Research ArticleImmunotherapy

Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies

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Science Translational Medicine  06 Jan 2016:
Vol. 8, Issue 320, pp. 320ra3
DOI: 10.1126/scitranslmed.aad5222

Young blood for immunotherapy

Adoptive cell therapies with engineered T cells have shown promising results in patients whose cancer is recalcitrant to other therapies. However, chemotherapy can inhibit both the expansion and function of T cells, limiting the success of this approach. Now, Singh et al. report that T cell populations enriched for early lineage cells expanded better in vitro. These cells could be specifically enriched by collecting T cells at time points diametric to chemotherapy administration and expanded in vitro with targeted culture methods. These data suggest that enriching early lineage cells may increase the number of patients who may benefit from engineered T cell therapy.

Abstract

Engineered T cell therapies have begun to demonstrate impressive clinical responses in patients with B cell malignancies. Despite this efficacy, many patients are unable to receive T cell therapy because of failure of in vitro expansion, a necessary component of cell manufacture and a predictor of in vivo activity. To evaluate the biology underlying these functional differences, we investigated T cell expansion potential and memory phenotype during chemotherapy in pediatric patients with acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). We found that patients with T cell populations enriched for early lineage cells expanded better in vitro and that patients with ALL had higher numbers of these cells with a corresponding enhancement in expansion as compared to cells from patients with NHL. We further demonstrated that early lineage cells were selectively depleted by cyclophosphamide and cytarabine chemotherapy and that culture with interleukin-7 (IL-7) and IL-15 enriched select early lineage cells and rescued T cell expansion capability. Thus, early lineage cells are essential to T cell fitness for expansion, and enrichment of this population either by timing of T cell collection or culture method can increase the number of patients eligible to receive highly active engineered cellular therapies.

INTRODUCTION

Immunodeficiency resulting from systemic chemotherapy for cancer has been recognized for decades. In the 1970s, attempts were undertaken to boost poor immune function in pediatric patients being treated for acute lymphoblastic leukemia (ALL) using vaccination with Bacille de Calmette et Guérin (1). The recognition that chemotherapy depleted cells of the myeloid lineage and resulted in dysfunctional innate immunity led to the development of exogenous granulocyte–stimulatory factor support, which has significantly reduced the morbidity associated with prolonged neutropenia (2). Analysis into the effect of systemic chemotherapy on T lymphocytes has elucidated a complex and dynamic impact on this cellular compartment. Early studies demonstrated that pediatric patients with ALL have lower absolute T cell counts (ATCs) as compared to normal controls even at diagnosis (3), and this deficit seems to be sustained through chemotherapy. Further studies demonstrated similar findings in children with sarcoma and non-Hodgkin lymphoma (NHL) (4). These small studies also found variability in which T cell subsets (among those recognized at the time) were affected by chemotherapy, demonstrating a preferential depletion of naïve T (TN) cells relative to memory cells after completion of therapy (46).

Much recent work has described the various phenotypes of circulating T cells and the roles these cells play in cellular immunity. Until recently, the paradigm of hierarchical T cell differentiation was one in which TN cells encounter antigen and differentiate into central memory (TCM) cells, effector memory (TEM) cells, and terminal effector (TEff) cells. Memory cells are responsible for initiating robust and efficient activation in response to antigen reexposure, and effector cells are responsible for mediating cytotoxic responses (7). The recent description of stem central memory T (TSCM) cells has shifted this paradigm (8). These stem-like cells have the ability of self-renewal and multipotency, are highly proliferative, and have enhanced antitumor activity in animal models when compared to other T cell subsets (9). They can give rise to all memory and effector subsets, but not TN cells, suggesting that a more accurate model of T cell differentiation may flow from TN cells to TSCM cells, with both of these cell types having the ability to give rise to memory and effector subsets (10). These qualities, namely, self-renewal, multipotency, and robust antitumor activity, make these cells an extremely attractive substrate to mediate engineered cellular therapy for cancer.

