A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy
© The Author(s) 2016
Received: 5 April 2016
Accepted: 11 July 2016
Published: 19 July 2016
Immunotherapy based on the adoptive transfer of gene modified T cells is an emerging approach for the induction of tumor-specific immune responses. Memory stem T cells, due to their enhanced antitumor and self-renewal capacity, have become potential candidate for adoptive T cell therapy of cancer. Methods to generate memory stem T cells ex vivo rely on CD3/CD28 costimulation and the use of cytokines such as IL-7 and IL-15 during the entire culture period. However, a strong costimulation may induce differentiation of memory stem T cells to effector memory T cells. Here we show that manipulation of the length of the costimulation and addition of IL-21 enhance the ex vivo expansion of memory stem T cells.
Purified naïve T cells from healthy donors were cultured in the presence of anti-CD3/CD28 coated beads, IL-7, IL-15 and/or IL-21 (25 ng/ml). T cells phenotype from the different memory and effector subpopulations were analyzed by multiparametric flow cytometry.
A short anti-CD3/CD28 costimulation of naïve T cells, combined with IL-7 and IL-15 significantly increased the frequencies of CD4+ and CD8+ memory stem T cells ex vivo, compared to a prolonged costimulation (34.6 ± 4.4 % vs 15.6 ± 4.24 % in CD4+; p = 0.008, and 20.5 ± 4.00 % vs 7.7 ± 2.53 % in CD8+; p = 0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment and expansion of CD4+ and CD8+ memory stem T cells with an increase in the absolute numbers (0.7 × 106 ± 0.1 vs 0.26 × 106 ± 0.1 cells for CD4+; p = 0.002 and 1.1 × 106 ± 0.1 vs 0.27 × 106 ± 0.1 cells for CD8+; p = 0.0002; short + IL-21 vs long).
These new in vitro conditions increase the frequencies and expansion of memory stem T cells and may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy of patients with cancer.
KeywordsImmunotherapy IL-21 Memory stem T cell
Adoptive T cell therapy (ACT) represents a highly promising strategy to treat cancer [1, 2]. While this approach has been mostly based on the use of terminally differentiated effector T cells, recent studies indicate that the use of less differentiated T cells with extensive replicative capacity has greater engraftment and antitumor effect [3–6]. In particular, the recently described memory stem T cells (TSCM), defined as CD62L+CCR7+CD45RA+CD95+CD45RO−CD28+CD27+IL2Rb+IL7Rα+, have enhanced proliferation, self-renewal, persistence and are able to differentiate into central memory, effector memory and effector T cells [6, 7]. Remarkably, studies in murine models have shown that TSCM have greater antitumor effect in vivo compared to other more differentiated T cell subsets [6, 8].
All these features make TSCM a promising T cell subset candidate for ACT of cancer. Although TSCM have been identified in the peripheral blood under basal conditions, the very low amount of these cells pose difficulty to their isolation, limiting their use for ACT of patients with cancer. Gattinoni et al. showed that CD8+ TSCM could be generated through the induction of the Wnt-β-catenin signaling . Further studies demonstrated the generation and expansion of CD8+ TSCM from naïve T cells [9–11] through CD3/CD28 costimulation in combination with IL-7 and IL-15 cytokines. However, prolonged in vitro costimulation decrease the expression of memory markers substantially (e.g., CD62L, CCR7 or CD27) leading to a swift to more differentiated T cells [12, 13]. For these reasons, identification of reproducible methods to generate and expand large numbers of TSCM for ACT of cancer remains a clinical priority.
IL-21, the most recent member of the common γ-chain (γc) receptor cytokine family (that includes IL-2, IL-7, and IL-15), has a role in innate and adaptive immunity , and contrary to IL-2, plays a key role in the development and maintenance of central memory T cells by the induction of an early differentiation phenotype . Importantly, T cells generated under IL-21 showed a superior antitumor effect in vivo in experimental models [15–17]. IL-21 acts synergistically with IL-7 and IL-15 to promote proliferation and survival of both memory and naïve T cells [18, 19]. However, to our knowledge the effects of IL-21 on the TSCM generation have not been studied so far.
