A human memory T-cell subset with stem cell-like properties

Abstract

Immunological memory is thought to depend upon a stem cell-like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T-cell population that displays enhanced self-renewal and multipotent capacity to derive central memory, effector memory and effector T cells. These cells, specific for multiple viral and self-tumor antigens, were found within a CD45RO⁻, CCR7⁺, CD45RA⁺, CD62L⁺, CD27⁺, CD28⁺ and IL-7Rα⁺ T-cell compartment characteristic of naïve T cells. However, they expressed increased levels of CD95, IL-2Rβ, CXCR3, and LFA-1, and exhibited numerous functional attributes distinctive of memory cells. Compared to known memory populations, these lymphocytes displayed increased proliferative capacity, more efficiently reconstituted immunodeficient hosts and mediated superior anti-tumor responses in a humanized mouse model. The identification of a human stem cell-like memory T-cell population is of direct relevance to the design of vaccines and T-cell therapies.

Long-lived self-renewing memory lymphocytes are a hallmark feature of the adaptive immune system in response to pathogens and tumors^1–3. Analogous to organ systems in which non-replicating, terminally-differentiated cells are continually replenished by the progeny of less differentiated stem cells, it has been suggested that memory cells might contain stem cell-like cells^4,5. Indeed, several characteristics of stem cells can be found to certain degrees in memory B and T cells, including selective transcriptional profiles⁶, the capacity to self-renewal and the multipotency to differentiate into progeny with diverse fates^4,5.

The memory T-cell compartment is heterogeneous and has been conventionally divided into two subsets based on the expression of the lymph node homing molecules CD62L and CCR7⁷. Central memory T cells (T_CM) express high levels of CD62L and CCR7 and were thought to be the stem cell-like memory subset, whereas CD62L⁻ CCR7⁻ effector memory T cells (T_EM) are considered committed progenitor cells that undergo terminal differentiation after a limited number of divisions^4,5. The identification in mice of a novel population of memory T cells with enhanced stem cell-like qualities compared to conventional T_CM cells adds complexity to this dichotomous view^8,9. These memory T cells, which were designated memory stem cells (T_SCM), exhibit a CD44^low CD62L_high phenotype like naïve T cells (T_N), but co-express stem cell antigen–1 (Sca-1) and high levels of the antiapoptotic molecule B cell lymphoma 2 (Bcl-2), the β chain of the IL-2 and IL-15 receptor (IL2-Rβ), and the chemokine (C-X-C motif) receptor CXCR3^8,9. Whether a similar memory T-cell population exists in human is currently under intensive investigation¹⁰.

A human CD8⁺ memory T-cell population has been described that shares phenotypic and functional characteristics with hematopoietic stem cells including the expression of the stem cell marker C-KIT and the ability to efflux cellular toxins through the ATP-binding cassette (ABC)–superfamily multidrug efflux protein ABCB1¹¹. However, recent data revealed that these cells are predominantly Vα7.2⁺ mucosal associated invariant T cells (MAIT)¹². More recently, Schenkel et al.¹³ speculated that CD4^dimCD8^bright T cells expressing high levels of β-catenin, a molecule associated with the generation of mouse T_SCM^9,14, represent human T_SCM, but the definitive identification of human T_SCM remains to be accomplished.

Identification of human T memory stem cells

We previously found that mouse T_SCM can be generated effectively in vitro by triggering Wnt signaling during T cell priming using Wnt3A or inhibitors of glycogen synthase kinase-3β (GSK-3β)^9,14,15. We sought to employ the same strategy to generate candidate human T_SCM by activating CD45RO⁻CD62L⁺ naïve CD8⁺ T cells in the presence of the GSK-3β inhibitor TWS119 (Fig. 1a). After two weeks, the majority of T cells cultured with TWS119 retained a CD45RO⁻CD62L⁺ naïve-like phenotype, whereas in the absence of GSK-3β inhibition, T cells uniformly upregulated the memory marker CD45RO (Fig. 1a). To determine whether the CD45RO⁻CD62L⁺ T cells generated in the presence of TWS119 were truly naïve cells or had acquired memory traits, we performed an extensive phenotypic analysis using established markers of T-cell activation and differentiation (Fig. 1b)¹⁶. The vast majority of molecules (CD45RA, CCR7, CD27, IL-2Rα, IL-7Rα, CD69, 41BB, CCR5 and CD57) showed a similar expression pattern between T_N and TWS119-generated naïve-like T cells (Fig. 1b). However, the naïve-like T cells expressed levels of CD95 and IL-2Rβ similar to conventional memory T cells (Fig. 1b). Thus, we hypothesized that the expression of CD95 and IL-2Rβ on otherwise phenotypically naïve T cells could identify human T_SCM cells.