Chimeric antigen receptor (CAR)–engineered T cell therapy has demonstrated significant success in the treatment of CD19+ B cell malignancies (1115). Our group has previously demonstrated that an enriched TCM population in the infused CAR T cell product correlates with enhanced in vivo persistence and improved antitumor activity (16) and that ex vivo expansion of T cells using CD3/CD28 stimulatory beads produces larger quantities of TCM cells as compared to other methods of ex vivo CAR T cell generation (17). Despite this robust expansion process, one of the primary reasons that patients seeking highly active cell therapy are ineligible for entry into clinical trials is an inability to produce a sufficient number of T cells during ex vivo manufacture. At the Children’s Hospital of Philadelphia and University of Pennsylvania, eligibility for entry into our CAR clinical trials requires production of >5 × 106 CAR+ cells/kg for infusion at the conclusion of clinical manufacture. As part of our initial clinical trial design, all collected T cells undergo a “test expansion,” in which a small aliquot of collected T cells is expanded in vitro to estimate expansion ability of the harvested product. We have found that greater than fivefold expansion in vitro during test expansion is highly predictive of successful clinical-scale expansion, with a positive predictive value of 70% and a negative predictive value of 95% (table S1). In our recently published CD19 CAR T cell trial, we demonstrated that cells that were produced in this method and that passed this expansion threshold were able to mediate complete remissions in 90% of patients with ALL (11), suggesting that in vitro expansion is highly correlated with clinical activity. To date, we have evaluated 83 patients at the Children’s Hospital of Philadelphia for entry into our CD19 CAR clinical trial, and 63 of these patients have passed test expansion, meaning 24% of patients evaluated were unable to receive this highly active therapy because of failed expansion. Exploration of methods to enhance ex vivo T cell expansion led us to investigate the interplay between expansion ability, memory phenotype, and timing of T cell collection during therapy.

We undertook a prospective and longitudinal analysis of T cell expansion and phenotype after each cycle of chemotherapy in pediatric patients with B cell malignancies. We found that samples from patients with leukemia have significantly greater expansion potential than those from patients with lymphoma, who demonstrate poor expansion throughout therapy. Examination of T cell phenotypes revealed that patients with ALL have enriched TN and TSCM cell subsets (“early lineage” cells), whereas those with lymphoma have very low early lineage cell counts. We stratified our analysis of ALL into standard-risk (SR-ALL) and high/very-high-risk (HR/VHR-ALL) disease and found that SR-ALL samples maintained robust expansion until late in therapy, whereas HR/VHR samples demonstrated successful initial expansions that acutely declined in association with cyclophosphamide and cytarabine administration. We also found that cyclophosphamide and cytarabine appeared to selectively deplete TN cells in both ALL risk groups, corresponding to the decline in HR/VHR-ALL T cell expansion. Finally, we demonstrated that culture with exogenous interleukin-7 (IL-7) and IL-15 during expansion significantly enhanced TSCM cell counts, and this enrichment significantly improved the expansion ability of lymphoma T cells. Together, these data demonstrate underlying variability in T cell function directly related to disease and chemotherapy and support a new approach to increase the number of patients eligible for engineered cell therapy moving forward.

RESULTS

Patients evaluated represent a wide spectrum of pediatric B cell malignancies

Fifty patients with newly diagnosed B cell malignancies were enrolled as part of a clinical trial under an approved Children’s Hospital of Philadelphia Institutional Review Board protocol (Table 1). Patient age at time of enrollment ranged from 8 months to 19 years, and patients were 58% male and 42% female. Disease groups included ALL, stratified as National Cancer Institute (NCI) SR (SR-ALL, n = 17) or HR/VHR (HR/VHR-ALL, n = 21), and NHL (n = 12). Lymphoma subtypes included Burkitt (n = 6), diffuse large B cell (DLBCL, n = 3), primary mediastinal (n = 1), primary lymphoma of bone (n = 1), and follicular lymphoma (n = 1); all NHL subtypes were grouped for analysis.

Table 1. Patient characteristics.

Fifty patients were included in our prospective analysis.

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T cell expansion varies by disease group and previous therapy

Peripheral blood was collected from each enrolled patient at diagnosis and after every cycle of chemotherapy. The chemotherapeutic regimens administered to SR- and HR-ALL patients during these cycles are shown in table S2. T cells were isolated and stimulated using beads coated with CD3 and CD28 agonist antibodies, as used in our clinical test expansions and GMP (good manufacturing practice) cell manufacturing process (18), and cell expansion was measured over time. As outlined above, a threshold of greater than fivefold expansion during test expansion was associated with a high likelihood of successful clinical expansion. We applied the same threshold to stratify our analysis into samples that “pass” expansion and those that “fail” expansion, meant to represent those patient samples that would have been eligible or ineligible, respectively, for inclusion in our clinical trials. Nearly 80% of patients with ALL met this threshold at diagnosis, with pass rates slowly declining over the course of therapy, reaching ~40% during maintenance phase (Fig. 1A). In distinct contrast were cells collected from patients with NHL, which demonstrated poor expansion, with only 25% passing at diagnosis, and few samples (12.5% of all remaining time points) demonstrating any expansion after initiation of therapy (Fig. 1A). The difference in pass rates between leukemia and lymphoma samples was significant at all time points (see table S3 for results of statistical analysis).

