Abstract

Introduction

Antigen-specific CD8+ T cells can be used to treat cancer and prevent infections in humans [1–5]. Adoptive cell transfer (ACT) using tumor-infiltrating T lymphocytes (TIL) is the most effective immunotherapy for patients with advanced metastatic melanoma, reaching a 72 % objective response rate [1, 5, 6]. However, the requirement for identifying pre-existing tumor responsive cells limits its broad application. The genetic engineering of peripheral blood-derived T cells with anti-tumor TCRs using gamma-retroviruses readily renders autologous PBL from any patient into tumor-killing T lymphocytes in vitro with resulting clinical responses [7–9].

We previously reported a methodology that combined lentiviral vector transduction of CD8+ T cells with plate-bound anti-CD3 antibody and IL-2 for activation that yielded large numbers (>1 × 10⁹) of TCR-engineered T cells within 12 days, and the majority of these cells displayed a central memory-like phenotype with pronounced in vitro anti-tumor activities [10]. It is well established that the phenotype of ex vivo-cultured T cells can be modulated by the duration of culture, the addition of pharmaceuticals, or the particular cytokine used for expansion [3, 11–14]. Cytokines sharing the common γ-chain receptor (γc) include IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These proteins play a variety of important roles during the immune response in vivo including long-term T-cell memory formation [15], and it has been reported that IL-15, IL-7, and IL-21 preserve/promote a less-differentiated phenotype compared to IL-2 [16–19]. Furthermore, during in vitro dendritic cell–mediated priming or expansion of anti-tumor CD8 T cells, the presence of IL-21 not only enriches for tumor-reactive CD8 T cells, but also leads to a less-differentiated phenotype [17, 20–22].

IL-12 is a non-γc cytokine, which also plays a pivotal role in the regulation of T-cell differentiation and memory formation [23–25]. IL-12 is naturally produced by dendritic cells, macrophages, and human B-lymphoblastoid cells and is synthesized in response to antigenic stimulation. It is a T-cell-stimulating factor, which can enhance the production of IFN-γ and TNF-α from T and NK cells, and reduces IL-4-mediated suppression of IFN-γ. It has been reported that priming of naïve CD8 T cells with IL-12 selectively enhances the survival of CD8/CD62L^high cells and results in superior anti-tumor activity in a tolerogenic murine model [26]. Moreover, we recently reported that the synthesis of IL-12 by TCR gene–engineered T cells in the tumor microenvironment greatly enhanced eradication of established B16 melanoma tumors in the pmel adoptive cell therapy model [27, 28].

In this study, we systematically analyzed the effect of cytokines or their combinations on the differentiation and proliferation of TCR-transduced, in vitro-cultured, anti-tumor CD8 T cells. We propose a recipe for the sequential and combined use of cytokines to generate less-differentiated anti-tumor T cells. A cocktail of defined cytokine combinations not only yielded less-differentiated anti-tumor CD8 T cells, also re-programmed highly differentiated TIL into an apparently less-differentiated state.

Materials and methods

Results

A cocktail of defined cytokine combinations affects the phenotype and function of in vitro-cultured anti-tumor CD8+ T cells

Multiple pilot clinical studies have suggested that the engineering of T cells with anti-tumor antigen-specific receptors (TCR or CAR) can mediate regression of established tumors in cancer patients [7–9, 36, 37]. The target cell for the experiments reported herein was CD8+ T cells. For stimulation, we used plate-bound OKT3 as we had previously demonstrated that this was superior to CD3/CD28 bead stimulation for expansion of lentiviral vector–engineered CD8+ T cells [38]. As illustrated in the schema (Fig. 1a), all cultures were initiated in the presence of IL-2, but starting at day 4, the cells were supplemented with different cytokines or their combinations, and then at day 7, the cultures were split into two parallel cultures in which one arm kept the same combination of cytokines plus IL-12, while in the other arm IL-12 was removed (Fig. 1a). After a total of 12 days of in vitro culture, the phenotype of these two parallel cultures was analyzed (Fig. 1b, c). Representative FACS plots from three donors were as illustrated in Supplementary Fig. 1. Compared to cells cultured exclusively in IL-2, all cytokine combinations (except for the combination of IL-12 + IL-15 and control cultures) led to a significant (p < 0.01) increase in CD28+ cells, and the combinations of cytokines with IL-12 also led to a higher level of CD62L expression (Fig. 1b, p < 0.01).