We explored whether manipulating the length of CD3/CD28 costimulation and/or the addition of IL-21 could enrich for the TSCM population within T cells cultured under IL-7 and IL-15. We found that both, a very short costimulation and the addition of IL-21 increase the frequencies of CD4+ and CD8+ TSCM and enhance their proliferative capacity, resulting in a significant increase of TSCM numbers at the end of the culture. Our work reveals a new method to ex vivo enrich and expand TSCM, which may have relevant clinical implications for ACT of patients with cancer.
T-cell isolation, sorting and culture
Peripheral blood mononuclear cells (PBMCs) were obtained from healthy donors (n = 6) after informed consent, isolated by centrifugation through a ficoll-hypaque gradient (lymphoprep, Axis-shield, UK), and frozen until use. Naïve T-cells were enriched with an on demand naïve T-cell enrichment kit (Stem cell technologies, Canada) including CD14, CD16, CD19, CD20, CD36, CD45RO, CD56, CD66b, CD57, CD94, CD123, CD244, TCRγ/δ, glycophorin A and dextran-coated magnetic particles and according to manufacturer’s instructions.
TSCM, TCM, and TEM were FACS-purified on a FACS CANTO II cell sorter (BD Bio- sciences, USA) using the fluorescent antibodies anti-CD4, anti-CD8, anti-CD45RA, anti-CD45RO, anti-CD27, anti-CD95 and anti-CCR7.
Primary cells were cultured in RPMI-1640 medium supplemented with 10 % heat inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 µg/ml) (ThermoFisher Scientific, USA). All cell cultures were performed at 37 °C in a fully humidified atmosphere with 5 % CO2 in air.
Purified naïve T cells (higher than 95 %) were activated with anti-CD3/CD28 magnetic beads (Life Technologies, USA) in 1:2 bead/T-cell ratio, and then cultured with: IL-7 and IL-15 at 25 ng/ml each  (Peprotech, USA) or IL-7, IL-15 and IL-21 at 25 ng/ml each (Peprotech, USA). 2,5 × 105 cells/ml per well were plated in a 24-wells plate. After day 6, cells were cultured at a concentration of 1 × 106 cells/ml and plated in 12 or 6-well plates (depending on cell number). Cytokines and medium were replaced every 3–4 days, and cells were counted every 3–4 days by Trypan blue dye exclusion.
All donor’s samples were obtained according to a protocol approved by the Ethics Comittee of Hospital Santa Creu I Sant Pau.
Lentivirus production and transduction
A second generation lentival expression vector pLS-CG was used to generate GFP-encoding lentivirus. Lentivirus was produced in 293T cells cotransfected with the lentiviral vector and the packaging vectors pCMV-dR8.91 and pCMV-G using jet PRIME (Polyplus Transfection). Medium was changed 4 h after transfection, and lentivirus collected and concentrated after 48–72 h. Transduction was performed 48 h after initial stimulation by 1000 g spinoculation for 2 h at 32 °C in the presence of polybrene (5 μg/ml) (Sigma-Aldrich, USA). GFP expression was detected by flow cytometry in TSCM at day 8 of culture.
Flow cytometry, and antibodies and intracellular staining
Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).
For intracellular staining, cells were stimulated with 50 ng/mL PMA and 1 μg/ml ionomycin (Sigma-Aldrich, USA) during 6 h. Brefeldin 1× (eBioscience, USA) was added for the last 4 h. Cells were stained with surface antibodies, washed and fixed with BD Cytofix/Cytoperm (BD Biosciences, USA), and intracellular staining was performed with anti–IL-2, anti-IFN, granzyme-A, and perforin specific-antibodies (miltenyiBiotech, Germany). All antibodies were titrated before use.
Statistical analysis was performed with GraphPad Prism 6 (GraphPad Software, USA). Data are shown as the mean ± SEM. Differences were tested for statistical significance using one-way ANOVA test. A p value <0.05 was considered significant.
Short CD3/CD28 costimulation enriches for memory stem T cells (TSCM) cultured with IL-7/IL-15
Similar results were obtained in the CD8+ TSCM population (Fig. 2b). A short costimulation generated a significant increase of CD8+ TSCM frequencies compared to a long costimulation (20.5 ± 4.00 % vs 7.7 ± 2.53 % at day 10, respectively; p = 0.02). Day 10 was selected as an endpoint for culture since a decline in TSCM numbers was observed after this time (data not shown) and over this time period TSCM expand to numbers considered to be sufficient for clinical translation.