Fig. 1. Percentage of peripheral blood samples passing test expansion.

(A) Percentage of samples that pass test expansion from patients with ALL and NHL. (B) Percentage of samples that pass test expansion from patients with SR-ALL and HR/VHR ALL. (C) ATCs from peripheral blood at time of collection from all patients with leukemia or lymphoma. (D) ATCs from peripheral blood at time of collection from all patients with SR- or HR/VHR-ALL. Significant differences are denoted with an “*” above each column and represent P < 0.05. Statistical analysis can be found in table S3.

Stratification of ALL samples into NCI SR and HR/VHR groups revealed that T cells from patients with SR-ALL demonstrated robust and maintained expansion capability, with an initial decline early in therapy and a modest decline during the maintenance phase (Fig. 1B). Although HR/VHR-ALL samples expanded well early in therapy, pass rates declined rapidly after consolidation phase (Fig. 1B), resulting in statistically significant differences in rate of passing test expansion when compared to SR-ALL (table S3).

We examined the relationship between ATC and expansion potential to evaluate whether this observed functional difference was simply a reflection of variability in T cell quantity. ATC for leukemia samples declined after induction through maintenance therapy (Fig. 1C), demonstrating a similar trend as the expansion rates. Lymphoma samples at some time points had modest peripheral T cell counts (300 to 400 cells/μl); however, even during these cycles, T cells demonstrated poor expansion. Whereas leukemia samples demonstrated significantly greater expansion compared to lymphoma throughout therapy (Fig. 1A), no such statistical differences were observed in ATC between these two diseases. Examination of the risk group–stratified ALL samples demonstrated a progressive decline in SR-ALL ATC with a significant fall after delayed intensification (Fig. 1D). This progressive decline in ATC correlated with the progressive decline in expansion (Fig. 1B). HR/VHR-ALL ATC experienced a sharp decline after consolidation therapy, correlating with the abrupt decline in expansion after consolidation (Fig. 1B). HR/VHR-ALL ATC remained low for the remainder of therapy, with expansion potential demonstrating a progressive decline. These findings suggest that in ALL, ATC may have some bearing on expansion potential; however, this association was not directly correlated at each cycle. For NHL, ATC seemed to have no bearing on expansion potential.

Early lineage T cells are associated with enhanced expansion

Given that T cell quantity had no association with expansion in NHL and only a general correlation in ALL, we next evaluated the memory phenotypes present within these samples as potential contributors to the observed functional differences. Cell lineages were defined by well-described cell surface marker patterns (see Materials and Methods), and we divided our analysis into three comparison groups, displayed at the top of each column in Fig. 2 (pass versus fail, Fig. 2, A to E; leukemia versus lymphoma, Fig. 2, F to J; SR-ALL versus HR/VHR-ALL, Fig. 2, K to O).

Fig. 2. Memory phenotypes of T cells harvested from the peripheral blood of patients undergoing chemotherapy.

(A to O) Absolute cell counts are demonstrated from samples who passed and failed the greater than fivefold expansion threshold [column 1, (A) to (E)], patients with ALL or NHL [column 2, (F) to (J)], and patients with SR- and HR/VHR-ALL [column 3, (K) to (O)]. Significant differences are denoted with an “*” above each column and represent P < 0.05. Statistical analysis can be found in table S4, and a summary table can be found in table S5.

Comparison of samples that passed test expansion to those that did not (Fig. 2, A to E) revealed that successful expansion was associated with significant enrichment for TN cells at all time points except after delayed intensification, and enrichment of TSCM cells at all time points except diagnosis and after delayed intensification (Fig. 2, A and B). TN counts declined progressively through therapy in both passing and failing samples, whereas TSCM counts remained relatively stable. Passing samples were also enriched for TCM early in therapy (Fig. 2C) and TEM cells at a single time point (Fig. 2D); TEff counts were relatively equivalent in both groups (Fig. 2E). This overall pattern suggested that samples passing test expansion were significantly enriched for early lineage phenotypes as compared to those who failed after nearly every cycle of chemotherapy.

Figure 2 (F to J) reflects T cell phenotypes constituting the ATCs represented in Fig. 1C. We observed a significant enrichment of TN cells in ALL samples at diagnosis, after delayed intensification and during maintenance, and a significant enrichment of TSCM cells after all cycles except after induction and interim maintenance. The lymphoma samples, in contrast, had low TN and TSCM counts throughout therapy, with a relative preservation of memory and effector phenotypes (Fig. 2, H to J), suggesting that the circulating T cell population in patients with lymphoma is composed predominantly of differentiated T cells with limited expansion potential and scarce early lineage subtypes even at diagnosis. ALL samples were enriched for early lineage cells, correlating to enhanced expansion throughout therapy.