Figure 1. Identification of T_SCM cells in human blood.

a, Flow cytometry analysis of sorted human CD45RO⁻CD62L⁺ naïve CD8⁺ T cells prior to and 14 days after stimulation with α-CD3/CD2/CD28-coated beads and IL-2 in the presence or absence of 5µM TWS119. Numbers indicate the percentage of cells in the CD45RO⁻CD62L⁺ gate. b, Flow cytometry analysis of TWS119-generated CD45RO⁻CD62L⁺ naïve-like CD8⁺ T cells overlaid with CD45RO⁻CD62L⁺ naïve and memory (non-CD45RO⁻ CD62L⁺) cells from a healthy donor (HD). c, Flow cytometry analysis of PBMC from a healthy donor. Dot plots show the gating strategy to identify CD95⁺, IL2Rβ⁺ T_SCM cells. d, Percentages of circulating CD8⁺ T-cell subsets in 29 healthy donors. e, Flow cytometry analysis of PBMC from a representative healthy donor. Overlaid histogram plots show expression levels of a given molecule in different CD8⁺ T-cell subsets. CD8⁺ T-cell subsets were defined as follows: T_N cells, CD3⁺CD8⁺CD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL7Rα⁺CD95⁻; T_SCM cells, CD3⁺CD8⁺CD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL7Rα⁺CD95⁺; T_CM cells, CD3⁺CD8⁺CD45RO⁺CD45RA⁻ CCR7⁺CD62L⁺; T_EM cells, CD3⁺CD8⁺CD45RO⁺CD45RA⁻CCR7⁻CD62L⁻.

To determine if candidate T_SCM cells occur naturally we used polychromatic flow cytometry (PFC)¹⁷. Based on previous data¹⁷, we employed a highly stringent criterion of 7 markers to accurately define T_N. Strikingly, a CD95⁺IL-2Rβ⁺ subset could be found in CD45RO⁻CCR7⁺CD45RA⁺CD62L⁺CD27⁺CD28⁺IL-7Rα⁺ naïve-like CD8⁺ (Fig. 1c) and CD4⁺ (Supplementary Fig. 1a) T cells. In 29 healthy donors, these cells, referred to hereafter as T_SCM cells, represented about 2–3% of all circulating CD8⁺ and CD4⁺ T lymphocytes (Fig. 1d and Supplementary Fig. 1b). Further phenotypic analysis of T-cell differentiation markers revealed that T_SCM also expressed higher levels of BCL-2, LFA-1, CXCR3, CXCR4, and lower levels of CD38 and CD31 compared to T_N cells (Fig. 1e and Supplementary Fig. 1c). Notably, T_SCM cells expressed all of the core phenotypic markers (IL-2Rβ, BCL-2 and CXCR3) of their mouse counterparts^8,9, except SCA-1 which does not have a human ortholog. These T cells were phenotypically different from those described by Turtle et al.¹¹ and were not MAIT¹² (Supplementary Fig. 2). Similar to conventional memory, T_SCM cells were detected at low frequencies (<1%) in cord blood (Supplementary Fig. 3). The phenotype of T_SCM cells indicates a tropism for lymphatic tissues, but full anatomical characterization of T_SCM-cell niches remains to be addressed.

T_SCM possess attributes of conventional memory T cells

Due to the concomitant expression of numerous markers of naiveté as well as key molecules of memory differentiation, it remained unclear whether T_SCM cells were functionally naïve or memory T cells. Naive T cells have high levels of TCR rearrangement excision circles (TREC), which are diluted during clonal proliferation¹⁸. Like T_CM and T_EM cells, we found that T_SCM cells had low levels of TREC, indicating that they had undergone several rounds of division (Fig. 2a).

Figure 2. T_SCM cells possess attributes of conventional memory T cells.