According to previous data , when TSCM are cultured in vitro they may also acquire the expression of CD45RO, while preserving CD45RA and CCR7+CD27+CD95+ expression (i.e., a TSCM-like phenotype). We found no differences in the percentage of both CD4+ and CD8+ TSCM-like cells across different time points over the culture period (19.4 ± 3.06 % vs 24.4 ± 2.6 % in CD4+; p = 0.252 and 49.95 ± 3.6 % vs 53.36 ± 1.04 % in CD8+; p = 0.35).
When total TSCM (i.e., TSCM + TSCM-like population) were analyzed (Fig. 2c, d), a higher percentage was observed in the CD4+ population after a short stimulation, reaching 54.02 ± 3.837 % at day 10 vs 38.49 ± 1.48 % in the long stimulus condition (p = 0.0054) (Fig. 2c). In the CD8+ population, a trend to higher percentages were found after short costimulation compared to a long costimulation (70.45 ± 4.1 % vs 60.2 ± 3.29 %; p = 0.08) (Fig. 2d).
IL-21 increases the frequencies of TSCM generated under short CD3/CD28 costimulation
Finally, we analyzed if IL-21 could increase the percentage of total TSCM (i.e., TSCM + TSCM-like population). At day 10, significant higher frequencies were noted in the CD4+ subset in the short costimulation in the presence of IL-21 (62.92 ± 5.83 % vs 54.02 ± 3.83 %; short + IL-21 vs short, respectively; p = 0.025) (Fig. 3c). Although the differences between short and long stimulation in the CD8+ subset are enhanced by the addition of IL-21 (74.15 ± 5.26 % vs 60.2 ± 3.29 %; short + IL-21 vs short respectively; p = 0.04), no significant differences were observed in the short stimulation condition by the addition of IL-21 regarding total TSCM (74.15 ± 5.26 % vs 70.45 ± 4.13 %; p = 0.56) (Fig. 3d).
Short costimulation combined with IL-21 enhance TSCM expansion
TSCM shows different functional properties
To further define the functional features of T-cells generated under these conditions, we analyzed the expression of IL-2, IFN-γ, granzyme-A and perforin on FACS-purified TSCM, TCM and TEM at day 10 of culture. We found that both CD4+ and CD8+ TSCM secrete higher levels of IL-2 compared to TCM (CD4: 84.65 ± 0.75 % vs 83.3 ± 0.7 %; p = 0.48; CD8: 68.86 ± 1.9 % vs 62.56 ± 0.13 %, p = 0.03) and TEM (CD4: 84.65 ± 0.75 % vs 80.5 ± 0.75 %; p = 0.057; CD8: 68.86 ± 1.9 % vs 57.8 ± 1.12 %, p = 0.0022) (Additional file 1: Figure S1 A, B). Moreover, we observed lower proportion of CD4+ and CD8+ TSCM that secrete IFN-γ compared to TCM (CD4: 8.18 ± 0.16 % vs 13.6 ± 1 %; p = 0.016; CD8: 18.66 ± 4.2 % vs 48.76 ± 8.2 %, p = 0.0397) and TEM (CD4: 8.18 ± 0.16 % vs 21.55 ± 0.25 %; p = 0.0012; CD8: 18.66 ± 4.2 % vs 60.86 ± 6.4 %, p = 0.0091) and granzyme-A compared to TCM (CD4: 34.83 ± 8.6 % vs 55.66 ± 1.53 %; p = 0.0631; CD8: 7.2 ± 3.3 % vs 75.8 ± 0.3 %, p = 0.0004) and TEM (CD4: 34.83 ± 8.6 % vs 78.13 ± 2.36 %; p = 0.0033; CD8: 7.2 ± 3.3 % vs 78.1 ± 0.5 %, p = 0.0004). We also observed that CD4+ and CD8+ TSCM stained mainly negative for perforin (0.024 ± 0.002 % and 0.9 ± 0.3 %, respectively) (Additional file 1: Figure S1 C–H).