We again divided our analysis of samples from patients with ALL into SR-ALL and HR/VHR-ALL. These phenotypic studies, now reflecting expansion and ATC data represented in Fig. 1 (B and D), respectively, demonstrated that in both populations, TN cells are elevated initially but demonstrated sharp declines during therapy; for SR-ALL, this decline occurred after delayed intensification, and for HR/VHR-ALL, this occurred after consolidation (Fig. 2K). This variation in timing of TN decline resulted in a significant difference in absolute TN counts after consolidation and after interim maintenance; this significance was lost when SR-ALL TN counts declined after delayed intensification. The timing of depletion of TN cell counts correlated directly to the timing of decline in ATC demonstrated in Fig. 1D. TSCM cells, on the other hand, remained relatively stable in SR-ALL samples, although they progressively declined in HR/VHR-ALL samples (Fig. 2L). This decline again resulted in a significant difference after consolidation and interim maintenance that was lost after delayed intensification. Figure 1B demonstrated a significant difference in expansion that also emerged (and was maintained) after consolidation, consistent with the differences observed in TN and TSCM cell counts, suggesting that depletion of these early lineage cells abrogated successful expansion. Examination of trends in later lineage cells revealed several additional observations. TCM counts remained relatively stable in SR-ALL samples and demonstrated a decline after delayed intensification in HR/VHR-ALL samples, but this decline was not maintained (Fig. 2M). TEM cell counts were low throughout therapy in both disease groups, with a high degree of variability (Fig. 2N). Finally, TEff cell counts demonstrated a progressive decline in SR-ALL samples but remained relatively stable in HR/VHR-ALL samples (Fig. 2O; for results of statistical analysis, see table S4). The results from all three comparison groups are summarized in table S5.

Both SR- and HR/VHR-ALL started with equivalent early lineage cell counts at diagnosis, and the pattern of depletion led us to examine the chemotherapeutic regimens given. Two agents, cyclophosphamide and cytarabine, are given to each risk group at the times of the observed decline in TN cells; namely, SR-ALL patients received them during delayed intensification and HR/VHR-ALL patients received them with consolidation. These agents are not given at any other time for either treatment protocol, and no other agents were associated with this consistent and specific depletion.

Enrichment of TSCM cells rescues expansion

Several previous studies (8, 9, 19, 20) have demonstrated enrichment of the TSCM population by addition of IL-7 and IL-15 cytokines thought to support less-mature T cell phenotypes. Given the association between early lineage cells and expansion observed in the previous studies, we evaluated the effect of these cytokines on cell phenotype and expansion ability. Samples were collected and split into two cultures, one with and one without cytokines. We stratified this analysis only by disease type, not by risk category or cycle of therapy. For reference, we included the percentage of all samples passing test expansion for each disease group, which included all samples previously collected that were not part of our split-culture experiments (that is, samples analyzed in Figs. 1 and 2). Samples collected from patients with both leukemia and lymphoma demonstrated a significant enrichment in TSCM populations after culture with IL-7 and IL-15 (Fig. 3A; for results of statistical analysis, see table S6). This expansion of TSCM came at the expense of nearly every other cell type, with the exception of an increase in the TEff population in leukemia samples. This increase in TSCM cells was associated with enhanced expansion in both leukemia and lymphoma samples (Fig. 3B). Enhanced expansion was most pronounced in samples from patients with lymphoma, which demonstrated a statistically significant improvement (P = 0.003), reflecting a 57.7% overall pass rate when enriched for TSCM cells and a 15.3% pass rate when cultured without cytokines. This pass rate approached that demonstrated by our leukemia samples (60.3%).

Fig. 3. Effect of IL-7 and IL-15 on T cell phenotype and expansion.

Collected samples were split into two stimulatory cultures, either with or without IL-7 and IL-15 as described. (A) Percent change in absolute memory phenotype cell count in patients with leukemia and lymphoma after culture with cytokines. Significant differences are denoted with an “*” above each column and represent P < 0.05. (B) Percent of samples passing test expansion was evaluated after culture with or without cytokines. “All samples” represents every sample expanded without cytokines, some of which were collected before our split-culture protocol. Statistical analysis can be found in table S6.