a, TCR excision circle (TREC) copy number in sorted CD8⁺ T-cell subsets relative to T_N cells. Data are represented as mean ± s.e.m. of 4 donors. b, Intracellular cytokine staining of PBMC from a representative healthy donor after stimulation with SEB. Graphs show naïve-like (NL) gated T cells. NL, CD45RO⁻CCR7⁺CD45RA⁺CD27⁺CD28⁺. Numbers represent the percentage of CD95⁺ (T_SCM) and CD95⁻ (T_N) cells producing a single cytokine. c, Percentages of CD8⁺ T-cell subsets producing IFN-γ, IL-2 and TNF-α in 6 healthy donors (obtained as described in panel b). d, Pie charts depicting the quality of the cytokine response in CD8⁺ T-cell subsets in 6 healthy donors as determined by the Boolean combination of gates identifying IFN-β⁺, IL-2⁺ and TNF-α⁺ cells. e, CFSE dilution in sorted CD8⁺ T-cell subsets after stimulation with 25 ng ml⁻¹ of IL-15 for 10 days. Data are shown after gating on CD8⁺ cells. PD: percentage divided; PI: proliferation index. f, Percentage divided cells and g, Proliferation index of different CD8⁺ T-cell subsets after stimulation as in panel e. Data are represented as means ± s.e.m of 9 donors. h, Flow cytometry analysis of PBMC from HLA-A2⁺ donors. Graphs show tetramer-binding cells vs. CD95 expression in the NL (CD45RO⁻CCR7⁺CD45RA⁺CD27⁺IL7Rα⁺) gate. i, Percentage of tetramer-binding cells expressing CD95 in the NL gate determined as in panel h. Data represent the donors tested for tetramer specificity. HD: healthy donor; MP: melanoma patient. j, Frequency of two immunodominant CMV-specific TCRβ clonotypes relative to all CMV-specific TCRβ clonotypes in pp65 -specific T-cell subsets isolated over a period of 22 months from a representative donor. The figure legend shows the CDR3β amino acid sequences. Changes in the frequencies of immunodominat clonotypes are depicted as the thickness of the bars, with the magnitude scale shown on the right. * = P < 0.05; ** = P < 0.01; *** = P < 0.001; ns= not significant (t test, c,f,g,I and χ² permutation test, d).

Memory T cells can also be distinguished from T_N by their ability to rapidly acquire effector functions upon antigen rechallenge¹⁹. We found that within 4h after exposure to Staphylococcus Enterotoxin B (SEB), a significant fraction of CD95⁺ naïve-like CD8⁺ T cells produced IFN-γ, IL-2 and TNF-αs while T_N cells remained relatively quiescent (Fig. 2b,c). Thus, T_SCM cells rapidly acquired effector functions following superantigen stimulation like conventional memory T cells (Supplementary Fig. 4). Interestingly, the fraction of responding cells, as well as T-cell polyfunctionality, progressively increased from T_N cells →T_SCM cells →T_CM cells →T_EM cells (Fig. 2c,d), consistent with the hypothesis that T_SCM cells are the least differentiated subset. Similar findings were observed for CD4⁺ T cells (Supplementary Fig. 1d and e). The rapid responsiveness of T_SCM cells was also observed after polyclonal stimulation with α-CD3/CD2/CD28 beads (Supplementary Fig. 5). Consistent with the intracellular cytokine staining result, sorted T_SCM cells, but not T_N cells, secreted IFN-γ, IL-2 and TNF-α in response to α-CD3/CD2/CD28 stimulation (Supplementary Fig. 6). Thus, T_SCM possess the memory capability of rapid acquisition of effector functions following TCR stimulation.

Unlike T_N, memory T cells undergo robust proliferation in the presence of the homeostatic cytokines IL-15 and IL-7^20–22. We found that, similar to conventional CD8⁺ memory T cells, T_SCM cells divided extensively in response to IL-15 (Fig. 2e). While the majority of T_EM cells proliferated (Fig. 2f), they underwent fewer divisions, revealing a lower proliferative potential compared to other memory subsets (Fig. 2g). By contrast, T_SCM cells underwent numerous cell divisions (Fig. 2g), although a greater fraction of these cells remained undivided (Fig. 2f). This behavior is reminiscent of stem cells, which are quiescent but can give rise to progeny capable of extensive proliferation and differentiation. Similar findings were observed in the CD4⁺ T-cell compartment in response to IL-7 (Supplementary Fig. 1f–h). Thus, T_SCM have the replicative history and ability to respond rapidly to antigenic and homeostatic stimuli, characteristics of memory T cells.

The frequency of naïve CD8⁺ T-cell precursors for a given epitope has been estimated to be between 6 × 10⁻⁷ and 5 × 10⁻⁶, a range below the limit of peptide-MHC class I (pMHCI) tetramer detection²³. We reasoned that if we could measure tetramer-binding, naïve-like T cells, they would be enriched in the CD95⁺ T_SCM-cell compartment. In donors with detectable naïve-like CD8⁺ T cells specific for influenza or cytomegalovirus (CMV) epitopes, the vast majority of tetramer-binding cells expressed high levels of CD95 (Fig. 2h,i and Supplementary Fig. 7). By contrast, virtually all MART-1-specific naïve-like T cells in healthy donors did not express CD95, indicating that these cells were truly naïve (Fig. 2h,i and Supplementary Fig. 7). Notably, a significant fraction of MART-1-specific CD8⁺ T cells displayed a CD95⁺ phenotype in 7/11 patients with metastatic melanoma (Fig. 2h,i and Supplementary Fig. 7). Thus, tetramer-binding T cells found in the “naive-like” T-cell compartment could be derived from either increased thymic output (CD95⁻), as reported for MART-1 in healthy donors²⁴, or from antigenic encounter, expansion and differentiation (CD95⁺). These experiments also revealed that T_SCM represented a substantial fraction of the corresponding total antigen-specific CD8⁺ T-cell memory responses, averaging 0.6% for CMV pp65_495–503, 4.2% for influenza M1_58–66 and 7.6% for MART-1_26–35, and that their frequency tended to correlate with that of conventional memory T cells (Supplementary Fig. 8).