Finally, we analyzed the phenotype and self-renewal capacity of FACS-purified TSCM cells after being expanded for 10 days. We observed that, in contrast to TCM and TEM subsets, both CD4+ and CD8+ TSCM maintained their phenotype and proliferative capacity in culture for another 10 days after sorting. Remarkably, 93 % of the CD4+ and 88 % of CD8+ T cells displayed a TSCM phenotype after 20 days in culture with IL-7, IL-15 and IL-21, while TCM and TEM largely lost their proliferative capacities and became differentiated (Additional file 2: Figure S2).
CD4+ and CD8+ TSCM are efficiently transduced by lentiviral vectors
The generation of TSCM for ACT has become a clinically relevant issue since preclinical studies have shown that the use of the less-differentiated TSCM is associated with superior T-cell engraftment, persistence, and antitumor efficacy [3–6]. Although ACT to treat cancer have experimented significant progress in the past few years, particularly with the chimeric antigen receptor (CAR) approach, improvements on their clinical efficacy are clearly needed. Remarkably, although no ACT clinical trials with TSCM have been done in cancer patients so far, studies in patients with lymphoma receiving CAR-T cells showed that the infusion of >5 % of cells with a TSCM phenotype correlated with greater persistence and expansion in vivo, properties associated with clinical efficacy .
Previous studies have shown that the combination of IL-7, IL-15 and CD3/CD28 costimulation led to the generation of TSCM from naïve T cells . Interestingly, cells generated under this condition also express CD45RO, which indicates that the combination of prolonged CD3/CD28 costimulation with IL-7 and IL-15 induce a more differentiated TSCM (TSCM-like) than the originally described . This could be related to the stimulation through CD3 and CD28, which expands T cells efficiently but also causes a drastic change in the cell phenotype, including downregulation of CD45RA, CCR7 and CD62L [6, 13, 21]. Although both subsets have comparable gene expression profiles, it remains to be proven that the TSCM-like subset (i.e., those cells expressing CD45RO in addition to CD45RA and CCR7) retain similar antitumor efficacy than the less differentiated TSCM. In this regard, when CAR-T cells were cultured with IL-7/IL-15, only the fraction CD45RA+/CCR7+ retained the ability to expand after repetitive antigen stimulations . Accordingly, strategies directed to preserve the phenotype of TSCM during their ex vivo generation should be regarded a clinical priority.
In this study, we show that a very short (48 h) CD3/CD28 costimulation increases the percentage of TSCM within the CD4+ and CD8+ subsets in detriment of a more differentiated phenotypes. Moreover, the contribution of the less differentiated TSCM to the total TSCM frequency is superior in the short costimulation condition compared to a long costimulation, in line with the concept that prolonging the CD3/CD28 stimulation in the culture is associated with T cell differentiation [21, 22]. In our condition, TSCM proliferation was not affected by the absence of a prolonged CD3/CD28 signal, suggesting that a continued costimulatory signal would not be necessary to promote an efficient ex vivo TSCM expansion in the presence of IL-7 and IL-15 [23–25].
The T-cell receptor signal strength impacts on T cell expansion and differentiation . Methods for the generation of effector T cells based on CD3/CD28 stimulation have relied on the use of a high bead:cell ratio (e.g., 3:1). We observed that a high bead-to-cell ratio reduced the frequency of TSCM and did not generate a strong T cell proliferative response, a fact that may be associated with activation-induced T cell death [21, 27]. In contrast, in our hands, a low bead-to-cell ratio (e.g., 1:2) coupled to a short costimulation was the best condition for promoting the generation of higher TSCM frequencies while maintaining their proliferative capacity. This is in line with recent studies showing that low or equal bead:cell ratios give the higher yields of T cells when cultured in IL-7 and IL-15 . Moreover we observed that the short TCR triggering do not reduced the transduction efficiency of TSCM, probably due to the presence of the IL-7, IL-15 or IL-21 cytokines, which promote long-term survival of resting T cells, while maintaining their maturational status .