DISCUSSION

This study represents a prospective and longitudinal collection of data from the same patients over time, reflecting the immune systems and T cell subsets of pediatric cancer patients from diagnosis through chemotherapy, rather than normal donors or a cross-sectional study design. Although previous studies have assessed the effect of chemotherapy on T cells (36), these studies were small and were largely performed before the use of current chemotherapeutic regimens. Additionally, samples were collected at only a few time points, analyzed for only a few T cell subsets, and most importantly did not assess T cell function. We demonstrate that enrichment of early lineage subsets is associated with significantly improved in vitro expansion and that patients with ALL have T cells with enhanced expansion capability as compared to patients with NHL. The poor expansions from our NHL samples are strongly associated with an initial and maintained deficit of early lineage cells, highlighting a fundamental difference in the T cell compartments at the time of diagnosis, implicating a disease-driven depletion of early lineage cells in lymphoma that is not present in leukemia. Stratification of the ALL samples into SR and HR/VHR groups reveals an initially robust expansion in both, with a rapid decline in HR/VHR-ALL after consolidation therapy correlated with the depletion of TN cells. Finally, we demonstrate that inclusion of IL-7 and IL-15 to our stimulatory cultures rescued the poor expansion observed in lymphoma samples with enrichment of the TSCM subset. This enhanced proliferation resulted in a fourfold increase in the number of samples passing our expansion threshold, greatly enhancing the number of patients potentially eligible to receive engineered T cell therapy.

The studies presented in this work investigated the underlying T cell biology responsible for the variations in cell expansion observed in our clinical experience and thus reflect T cell memory phenotypes at the time of cell collection. We have previously demonstrated that T cell phenotype at time of cell infusion has a significant impact on both persistence and antitumor activity (17), observing that TCM cells persist longer and mediate more successful and sustained antileukemic responses (11). Although antigen-driven antitumor activity of engineered T cells was not addressed in this study, we evaluated the memory phenotypes of all samples at the end of test expansion and observed that samples passing expansion produce a population ~50% TCM, whereas those failing produce a cell product that is ~55% TEM and TEff, with a TEff pool twice as large as those passing (19.6% versus 10.5%; fig. S1). To fully evaluate which lineages gave rise to these post-expansion subsets, we sorted T cells from normal donors and expanded them in vitro using the same protocol as our test expansion. We found that the TCM population present at the end of T cell expansion is derived primarily from TN and TSCM cells (table S7). Thus, early lineage T cells present at time of collection not only predict in vitro expansion potential but also directly give rise to T cells that persist long term and mediate robust antitumor responses in patients.

The correlation between ATC and expansion potential (Fig. 1) revealed several interesting observations. Although there was no quantitative difference between ALL and NHL ATC, there was a clear functional difference. Further examination of T cell subsets (Fig. 2) revealed a quantitative difference in specific lineages, reflecting that the primary defect in lymphoma is not overall T cell quantity but early lineage cell quantity—these T cell populations were nearly devoid of early lineage cells and instead had large memory and effector pools. This supports the notion that more of the “wrong” cells will not drive expansion and would instead result in a less functional cell product. Both risk-stratified and unstratified ALL expansions did demonstrate a general correlation with ATC. Examination of the stratified ALL groups revealed that HR/VHR-ALL samples have a sharp decline in ATC after consolidation therapy, which is maintained through therapy and associated with a progressive decline in expansion. Data presented in Fig. 2 demonstrated a similarly timed decline in TN cell counts after consolidation in HR/VHR-ALL samples, suggesting that the depletion in ATC observed in Fig. 1D reflected the depletion of the TN cell population. Figure 2K demonstrated that SR-ALL samples had a decline in TN after delayed intensification, again correlating to the decline in ATC for this group. Thus, although ATC may appear to correlate with expansion in SR- and HR/VHR-ALL, more granular examination reveals that this correlation is in fact with absolute TN count. These data also lead to the key observation that cyclophosphamide and cytarabine selectively depleted TN cells. Most interesting is that although TN cells remain depleted in both risk groups after their initial loss, the effect on expansion is immediate in HR/VHR-ALL but not observed in SR-ALL until maintenance. As discussed below, this could be the result of a maintained “cumulative” early lineage population (in the form of a maintained TSCM population) or may reflect the impact that early administration of this chemotherapy has on circulating T cells compared to late administration. HR/VHR-ALL samples may be more adversely affected by these agents as a result of continued high-intensity chemotherapy after consolidation, whereas SR-ALL samples have only maintenance therapy to follow. The overall trends of these data are summarized in table S5.