To determine whether T_SCM clonotypes represent a long-lived population or merely recently activated cells transitioning from a naïve to a conventional memory state, we analyzed TCRβ sequences of CMV-specific T-cell subsets from the same donor spanning a time period of 22 months. Like conventional memory T cells, we found dominant persisting clonotypes in T_SCM cells, indicating that they represent a stable memory T-cell population (Fig. 2j and Supplementary Table 1). These findings demonstrate that T_SCM cells are long-lived memory T cells with multiple viral and self–tumor specificities.

T_SCM cells represent the least differentiated T-cell memory subset

We sought to compare the transcriptome of T_SCM cells with naive and conventional memory T-cell subsets and validate key findings with PFC (Fig. 3 and Supplementary Fig. 9). Nine hundred genes were differentially expressed among the four CD8⁺ T-cell subsets (P < 0.01, FDR < 5%) (Supplementary Table 2). Unsupervised hierarchical clustering revealed that T_SCM cells had a distinct gene expression profile more closely related to conventional memory T cells than T_N cells, further corroborating that T_SCM cells are a unique T-cell memory subset (Fig. 3a). Consistent with the data reported by Willinger et al.²⁵, the majority of genes (565/900) progressively increased (effector-associated genes) or decreased (naïve-associated genes) in the exact order: T_N cells →T_SCM cells →T_CM cells →T_EM cells (Supplementary Table 3). For example, transcripts of key regulators of effector differentiation and senescence, such as Eomesodermin²⁶, T-box 21²⁷ and PR Domain Containing 1, with ZNF Domain²⁸, as well as genes encoding for cytotoxic molecules (e.g. Granzyme A and Perforin) and markers of T-cell senescence (e.g. Killer Cell Lectin-like Receptor Subfamily G, Member 1 (KLRG1)²⁷), were increasingly expressed from T_N cells to T_EM cells (Fig. 3b). Conversely, the expression of transcription factors that inhibit T-cell activation and differentiation such as Lymphoid Enhancer-binding Factor 1⁹ and Forkhead Box P1²⁹ and LAG1 Homolog, Ceramide Synthase 6, which promotes cellular quiescence by regulating intracellular ceramide levels³⁰, progressively decreased from T_N cells to T_EM cells (Fig. 3b). These data are consistent with a linear model of T-cell differentiation, in which T_SCM are the least differentiated memory T-cell subset.

Figure 3. T_SCM cells represent a distinct, less differentiated T-cell memory subset.

a, Heat map of differentially expressed genes (P < 0.01 One-Way Repeated Measures ANOVA, FDR < 5% Benjamini-Hochberg's method) among CD8⁺ T-cell subsets. Red and blue colors indicate increased and decreased expression respectively. b, Robust Multichip Analysis (RMA)-normalized intensity of selected display of genes progressively downregulated (naïve associated genes) or upregulated (effector associated genes) from T_N cells →T_SCM cells →T_CM cells →T_EM cells. Data are represented as means ± s.e.m of 3 donors. c) Multidimensional scaling (MDS) analysis of differentially expressed genes (P < 0.01, FDR < 5%). Numbers represent the differentially regulated genes among each CD8⁺ T-cell subset (P < 0.01 (t test) and > 2 fold change in expression level). d, Heat map of differentially-expressed genes among T_SCM and T_CM cells (P < 0.01 (t test) and > 2 fold change in expression level). Red and blue colors indicate increased and decreased expression, respectively. Full gene names are listed in the Supplementary Information.

