by density-gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, St Louis, MO, USA) followed by negative selection of T cells using a Pan T Cell Isolation Kit (Miltenyi Biotech, Bergisch Gladbach, Germany).

4.2. Cell Culture and T Cell Transduction

 

T cells were stimulated with  Dynabeads Human T-Activator CD3/CD28  (Thermo Fisher Scientific, San Diego, CA, USA) and were expanded in Click’s media (47.5% Click’s media (Irvine  Scientific, Santa Ana, CA, USA), 47.5% RPMI-1640, 5% human serum, 2 mM L-glutamine,  100 IU/mL penicillin and 100 µg/mL streptomycin)  supplemented with  100 IU/mL IL-2 (“IL-2”), 10 ng/mL IL-15 (“IL-15”), 10 ng/mL IL-15 + 10 ng/mL IL-7 (“IL-15/IL-7”) or no cytokine (“None”). At 48 h following stimulation,  T cells were transduced with a lentivirus vector encoding ARI2h [17]. Subsequently, T cells were split, and cytokines were refreshed every 1–2 days for a further 7 days of culture, unless indicated otherwise. ARP-1 and U266 cell lines were obtained, cultured and modified  to express GFP-firefly luciferase (GFP-ffLuc), as previously  described [17]. All cultured cells were incubated at 37 ◦ C with 5% CO2. Live cells were counted using Trypan blue exclusion.

4.3. Flow Cytometry

Cell surface proteins on CAR-T cells were stained with  the following antibodies: CD4-APC/H7, CD8a-PE/Cy7, CCR7-PerCP/Cy5.5, CXCR3-AF488, CD69-FITC, CD3-APC (all from BD Biosciences,  San Jose, CA, USA); CD8a-FITC, CD28-FITC, CD27-PE, LAG-3-PE, TIM-3-FITC, TIGIT-PerCP/Cy5.5,  CD45RA-APC (all from Biolegend, San Diego, CA, USA); PD-1-APC (eBioscience, Thermo Fisher Scientific, San Diego, CA, USA). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY, USA) followed  by a BV421-conjugated anti-Fc antibody (Biolegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in 1% (v/v) FCS in PBS or 1% (v/v) paraformaldehyde  prior to acquisition. Live cells were gated based on forward and side scatter.

 

For intracellular staining of γH2AX,  surface-stained cells were fixed in 1% (v/v) paraformaldehyde, permeabilized in 90% (v/v) methanol at −20 ◦ C overnight, and then stained with PE-conjugated anti-H2AX (pS139) antibody (BD Biosciences). Immediately prior to analysis, cells were washed and resuspended in 1% (v/v) FCS in PBS containing

 

3.3 µg/mL 7-AAD.

 

All  flow  cytometry  data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software, version 7.6.2 (TreeStar, Ashland, OR, USA).

 

4.4. Analysing CAR-T Cell Ribosomal S6 Protein and p38 MAPK Phosphorylation CAR-T cells were incubated with  an equal number of ARP-1 cells, or media, for 6 h at 37 ◦ C, prior  to staining with  fluorophore-conjugated antibodies.  In some cases, CAR-T cells were pretreated with  100 nM rapamycin (Selleckchem, Houston, TX, USA) or 500 nM BIRB-796 (Axon Medchem, Groningen, Netherlands) for 30 min before the assay to act as negative controls for ribosomal S6 protein and p38 MAPK phosphorylation, respectively. Intracellular staining of ribosomal S6 protein and p38 MAPK was carried out as described above for γH2AX, but instead using PE-conjugated anti-S6 (pS235/pS236) and AF647-conjugated anti-p38 MAPK (pT180/pY182) antibodies (both from BD Biosciences).

 

4.5. Degranulation  Assay and Granzyme B Expression

 

CAR-T cells and ARP-1 cells were cultured at a 0.125:1 (degranulation assay) or 1:1 (granzyme B expression) ratio for 6 h at 37 ◦ C. For the degranulation  assay, cells were additionally incubated with  CD107a-AF647 antibody (BD Biosciences). One hour after the co-culture was initiated,  GolgiPlug (1:1000, BD Biosciences) was added to the cells. For intracellular staining of granzyme B, cells were fixed and permeabilized using the FIX&PERM Cell Fixation and Permeabilization Kit (Nordic-MUBio, Susteren, Netherlands) according to the manufacturer ’s instructions and stained with an AF647–conjugated anti- granzyme B antibody (BD Biosciences) during the permeabilization step.