It is interesting that depletion of TN cells correlated most closely with decreased expansion in HR/VHR-ALL samples, whereas enhancement of the TSCM population had a more significant impact on expansion in NHL samples. It is possible that these two lineages play different roles in these two different diseases and that TN are more important for expansion in patients with ALL and TSCM are more important in patients with lymphoma. Our conclusion, however, is that the cumulative quantity of these two cell types together is the most relevant determinant of expansion. This may also explain why SR-ALL samples maintain robust expansion after delayed intensification, after their TN population has been depleted—the TSCM population persists and can continue driving robust expansion. As noted above, both TN and TSCM contribute to the TCM population present at the end of expansion, further supporting the notion that these two lineages may exert a combined effect. The reason for the decline in SR-ALL expansion during maintenance is not clear; it may reflect a delayed effect of cyclophosphamide and cytarabine.

Cyclophosphamide is a well-known lymphodepleting agent used as chemotherapy, in conditioning for bone marrow transplantation, and for treatment of autoimmune disease. Its direct effects as an alkylating agent in causing apoptosis of lymphocytes have been well characterized (21). Recently, cyclophosphamide has also been demonstrated to mediate direct immunosuppression by down-regulation of CD3, CD28, and major histocompatibility complex class II genes, molecules essential to T cell activation, and up-regulating expression of immunosuppressive cytokines such as IL-1β (22). The antimetabolite agent cytarabine has also been well described as highly cytotoxic to cells with rapid turnover, and its efficacy in depletion of normal and malignant lymphocytes has been clearly demonstrated (2325). From our data, it is not clear which agent is primarily responsible for the TN cell depletion observed, or whether this is a combinatorial effect. Close examination of Fig. 2 (K to O) demonstrates that these agents are selective for early lineage cells because TCM, TEM, and TEff cells do not demonstrate nearly the same degree of depletion.

Previous reports have demonstrated that patients with ALL have fewer circulating T lymphocytes at time of diagnosis as compared to healthy controls (3). Although T cell function was not investigated, the authors demonstrated that the memory compartment was selectively enriched by chemotherapy and speculated that memory cells might serve as mediators of protective immunity (6). Our data demonstrate that not all patients with ALL have depletion of their early lineage subsets at diagnosis, and in fact, patients with SR-ALL retain high TN and TSCM levels through delayed intensification. Knowledge of the TN and TSCM bulk before T cell expansion ex vivo may be quite important because culturing cells from patients with leukemia with IL-7 and IL-15 enhanced not only the TSCM subset but also the TEff subset. TEff are believed to produce a great deal of cytokine without significant antitumor effect, and thus, increasing this population before infusion may risk more side effects without more benefit, or even reduced benefit. Moving forward, we propose an initial assessment of TSCM bulk before expansion to stratify those samples that would benefit from cytokine support.

Several groups have previously demonstrated the enrichment of TSCM cells by culture with the homeostatic cytokines IL-7 and IL-15 (8, 9, 20). IL-7 is involved in support of progenitor T lymphocytes as they differentiate from common lymphocyte progenitor cells. It is produced by thymic epithelium, a tissue particularly sensitive to the effects of systemic chemotherapy (26), thus generating a lack of IL-7 support in patients receiving chemotherapy. This lack of IL-7–mediated support may make early lineage cells more susceptible to the effects of chemotherapy. IL-15 has been shown to be involved in support of “memory” T cells (27). TSCM cells share several phenotypic and functional characteristics with traditional memory subsets, and recent data have demonstrated its preferential role in protection of younger cells (8), suggesting that IL-15 is also directly involved in support of these stem-like memory cells.

Many of the current engineered cell therapy trials use beads that act as artificial antigen-presenting cells with CD3 and CD28 costimulation, and the use of these beads produces engineered T cells capable of remarkable in vivo proliferation and clinical activity (11, 13, 15, 16, 28). CD3/CD28 stimulation alone, however, is insufficient to stimulate the TSCM subset (8). The addition of IL-7 and IL-15 can increase this population in samples where early lineage cells are lacking or otherwise deficient, such as in patients with lymphoma or those that have previously received specific lymphotoxic chemotherapies. It is important to note that this observed rescue of ex vivo expansion reflects pooled data collected from samples harvested at various times during chemotherapy, both early and late into therapeutic course. Samples collected during each cycle of therapy were equally represented in this analysis, which we speculate accounted for the lower overall pass rates for samples cultured without cytokines in our split-culture experiments as compared to “all samples,” which include a greater proportion of samples collected from patients early in therapy. Thus, it is possible that cells collected earlier during therapy may have pass rates higher than 60% when cultured with cytokines.