Multidimensional scaling (MDS) analysis³¹ confirmed that T_SCM cells was the memory T-cell subset most similar to T_N (Fig. 3c). Indeed, only 75 genes were differentially expressed between T_N and T_SCM (P < 0.01 and > 2-fold change in expression) compared to 157 and 226 for T_CM and T_EM cells, respectively (Fig. 3c, and Supplementary Tables 4–6). T_SCM and T_CM cells were the most closely related T-cell subsets, with 20 differentially expressed genes (Fig. 3c,d and Supplementary Tables 4–9). Among these genes, T_SCM cells, like T_N cells, expressed low levels of Heterogeneous Nuclear Ribonucleoprotein L-like, a key regulator of the alternative splicing of the CD45 pre-mRNA required for efficient CD45RO expression³², confirming the purity of the sorting. When considering this subset of 20 genes, T_SCM cells have a pattern of expression similar to T_N cells, while T_CM cells clustered with T_EM cells (Fig. 3d), further underscoring that T_SCM cells are less differentiated than T_CM cells.

Enhanced self-renewal and multipotency of T_SCM cells

The abilities to self-renew and differentiate into specialized cell types are defining qualities of stem cells. To determine whether T_SCM have these stem cell-like properties, we evaluated their capacity to self-renew with homeostatic signals as well as their multipotency after TCR activation. After exposure to IL-15, the vast majority of T_SCM cells maintained CD45RA⁺, and retained significantly higher levels of CD62L and CCR7 than T_CM cells (Fig. 4a and Supplementary Fig. 10). At the end of stimulation about 60% of cells derived from T_SCM maintained their phenotypic identity (CCR7⁺CD62L⁺CD45RA⁺CD45RO⁻) while only 30% of T_CM cells retained their input phenotype (CCR7⁺CD62L⁺CD45RA⁻CD45RO⁺) (Fig. 4b). T_SCM cells also displayed greater self-renewal capacity compared to T_CM cells following a secondary exposure to IL-15 (Supplementary Fig. 11).

Figure 4. Enhanced self-renewal and multipotency of T_SCM cells.

a, Percentage of CD8⁺ T cells expressing CCR7 and CD62L (right panel) and CD45RA (left panel) relative to cell division after exposure to 25ng ml⁻¹ of IL-15 for 10 days. Slopes (S) were compared using a Wilcoxon rank test, *= P = 0.0391. $\mathrm{s}=\frac{{\displaystyle \sum _i^n}(\mathit{\text{gi}}-\overline{g})(\mathit{\text{pi}}-\overline{p})}{{\displaystyle \sum _i^n}{(\mathit{\text{gi}}-\overline{g})}^2}$ where g = generation number and p = percentage of CD62L⁺CCR7⁺ cells (n = 8). b, Percentage of CFSE-diluted CD8⁺ T cells that retained the parental phenotype following stimulation with 25ng ml⁻¹ of IL-15 for 10 days. c, Percentage of CD8⁺ T cells expressing CCR7 and CD62L (right panel) and CD45RA (left panel) relative to cell division after stimulation with α-CD3/CD2/CD28-coated beads for 6 days. d, Percentage of CFSE-diluted CD8⁺ T cells with a given phenotype following stimulation with α-CD3/CD2/CD28-coated beads for 6 days. e, Stemness index of CD8⁺ memory T-cell subsets. Stemness index was calculated by multiplying self-renewal (SI) and multipotency (MI) indexes. SI was calculated as follows: SI= 2^PIP_RP, PI=Proliferation index, P_RP= Percentage of cells retaining the input phenotype and MI was calculated as the net entropy of the progeny T-cell subsets $\text{MI}={\displaystyle \sum _i^n}-pi\text{ln}pi$ where p = percentage of a given T-cell subset generated following α-CD3/CD2/CD28 stimulation. Data are represented as mean ± s.e.m of 4 donors; * = P < 0.05 (t test) (n = 4).

Following α-CD3/CD2/CD28 stimulation, however, T_SCM cells gradually upregulated CD45RO over several cell divisions while acutely downregulating CD62L and CCR7 (Fig. 4c and Supplementary Fig. 10). These dynamic changes in phenotype resulted in a diverse progeny comprising about 50% of T_CM cells and 4% of T_EM cells (Fig. 4d). Most importantly, 15% of T_SCM-derived cells maintained a CCR7⁺CD62L⁺CD45RA⁺CD45RO⁻ phenotype even after this potent stimulus, indicating that T_SCM cells have the multipotent capacity to derive all memory T-cell subsets (Fig. 4d). By contrast, T_CM cells retained a central memory phenotype or differentiated into T_EM cells, but did not generate T_SCM (Fig. 4d). Consistent with their advanced differentiation state, T_EM cells did not reacquire CD62L or CCR7 and did not dedifferentiate into T_CM or T_SCM cells after either IL-15 or α-CD3/CD2/CD28 stimulation (Fig. 4a–d). Taken together, these findings indicate that T_SCM cells have the stem cell-like properties of self-renewal and multipotency in vitro (Fig. 4e).