 

4.6. SA-β-Gal/Mitochondrial  Staining

 

T-cell SA-β-gal activity was measured using C12FDG (Sigma Aldrich) as previously described [43]. For each condition, SA-β-gal activity was calculated  as the MFI of C12FDG- stained cells, minus the background fluorescence detected in unstained cells. To stain mitochondria and to measure mitochondrial membrane potential, surface-stained cells were incubated with  25 nM TMRM and 10 nM MitoTracker  Deep Red FM (both from Thermo Fisher Scientific) for 30 min at 37 ◦ C prior to analysis by flow cytometry.

 

4.7. In Vitro Cytotoxicity Assay

 

A luminescence-based method, in which the expression of luciferase in cell lines acts as a marker of target cell viability, was employed for all in vitro cytotoxicity assays. For

 

24-hour assays, 25,000 GFP-ffLuc-expressing ARP-1 (ARP-1-GFP) or U266 (U266-GFP) cells were plated per well of a white 96-well flat-bottomed plate, and T cells were added at the T cell:tumor cell (effector:target) ratios indicated in the figure legends and in triplicates. For long-term  challenges, ARP-1-GFP cells were co-cultured  with  T cells at a 0.125:1 effector:target ratio in a 24-well plate, and 100 µL was transferred to each of triplicate wells of a white 96-well flat-bottomed plate prior to analysis. Just before analyzing luminescence,100 µg/mL D-luciferin (PerkinElmer, Waltham, MA, USA) was added to the co-cultured cells and the plate was shaken gently for 10 min in the dark. Luminescence measurements were read on a plate reader. The percentage of surviving tumor cells was calculated as 100× (signal from the sample well—background signal)/(signal  from the well containing tumor cells alone—background signal).

 

4.8. Long-Term Challenges

 

T cells were added to ARP-1-GFP cells at a 0.5:1 ratio and after 4 days, 0.5–1 × 106 T cells were analyzed by flow cytometry. Rechallenges were performed by adding T cells from the first challenge to fresh ARP-1-GFP cells at the same 0.5:1 ratio.

 

4.9. Analysis of Cytokine Production

 

Supernatants collected from  a 6-hour co-culture of T cells and an equal number of ARP-1 cells were stored at −80 ◦ C until  analysis. The abundance of 34 different  cy-tokines/chemokines was determined using the ProcartaPlex Multiplex Immunoassay Kit (Thermo Fisher Scientific), following  the manufacturer ’s instructions. Data were analyzed using ProcartaPlex Analyst software (Thermo Fisher Scientific) and a heatmap of the deter- mined protein concentrations was generated using Morpheus software (Broad Institute; https://software.broadinstitute.org/morpheus (accessed on 24 February 2021)). Addition- ally, levels of IFNγ and IL-2 were measured by ELISA using commercially available kits (Biolegend).

 

4.10. In Vivo Murine Experiments

 

In vivo  mouse experiments were approved  by the Ethical Committee of Animal Research (Hospital  Clínic, Barcelona, Spain).  Mouse experiments were performed  as previously described [17]. In brief, irradiated 8-week-old male NOD-SCIDIL2gc− /− mice received an intravenous injection of 1 × 106 ARP-1-GFP cells, and 3 weeks later, 8 × 106 UT T cells or 8 × 106 (6 × 106 CAR+) CAR-T cells that had been expanded with IL-2, IL-15 or IL-15/IL-7, as described above, were administered to the mice via intravenous injection as well. Thereafter, the bioluminescence signal, corresponding to tumor growth, and the bodyweight of each animal was measured weekly.  Beginning 2 weeks after the T-cell infusion, mice were removed from the study when they had a bioluminescence signal that exceeded 20,000 p/sec/cm2/sr.

 

4.11. Statistical Analysis

 

GraphPad Prism version 8.0.1 (GraphPad Prism Software, La Jolla, CA, USA) was used for data analysis. Multiple comparisons were performed using a repeated measures one-way ANOVA, corrected with a Tukey or Dunnett post hoc test. Comparison of mouse survival between different groups was analyzed using the log-rank (Mantel–Cox) test.

 

5. Conclusions

 

The generation of a rapidly  increasing number of new CAR-T cells, especially for hematological malignancies, makes the need to develop improvements to this therapeutic even more timely. Here, we have presented data that could be applied to the development of all types of CAR-T cells, and thus improve treatment protocols for multiple cancers. For BCMA-targeting CAR-T cells in particular, we have demonstrated that a more favorable phenotype and function can be achieved by using IL-15 alone, in place of IL-2 or IL-15/IL-7, during the ex vivo expansion stage. This new finding is a further advancement to a CAR-T cell that is already used in the clinic for R/R MM, and thus has the potential to improve the limited therapeutic options available for these patients.