Although previous reports demonstrating the enrichment of TSCM cells by IL-7 and IL-15 opened the door to this novel cell culture method, they were all performed using T cells from normal donors. We demonstrate enrichment of TSCM cells from patients with hematologic malignancies receiving chemotherapy, taking this proof of concept to clinical application in a patient population known to have dysfunctional T cells. This confirmation represents an essential step in the translation of this culture method to patients undergoing evaluation for engineered cell therapy.

The relevance of early lineage populations extends beyond cell expansion and ability to generate a viable clinical product, as other groups have demonstrated enhanced antitumor activity of TSCM cells in xenograft models of malignancy (8). Studies have also demonstrated that quantity of TSCM cells correlates with persistence in patients receiving CD19 CAR T cells (9). We would hypothesize that culture of T cells collected from patients with both HR/VHR-ALL and NHL with IL-7 and IL-15 would enhance the TSCM populations (as shown in Fig. 3B), allow those patients to achieve a target T cell dose composed of highly replicative T cells, and thus increase the number of patients able to receive highly active CD19-directed CAR therapy. Whether TSCM or TCM mediate more successful antitumor responses in humans remains to be seen. What is clear, however, is that current manufacture methods are insufficient to produce the cells needed for trial consideration for most patients with NHL, and this method of clinical cell culture significantly improves expansion while generating a cell population with potentially robust antitumor activity.

Several limitations exist in this study. We report here on a prospective observational study of children with B cell malignancies to determine T cell proliferative capacity. Children received standard-of-care therapy; however, individual children may have had longer or shorter delays between cycles, which may have influenced results. Although our sample size (n = 50) has allowed us to discern statistically significant differences, the interactions between patient, therapy (especially length and intensity of therapy), diagnosis, and age are complex. Our data suggest that successful expansion predicts production of a highly TCM-rich cell product. This product is in turn associated with high antitumor activity and persistence. These associations provide insight into the composition of highly active cell therapy products. A larger study would be required to ascertain whether test expansion might serve as a potency assay that would actually predict the response of an individual product in clinical use.

Our clinical trial results indicate that successful expansion under our manufacture conditions produces a highly effective engineered T cell therapy. We have identified that early lineage T cells are essential for ex vivo expansion in patients with B cell malignancies, and demonstrate a method to enrich TSCM in clinical cultures. These findings allow for the possibility to greatly increase the number of patients eligible for clinical trials and to investigate the clinical utility of TSCM-enriched CAR T cells for cancer therapy.

MATERIALS AND METHODS

Study design

This is an ongoing prospective, longitudinal, observational study of patients with B cell malignancies receiving chemotherapy. Patients were enrolled onto Children’s Hospital of Philadelphia Institutional Review Board–approved clinical trial CHP-12-009915. Peripheral blood samples were collected during scheduled venipuncture at the times indicated; samples collected during maintenance phase were collected after one cycle of therapy. Peripheral blood samples then underwent T cell isolation and stimulation as described below. Samples were deidentified and encoded before experimental procedures were performed, and thus, data collection was blinded. Given the observational nature of this study, it was not randomized. The primary objective of this study is to identify the time before, during, or after chemotherapy when T cell expansion is most likely to be successful. The secondary objectives are to (i) determine whether there is a relationship between success of expansion and absolute lymphocyte count or ATC and (ii) determine whether certain chemotherapy agents have an impact of ability to expand T cells.

In this ongoing study, initial sample size was defined as >20 patients to enable comparison to normal donors. We predicted 75% of study samples and 100% of normal donors would undergo successful expansion (based on previously collected data). We thus needed 20 patient samples to generate a 95% confidence interval of 53 to 97%.

Inclusion criteria were defined as follows: males or females aged 6 months to 21 years; diagnosis of CD19-positive B cell malignancy; no previous gene therapy (patients may have received CD19 CAR T cell therapy if it was followed by myeloablative stem cell transplant); parental/guardian-informed consent; and, if appropriate, child assent. Exclusion criteria were defined as follows: active hepatitis B or C infection; HIV infection; any uncontrolled medical condition that would preclude participation; and parents or guardians who, in the opinion of the investigator, may be noncompliant with study procedures.