Previous studies have shown that IL-21 synergizes with either IL-7 or IL-15 to regulate CD8+ T cell expansion and to maintain a memory phenotype [18, 19, 29]. However, the potential role of IL-21 in the ex vivo generation of TSCM has not been described. Here, we show for the first time that the addition of IL-21 significantly increases the percentage of CD4+ and CD8+ TSCM cultured with a short CD3/CD28 stimulation and IL-7/IL-15. Remarkably, addition of IL-21 to that condition further increased the proliferation rate of TSCM, which translated into an increase of absolute numbers of TSCM at the end of the culture. Although previous studies have shown that CD3/CD28 costimulation in combination with IL-7 and IL-15 promote a substantial increment in TSCM expansion , the reduction in the length of costimulation and, particularly, the addition of IL-21 produces even higher numbers of TSCM that could be sufficient for clinical translation. Interestingly, this effect was noted in both CD4+ and CD8+ TSCM, and although the proliferation rate was greater in the CD8+ subset compared to that in CD4+, it is remarkable that the CD4+/CD8+ ratios of TSCM were preserved over the culture period.
According to previous studies showing a higher expression of IL-21 receptor in activated naïve T cells , and that the effect of IL-21 might depend on the T cell differentiation status [21, 31], we observed that the impact of IL-21 was more pronounced in those T cells with a phenotype close to naïve T cells, resulting in an enrichment of less differentiated TSCM within the total TSCM subset. Moreover, we have noticed a higher expression of IL-21R in naïve and TSCM cells compared to more differentiated T cells subsets (57.5 % of TSCM vs 22 % of TCM in CD4+ and 42.6 % of TSCM vs 5 % of TCM in CD8+) (data not shown). Interestingly, this difference is more pronounced at day 4 of the culture (in particular in CD8+ T cells), which could increase the effect of IL-21 in the following days.
It has been shown that self-renewal depends on STAT3 (Signal transducer and activator of transcription 3) signaling cytokines, including IL-6, IL-10 and IL-21, which inhibit exhaustion and differentiation of T cell . Similarly, T cells expanded in the presence of IL-21 (the unique common γ-chain cytokines to sustain STAT3 activation) prevent the loss of CD45RA, CD62L, CD27, and IL-7Rα, increasing the ability of the cell to release IL-2 [33, 34]. Interestingly, we noticed that TSCM, TCM and TEM cultured in the presence of IL-21 express high level of IL-2. In addition, TSCM express low levels of IFN-γ, granzyme-A and perforin compared to TCM and TEM. Thus, TSCM display phenotypic and functional characteristics of early-differentiated cells.
We also observed that highly purified naïve T cells are a more suitable starting population for TSCM generation in contrast to the use of “bulk” peripheral blood mononuclear cells [10, 20]. The use of purified naïve T cells may overcome the variability of this subset observed between donors when using whole peripheral blood lymphocytes. Another important issue is the potential deleterious effect that more differentiated memory T cells present in the starting cell population may have on the generation of TSCM. Accordingly, in our experience, TSCM are more efficiently generated when naïve T cells are used as starting population (data not shown), in line with previous data from Bonini´s group .
In summary, we demonstrated that when naïve T cells are cultured with IL-7 and IL-15, the use of a very short CD3/CD28 costimulation enhances CD4+ and CD8+ TSCM generation and expansion. Moreover, the addition of IL-21 to this culture conditions produces a further enrichment and greater expansion of CD4+ and CD8+ TSCM. Importantly, the conditions described here allow an efficient transduction of both CD4+ and CD8+ TSCM with lentivirus. These refinements on the in vitro conditions may be of clinical relevance for the generation of TSCM for ACT of patients with cancer, and can be easily adapted to preserve this cell subset in the final product under GMP conditions.
adoptive T cell therapy
- TCM :
central memory T cells
chimeric antigen receptor
- TEM :
effector memory T cells
- TEMRA :
effector memory-RA + T cells
fluorescence minus one
- TSCM :
memory stem T cells
multiplicity of infection
- TN :
naïve T cells
peripheral blood mononuclear cells
signal transducer and activator of transcription 3
CAF: Design, experimental and analytical performance. LEG: Experimental performance. SV: Design performance. JS: Analytical performance. JB: Study’s conception, design, and analytical performance. All authors read and approved the final manuscript.
The authors thank Dr. Sonia Guedan for helpful discussions and critical review of the manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All donor’s samples were obtained according to a protocol approved by the Ethics Comittee of Hospital Santa Creu I Sant Pau.
This work was supported by Fundació La Marató TV3, Cellex Research Foundation (Barcelona), AGAUR 2014SGR-1281, and ISCIII RD12/0036/0071.
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