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Fig. 1

The screening for optimal combination of cytokines to generating less-differentiated anti-tumor CD8 T cells. a The schema for cytokine cocktails and time line during in vitro culture. All cultures were initially stimulated and maintained in IL-2 until day 4. LVV, lentiviral vector transduction; w/o, without. b The phenotype of in vitro-cultured CD8 T cells in the presence IL-12 and other cytokines. The phenotype of cells was evaluated using a panel of antibodies at day 12 of culture. c The phenotype of in vitro-cultured CD8 T cells following withdrawal of IL-12 at day 7. The phenotype of cells was evaluated at day 12 of culture. The mean ± SD from three donors was calculated, and t test was used for statistical analysis. An asterisk indicates p < 0.01 compared to the IL-2 group

Removal of IL-12 at day 7 resulted in a significant down-regulation of CD45RO, and a significant up-regulation of CD127 in the IL-7 and IL-21 groups (Fig. 1c). Markers CD27 and CCR7 were higher in the group switched to IL-21 alone (Fig. 1c). In general, the in vitro-generated anti-tumor CD8+ T cells cultured in IL-12 plus other cytokines showed similar function when co-cultured with antigen-matched tumor lines (Supplementary Fig. 2). In cultures where IL-12 was withdrawn at day 7, the IL-21-treated group showed a reduced production of IFN-γ and IL-2 in co-culture assays (Supplementary Fig. 2). Except for the IL-21-treated group, all cultures underwent a similar fold expansion during 12 days of in vitro culture (Supplementary Fig. 2).

The resultant phenotype of CD8+ T cells grown in cultures where IL-12 was withdrawn at day 7 was investigated further by repeating the experiment in three additional donors (without the IL-15 group). FACS analysis demonstrated that compared to cells grown in IL-2 alone, the combination of IL-12 plus IL7 or IL21 generated CD62L^highCD28^high CD127^highCD27^highCCR7^high anti-tumor CD8 T cells (Fig. 2b); moreover, the increase in markers CD28, CD127, and CD62L was also seen at the mRNA level (Fig. 2c). The differentiation status of these cells was further analyzed by determining the mRNA levels for stem cell-associated genes and genes associated with T-cell ontogeny. In the group cultured with IL-21, we observed a significant increase in stem cell–associated genes (SOX2, NANOG, OCT4, and LIN28A) and a decrease in FOXP3 (Fig. 2c). The only effector T-cell gene seen to increase was IFN-γ, which was significantly up-regulated at the level of mRNA in cultures treated with IL-2 plus IL-12 (Fig. 2c). Co-culture of these cells with matched melanoma lines demonstrated increased production of IFN-γ (Fig. 3a) and IL-2 (Fig. 3b) in the IL-12 plus IL-7 and IL-2 plus IL-12 groups and reduced cytokine production (at day 13) in the IL12 plus IL-21 group. Cell growth was again similar with the exception of the IL-12 plus IL-21 group, which did not continue to expand (Fig. 3c).