Increased proliferative capacity, survival and anti-tumor activity of T_SCM cells

We previously found that mouse T_SCM cells have enhanced proliferative and survival capacities compared with T_CM and T_EM cells⁹. To evaluate the replicative responses of T_SCM cells we measured the levels of ³H-thymidine incorporation after TCR stimulation. As previously reported³³, T_CM and T_N cells displayed increased proliferative responses compared to T_EM cells, but they were outpaced by T_SCM cells (Fig. 5a). Although assessment of telomerase activity was uninformative (Supplementary Fig. 12), we sought to ascertain the long-term replicative and survival capacities of T_SCM. We adoptively transferred CD8⁺ T-cell subsets into highly immunodeficient NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice and evaluated T-cell engraftment one month after transfer. We co-transferred CD8-depleted peripheral blood mononuclear cells (PBMC) to provide a source of human cytokines and costimulatory molecules³⁴. Strikingly, we found that T_SCM engrafted with 10- to 100-fold more progeny than T_CM or T_N cells in both lymphoid and non-lymphoid tissues (Fig. 5b,c and Supplementary Fig. 13). Notably, T_EM cells, which are used in clinical trials for adoptive immunotherapy^35,36, had a poor proliferative and survival capability with engraftment comparable to the contaminating CD8⁺ T-cell population from the co-transferred CD8-depleted PBMC. This humanized mouse model, however, is inadequate to test T-cell self-renewal in vivo because all CD8⁺ T-cell subsets uniformly differentiated into effector cells (Supplementary Fig. 14) likely as a result of encounter homeostatic cytokines and with xenogeneic major histocompatibility antigens. Nonetheless, adoptive transfer in NSG mice prove that T_SCM cells have enhanced replicative and survival capabilities compared to naïve and conventional memory subsets.

Figure 5. Increased proliferative capacity, survival and antitumor activity of T_SCM cells.

a, ³H-thymidine incorporation by sorted CD8⁺ T-cell subsets after stimulation with α-CD3/CD2/CD28-coated beads. Data are represented as means ± s.e.m of 10 donors. Results are normalized to the number of seeded cells, as different cell numbers were obtained from different sorts. * = P < 0.05; ** = P < 0.01; *** = P < 0.001 (t test) b, Flow cytometry analysis of human T cells in the spleen, lymph nodes (LN) and liver of a representative NSG mouse at 4 weeks following adoptive transfer of CD4⁺ T cells (5 × 10⁶) with or without sorted CD8⁺ T-cell subsets (10⁶). Graphs show T cells after gating on human CD45⁺ cells. Numbers indicate the percentage of cells in the CD4⁺CD8⁻ or CD4⁻CD8⁺ gates. c, Total human CD8⁺ T-cell recovery in the spleens, LN and livers from 6 NSG mice 4 weeks following adoptive transfer of CD4⁺ T cells with or without sorted CD8⁺ T-cell subsets. A total of 6 mice per T-cell subset from two independent experiments (3 replicate mice per T-cell subset per experiment) are shown. Horizontal bars indicate median values. * = P < 0.05; ** = P < 0.01 (t test) d–f, In vivo bioluminescent imaging (d), percentage change of body weight (e), and survival of NSG mice (f) bearing M108-luciferase mesothelioma after adoptive transfer of CD4⁺ T cells (10⁶) with or without sorted CD8⁺ T-cell subsets (3 × 10⁶) expressing a mesothelin-specific chimeric antigen receptor. *** = P < 0.001 One-Way Repeated Measures ANOVA (e) and Log-rank (Mantel-Cox) Test (f).

T-cell proliferative and survival capacities correlate with the anti-tumor efficacy of adoptively transferred T cells^35–39. T cell receptor (TCR) or chimeric antigen receptor (CAR) gene engineering are currently used in the clinic to redirect the specificity of circulating T cells toward the desired target^36,40,41. We exploited this approach coupled to the pharmacological activation of Wnt signaling to generate high numbers of mesothelin-specific ex vivo-generated T-cell memory subsets (Supplementary Fig. 15) to test in a xenograft tumor model that we recently established³⁴. Mesothelin-specific T_SCM, T_CM or T_EM cells were co-transferred with mesothelin-specific CD4⁺ T cells into NSG mice bearing luciferase-expressing M108 mesothelioma established for 3 months in the peritoneum. To generate a treatment window, we administered 3×10⁶ CD8⁺ T cells and 10⁶ CD4⁺ T cells, 10-fold fewer cells than previously published³⁴. T_EM mediated poor anti-tumor responses as indicated by the increased intensity of the bioluminescent signal in the abdomen (Fig 5d) and the ascites-dependent weight gain (Fig. 5e). Furthermore, transfer of T_EM did not significantly extend the survival of the animals compared to CD4⁺ T cells alone (Fig. 5f). T_CM cells were more effective than T_EM cells and improved survival, although all mice died from tumor progression within 40 days after treatment (Fig. 5d–f). In stark contrast, T_SCM cells triggered tumor regression and cure in mice that otherwise died within two to three weeks (Fig. 5d–f). Late mortality of mice receiving T_SCM cells was ascribed to the development of xenogeneic graft-versus-host disease as manifested by loss of body weight (Fig. 5d,e). Thus, adoptively transferred T_SCM cells have enhanced anti-tumor activity and are more therapeutically effective than conventional T_CM and T_EM cells.