Ex vivo T cell expansion and culture

Lymphocytes were harvested from peripheral blood using Ficoll-Paque (GE Life Sciences) density centrifugation medium as per standard protocol. The harvested buffy coat was resuspended in T cell culture medium, and cells were plated on appropriate-sized culture vessels at a concentration of 5 to 10 × 106 cells/ml and incubated overnight at 37°C. After this 18- to 24-hour incubation, the supernatant from these cultures was collected and washed, leaving adherent cells in culture vessels and isolating only suspension cells. A sample of this supernatant was stained for CD3, CD4, and CD8 expression (see below) to calculate ATCs. This full culture was then combined with stimulatory microbeads coated with CD3 and CD28 agonist antibodies (Life Technologies; catalog #111.32D) at a ratio of three beads per T cell, and resuspended at a concentration of 106 T cells/ml for T cell expansion, with the same stimulation and culture conditions used in our clinical test expansions. Cells were counted, cell sizes were measured every other day, and beads were removed on day 7. Culture period ended when cell growth kinetics and volume indicated that the cells had rested down from activation. For cytokine studies, samples were collected as described and combined with beads, then split into two cultures. One culture (no cytokine) was treated as above, and the other (+IL-7 and IL-15) was treated with IL-7 (25 ng/ml) and IL-15 (10 ng/ml) (R&D Systems, #207-IL-025 and #247-IL-205).

In our initial clinical trials of CD19 CAR T cells, a lower limit of greater than fivefold expansion is included as part of our inclusion criteria. We applied this same threshold as a standard for comparison across groups because it bears clinical relevance for engineered cell therapy trials and carries a high negative-predictive value for ability to produce a clinical product that meets target goal (see table S1). We would point out that although these culture conditions are the same as used for our clinical test expansions, our manufacture methods for clinical products are slightly different (11).

Flow cytometry

T cells were stained for cell surface markers to differentiate T cell lineage. Previous work (8) has demonstrated that CCR7, CD62L, CD45RO, and CD95 can be used to differentiate the various T cell phenotypes by using the following expression patterns: TN—CCR7+, CD62L+, CD45RO, CD95; TSCM—CCR7+, CD62L+, CD45RO, CD95+; TCM—CCR7+, CD62L+, CD45RO+, CD95+; TEM—CCR7, CD62L, CD45RO+, CD95+; and TEff—CCR7, CD62L, CD45RO, CD95+. The antibodies used for this analysis as well as quantitation of T cell bulk described above were CD8–FITC (fluorescein isothiocyanate) (BD Biosciences, #347313), CD3–PE (phycoerythrin) (BD Biosciences, #555340), CD4–APC (allophycocyanin) (BD Biosciences, #555349), CCR7-FITC (BD Biosciences, #561271), CD95-PE (BD Biosciences, #556641), CD45RO (BD Biosciences, #559865), and CD62L-PE/Cy7 (cyanine 7) (BioLegend; 304822). Cells were resuspended in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline + 1% fetal calf serum), then incubated with CCR7 antibody for 20 min at 37°C, washed once, and then incubated with remaining antibody cocktails for 25 min at 4°C. Samples were then washed twice, and flow cytometry acquisition was performed on a BD FACSVerse flow cytometer (BD Biosciences, #653118). Analysis was performed using FlowJo software (Tree Star Inc.).

Statistical analysis

All statistical analysis was performed using Prism 4 (GraphPad Software) using analysis of variance testing for group comparisons. Fisher’s exact test was used for comparison of percentages. All comparisons that are reported as significant reached a P value <0.05 or as calculated after Bonferroni corrections for multiple comparisons.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/320/320ra3/DC1

Fig. S1. Post-expansion phenotype of samples passing and failing test expansion.

Table S1. Statistical analysis of test expansion thresholds in patients receiving CD19 CAR T cell therapy.

Table S2. Summary of chemotherapeutic regimens for SR- and HR-ALL.

Table S3. Statistical analysis of data presented in Fig. 1.

Table S4. Statistical analysis of data presented in Fig. 2.

Table S5. Summary of significant differences in Fig. 2.

Table S6. Statistical analysis of data presented in Fig. 3.

Table S7. Post-expansion phenotypes of sorted T cell memory lineages.

REFERENCES AND NOTES

  1. Acknowledgments: We thank C. June, A. Seif, and H. Bassiri for the review of this manuscript. Funding: Supported in part by grants from the Leukemia and Lymphoma Society, Weinberg Funds, Cookies for Kids’ Cancer, and a Stand Up To Cancer–St. Baldrick’s Pediatric Dream Team Translational Research grant (SU2C-AACR-DT1113) and Pennsylvania Department of Health (S.A.G.). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research. D.M.B. is a St. Baldrick’s Foundation Scholar. Author contributions: N.S., S.A.G., and D.M.B. designed the research; N.S., J.P., and D.M.B. performed the research; N.S. and D.M.B. wrote the paper; D.M.B. performed all statistical analysis; and all authors reviewed the paper. Competing interests: S.A.G. has a consulting agreement with and receives research funding from Novartis. All other authors declare no competing interests.
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