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Fig. 2

Cytokine combinations lead to a less-differentiated phenotype in vitro. a Schema of cytokine in vitro culture. All cultures were initially stimulated and maintained in IL-2 until day 4. LVV, lentiviral vector transduction; w/o, without. b Phenotype of in vitro-cultured anti-tumor CD8 T cells grown in defined cytokine combinations at day 12. Following FACS, the percentage of each marker was calculated (left) and the MFI presented (right). c Gene expression of in vitro-cultured anti-tumor CD8 T cells. Total RNA from the cells was extracted and reverse-transcribed; the level of mRNA representing each candidate gene was measured using Q-RT-PCR. The level of mRNA at IL-2 group was set up as 1; the level of mRNA from other groups was normalized accordingly. The mean ± SD from three donors was calculated, and t test was used for statistical analysis. An asterisk indicates p < 0.05 compared to the IL-2 group

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Fig. 3

Function of cytokine-treated anti-tumor antigen CD8 T cells. T cells grown as described in the schema in Fig. 2a and then tested for anti-tumor reactivity at day 10 or 13 in the different cytokine combinations as shown. Co-cultures were with melanoma lines (mel 526, MART-1 +/HLA-A2+ and mel 938, MART-1 +/HLA-A2−) followed by determination of a IFN-γ and b IL-2. c The number of viable cell in each cytokine combination was determined and the fold expansion plotted

CD8 T cells primed with defined cytokine combinations show enhanced engraftment in NOD/SCID/γc−/− mice

We have previously reported on the use of the NOD/SCID/γc−/− mouse model to monitor the engraftment of in vitro-generated anti-tumor T cells from melanoma patients [35]. We generated anti-tumor CD8 T cells using the defined cytokine regimen of IL-12 plus IL-7 or IL-21 between days 4 and 7, followed by removal of IL-12 and culture until day 10. The cells were harvested, washed, and injected intravenously into the mice. Forty days later, mice were killed and lymphocytes extracted from lymph nodes (Fig. 4). We detected the enhanced engraftment of human CD8 T cells in LN from groups treated with IL-12 plus IL-7 or IL-21 compared to the IL-2-treated group, and the difference of persistent cells was statistically different when calculating the number of viable human CD8 T cells (Fig. 4).

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Fig. 4

CD8 T cells cultured with IL-7 and IL-21 show enhanced engraftment in NOD/SCID/γc−/− mice. The CD8 T cells were transduced and maintained in the presence of IL-2. At day 4, the cells were split, cultured in IL-2 alone or IL-12 plus IL-7 or IL-21 until day 7, at which time the cells were split and the cultures continued without IL-12. At day 10, 1 × 10⁶ CD8 T cells mixed with 3 × 10⁵ unmanipulated CD4 T cells isolated from the same donor were injected intravenously per mouse. Forty days later, mice were killed, and lymphocytes were extracted from lymph nodes. FACS was performed using anti-human CD8 antibody on left; the total number of engrafted human CD8 were calculated and plotted on right. Control group indicates only CD4 T cells were injected. The mean ± SD from three mice was calculated

Cytokine combinations induce a less-differentiated phenotype in TIL from melanoma patients

TIL used for ACT are generally highly differentiated late-stage effector cells with a phenotype of CD27low, CD28low, CD45RA−, CD62L−, CCR7−, and CD127low [39]. Because of the substantially phenotypic difference resulting from the use of cytokines during in vitro culture of TCR gene-modified PBL, we tested whether the defined cocktail of cytokine combinations could also influence the phenotype of TIL. At day 15 following rapid expansion, we took TIL and cultured them in media supplemented with the cytokine combinations as shown for 6 days (Fig. 5a). At the end of 6 days in culture, cells underwent one- to threefold expansion, and the percentage of viable cells from 6 donors ranged from 60 to 85 % (Fig. 5b). The IL-21-treated cells showed the lowest viable cell number and the least expansion (Fig. 5b). TILs cultured under these different conditions showed dramatically different appearances. In cultures of TIL grown in IL-7 and IL-21, the cells exhibited a rounded morphology and grew in a diffuse pattern compared to cells in the IL-2 groups, which grew in clusters (Fig. 5c).