Discussion

We identified a long-lived human memory T-cell subset found within the naïve-like T-cell compartment. Once thought to be homogenous, the so-called “naïve T cell subset” is emerging as a complex amalgamation of cell types, including recent thymic emigrants^42,43, “super-naïve” CD44^{very low} T cells⁴⁴, and CXCR3⁺ or CCR4⁺ early memory cells⁴⁵. Evidence presented here indicates that T_SCM are a clonally expanded primordial memory subset arising after antigenic stimulation with increased proliferative and reconstitutive capacities. T_SCM cells have enhanced self-renewal, and the multipotency to generate all memory and effector T-cell subsets in vitro. These qualities are all consistent with stem cell-like behavior, but formal proof of “stemness” in vivo will have to await clinical trials involving the adoptive transfer of T_SCM and assessment of long-term self-renewal and repopulating potential. Nevertheless, the qualities of human T_SCM cells that we have measured are consistent with those of mouse T_SCM cells that displayed enhanced self-renewal and multipotency in serial transplantation experiments^8,9.

Given the young age at which the thymus involutes in humans relative to life expectancy, the presence of such a long-lived less differentiated memory T-cell population might ensure protection against pathogens throughout life. Indeed, the vast majority of naïve-like T cells in centenarians express high levels of CD95⁴⁶, a phenotype consistent with the T_SCM described here.

Expression of CD95 by long-lived memory T cells might seem perplexing because of its well established pro-apoptotic function. However, the outcome of FAS ligand-CD95 interaction might be dependent on the differentiation state of the cellular substrate, as observed in other organ systems⁴⁷. CD95 triggers apoptosis in terminally differentiated neurons through the formation of a death-inducing complex, but promotes cell proliferation in neural progenitors and cancer stem cells by inducing TCF/β-catenin signaling⁴⁷. Considering the emerging role of TCF/β-catenin signaling in the formation and maintenance of memory T cells^9,14,48,49, CD95 might not only demarcate memory T cells but also be functionally important for their self-renewal and persistence.

The finding that T_SCM cells have enhanced proliferative capacity and can sustain the generation of all memory and effector T-cell subsets has considerable implications for the design of T cell-based vaccines to target intracellular pathogens and cancer. Heterologous prime-boost approaches have been used to increase the frequency of memory T cells but they have the undesired effect of driving T cells toward a state of terminal differentiation^3,50,51, which compromises their ability to clear systemic infections^52,53 and eradicate tumors³⁷. The use of small molecules targeting the mTOR ^54,55 and Wnt^14,56 signaling pathways might improve vaccines by modulating T-cell differentiation and enriching for T_SCM and T_CM cells. Finally, by coupling TCR or CAR gene-engineering technology^36,40,41 with the pharmacologic modulation of T-cell differentiation⁵⁶, we have demonstrated a feasible and translatable strategy for the in vitro generation of highly effective T_SCM-like cells to use in adoptive T-cell therapy trials for the treatment of cancer and infectious diseases.

METHODS

Antibodies, flow cytometry and cell sorting

We obtained all human samples from healthy donors or patients enrolled in clinical trials approved by the NCI Institutional Review Board. We conjugated unlabeled antibodies (BD Biosciences) in our laboratory reported at http://www.drmr.com/abcon. We purchased fluorescently-conjugated antibodies from BD Biosciences, eBioscience, Biolegend, Invitrogen and Beckman Coulter. Except for MART-1^26–35 (27L) (Beckman Coulter), we produced recombinant pMHCI tetramers as described previously⁵⁷. We performed surface and intracellular staining as described previously²². We measured cytokine release using the cytometric bead array/human Th1/Th2/Th17 cytokine kit (BD Biosciences). We performed flow cytometry acquisition on a modified LSR II, equipped to detect 18 fluorescencet parameters. We compensated and analyzed data with FlowJo software (Treestar Inc.). We sorted T cell subsets using a modified FACSAria (BD Biosciences).