 

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.3390/cancers13143534/s1, Figure S1: Quantification of ARI2h SSC-A (related to Figure 1), Figure S2: IL-15-grown ARI2h BCMA-CARs are highly functional in long-term cytotoxicity assays and short- term generation of functional molecules (related to Figure 2), Figure S3: Further characterisation of ARI2h BCMA-CARs isolated from MM tumour-bearing mice (related to Figure 3), Figure S4: Memory phenotype, ribosomal S6 phosphorylation and CD69 expression of ARI2h BCMA-CARs (related to Figure 4), Figure S5: Expression of TIGIT and CD28 on ARI2h BCMA-CARs (related to Figure 5), Figure S6: Effect of BIRB-796 on p38 MAPK phosphorylation and ARI2h BCMA-CAR DNA damage and mitochondrial phenotype (related to Figure 6), Figure S7: Quantification of IFNγ and IL-2 from multiplex  immunoassay experiment (related to Figure 7).

 

Author Contributions: Conceptualization, A.M.B. and B.M.-A.; data curation, A.M.B.; formal anal- ysis, A.M.B. and B.M.-A.; funding  acquisition, C.F.d.L., A.U.-I. and B.M.-A.; investigation, A.M.B., M.B., V.L. and B.M.-A.; methodology, A.M.B. and B.M.-A.; project administration, A.U.-I. and B.M.-A.; resources, C.F.d.L., A.U.-I. and B.M.-A.; supervision, B.M.-A.; visualization,  A.M.B. and B.M.-A.; writing—original draft, A.M.B.; writing—review and editing, A.M.B., C.F.d.L., A.U.-I. and B.M.-A. All authors have read and agreed to the published version of the manuscript.

 

Funding: The La Caixa Foundation (CP042702), the Institute of Health Carlos III (projects: PI17/01043, PI19/00669 and ICI19/00025), the Fondo Europeo de Desarrollo Regional (FEDER) and the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR) (project: 2017 SGR 00792) provided funding for this study.

 

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki  and approved by the Ethical Committee of Clinical Research at Hospital Clínic, Barcelona. In vivo mouse experiments were approved by the Ethical Committee of Animal Research (Hospital Clínic, Barcelona, Spain).

 

Informed Consent Statement: Informed  consent was obtained from all subjects involved  in the study.Data Availability Statement: Data are available upon reasonable request.

 

Acknowledgments: We acknowledge Multiple Myeloma Research Center (Little Rock, AK, USA) for providing the ARP1 cell line and Amer Najjar for providing the plasmid coding for GFP-ffLuc.

 

Conflicts of Interest: The authors declare no conflict of interest.

 

by pro-inflammatory molecules produced by other immune cells, such as macrophages, that are inadvertently stimulated by the CAR-T-cell-derived  cytokines/chemokines. To understand whether ARI2hIL-15  produced reduced levels of CRS-related molecules, the supernatant of CAR-T cell/ARP-1  cell co-cultures was analyzed for the presence of 34 different  cytokines and chemokines. As a control, IFNγ and IL-2 were included in this panel, as they had previously been analyzed by ELISA (Figure S7A,B and Figure 2C,D).

 

Overall, ARI2hNone  cells produced very low levels of almost all analyzed molecules,further highlighting the fact that the addition of IL-2 or IL-15 is critical to the generation of functional ARI2h cells (Figure 7A). Cytokine/chemokine production was similar for ARI2hIL-2, ARI2hIL-15 and ARI2hIL-15/IL-7, although some differences were observed. The release of TNFα, an important  macrophage-activating molecule that is associated with CRS severity in MM patients treated with anti-BCMA CAR-T cells [5,13], was reduced in ARI2hIL-15  compared to ARI2hIL-2  (Figure 7B). In addition,  ARI2hIL-15  secreted reduced levels of IL-9, SDF-1α and IL-22 compared to ARI2hIL-2 cells (Figure 7C–E). All of these are associated with MM progression; high IL-9 levels are related to resistance to proteasome inhibitor therapy, SDF-1α activates MM cells, and an increase in IL-22-producing  TH22 cells is associated with a poor prognosis for MM patients [35–37].