In vitro generation of T_SCM-like cells

We stimulated CD45RO⁻CD62L⁺ cells with α-CD3/CD28 beads (Invitrogen) at 1:1 bead-to-cell ratio and 300 IU ml⁻¹ IL-2 (Chiron) in the presence of 5 µM TWS119 (Cayman Chemical) for two weeks.

Animal experiments

We conducted all animal experiments with the approval of the NCI or University of Pennsylvania Institutional Animal Use and Care Committees. We used NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ mice (NSG) (The Jackson Laboratory) as recipients for adoptive transfer experiments.

Generation of mesothelin-specific T cells

We stimulated T cells with α-CD3/CD28 beads at 1:3 cell to bead ratio and 20 IU ml⁻¹IL-2. We transduced T cells with lentiviral vectors encoding for the anti-mesothelin chimeric receptor SS1:BB:TCR-ζ³⁴ 24h after activation and added 5µM TWS119 12h after transduction. Cells were fed every other day with TWS119 and IL-2 for two weeks. We purified CD8⁺ T memory subsets using MACS positive selection Multisort kits (Miltenyi Biotec). Approximately 2 × 10⁶ CD8⁺ T cell subsets were mixed with 10⁶ unsorted CD4⁺ T cells and injected i.v. into tumor-bearing NSG mice.

Mouse xenograft studies

We engineered the M108-km1 human mesothelioma cell line³⁴ with a lentiviral vector to express firefly luciferase, yielding the M108-Luc cell line. Animals were injected i.p. with 8 × 10⁶ M108-Luc cells. We measured tumor burden by bioluminescent imaging and body weight.

Bioluminescence imaging

We injected mice i.p. with 150 mg kg⁻¹ body weight D-luciferin (Caliper Life Sciences) and imaged 10–12 minutes later using a Xenogen Spectrum system and Living Image v3.2 software (Caliper Life Sciences).

Determination of T cell replicative history

We determined the replicative history of sorted subsets by quantifying T cell receptor rearrangement excision circles (TREC) using real time qPCR as described previously¹⁸.

Clonotypic analysis of antigen-specific CD8⁺ T cell populations

We amplified TRB gene products of sorted NV9-specific CD8⁺ T cell subsets using a template-switch anchored RT-PCR and performed subcloning, sequencing and analysis as described previously⁵⁷. TCR nomenclature was translated directly from the IMGT database (The ImmunoGeneTics information system® http://imgt.cines.fr) using web-based alignment of molecular TRB transcripts.

Whole genome gene expression analysis

Total RNA from sorted CD8⁺ T cell subsets was isolated using an RNEasy Micro kit (Qiagen), processed using a WT expression kit (Ambion), fragmented and labeled using a WT Terminal Labeling Kit (Affymetrix), hybridized to WT Human Gene 1.0 ST arrays (Affymetrix) and stained on a Genechip Fluidics Station 450 (Affymetrix). We scanned arrays on a GeneChip Scanner 3000 7G (Affymetrix). We imported the raw data from. CEL files into the Partek Genomincs Suite using the RMA method. We identified differentially expressed genes (DEGs) by One-Way Repeated Measures ANOVA (P < 0.01) corrected by Benjamini-Hochberg's False Discovery Rate method (P < 0.05). For pair-wise comparisons, we further filtered genes for between-group alpha levels of P < 0.01 and a fold change criterion of > 2.0. We deposited array data at the Gene Expression Omnibus (GEO) public depository under the accession number GSE23321.

Analysis of T cell proliferation

We determined cell proliferation by ³H-thymidine incorporation and by 5-(and 6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. In the former case, we stimulated cells for 24 hours with α-CD3/CD2/CD28-coated beads (Miltenyi Biotec), then pulsed with ³H-thymidine (1 µCi; Perkin Elmer) for an additional 16 hours. We harvested supernatants and determined counts per minute with a β-scintillation counter (Perkin Elmer). In the latter, we labeled cells with 2 µM CFSE for 7 minutes at 37°C and stimulated them with αCD3/CD2/CD28-coated beads (Miltenyi Biotec) for 6 days or with rhIL-7 or rhIL-15 (both 25 ng ml⁻¹; Peprotech) for 14d and 10d, respectively. We determined proliferation index, and percentage of divided cells with FlowJo.

Statistical analysis

We performed statistical analyses using Prism (GraphPad Software) and Spice software. For most of the comparison we used a non-parametric Wilcoxon rank test to compare two groups. We employed One ANOVA to compare three or more groups and χ² permutation test for pie-chart comparison⁵⁸. We used non-parametric Spearman’s rank correlation test to measure statistical correlation between two groups and Kaplan-Meier method to analyze survival.