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Fig. 5

Effect of cytokine cocktails on TIL. a The schematic illustration of cytokine addition during 6-day in vitro culture. TILs were rapidly expanded until day 15 of growth, then harvested and washed, and the cytokines or their combinations were added as depicted. The concentration of IL-2 was 300 IU/ml, and the other cytokines were at 10 ng/ml. At day 3, the supernatant from the TIL culture was removed, and other cytokines were added without the use of IL-12. b The viability and fold expansion during 6-day in vitro culture. c The morphological variation of TILs cultured with different combinations of cytokines at day 6. Shown are representative photomicrograph fields, scale bar indicates 50 μm

Unexpectedly, the 6-day cytokine treatment in vitro led to a substantially phenotypic change in TIL. The expression of CD62L and CD127 increased significantly in three groups supplemented with IL-12, while the markers reflecting late-stage effector cells CD70 [35] and CD57 [40] significantly decreased in IL-7- and IL-21-treated groups (Fig. 6a). In some circumstances, the phenotypic changes were consistent with changes at mRNA level, that is, CD127 and CD62L, etc. (Fig. 6b). We also observed a significant enhancement of stem cell–associated genes (SOX2, NANOG, and OCT4) in groups supplemented with IL-7 or IL-21 (Fig. 6b).

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Fig. 6

The phenotype of TIL grown with different combinations of cytokines. a The phenotype of TILs at day 6 was analyzed by FACS using a panel of antibodies as shown. b Gene expression of cytokine-cultured TILs at day 6. The total RNA from the cells was extracted and reverse–transcribed; the level of mRNA representing each candidate gene was measured using Q-RT-PCR assay. The level of mRNA at IL-2 group was set up as 1; the level of mRNA from other groups was normalized accordingly. The mean ± SD from six donors was calculated. An asterisks indicate p < 0.01 compared to the IL-2 group

Discussion

The ultimate goal of adoptive cell transfer is to eliminate tumor cells and the generation of anti-tumor immunological memory. We have reported that in vitro genetically engineered anti-tumor T cells mimic in vivo-antigen-experienced T cells exhibiting distinct subsets, including Tcm-like cells [35]. Cytokines have been well studied for their ability to affect the in vitro and in vivo properties of T cells, but it is unlikely that a single cytokine can bestow onto T cells the multitude of properties associated with effective adoptive cell therapy. We thus sought to investigate both different cytokine combinations and their duration of addition on the in vitro properties of TCR gene–engineered human T cells. Cytokines sharing the γc receptor are required for the development or maintenance of memory T cells and have very diverse functions, but only IL-2 has undergone extensive study in humans. Antigen-specific CD8+ T cells from mice can be induced to differentiate during in vitro culture in the presence of IL-2. Depending on the length of culture, cells can have a phenotype associated with naïve, early effector, intermediate effector, and late effector cells [3]. Ironically, despite having the strongest anti-tumor effects in vitro, the highly differentiated effector T cells were significantly less effective in vivo compared to T cells at their early effector stage. In practice, the criteria used to select T cells for clinical application included IFNγ release or in vitro cytotoxicity, which can be negatively correlated with in vivo anti-tumor efficacy in mice.

While the role of cytokines is diverse, it has been reported that IL-7 and IL-21 are superior to IL-2 and IL-15 in promoting human T-cell-mediated rejection of systemic lymphoma in immunodeficient mice [19]. Furthermore, we demonstrated that IL-21 prevented differentiation of CD8+ T cells in vitro and the treatment with IL-21-cultured CD8+ T cells robustly eradicated large established tumors in a mouse model [13]. More recently, we reported significantly enhanced anti-tumor reactivity of IL-12 gene-modified T cells [27, 28]. Based on our data reported herein, we proposed a recipe for a defined cytokine combination to be used in the culture of retroviral vector gene–engineered T cells. Specifically, following the initial stimulation in culture with IL-2 as described [38], medium is changed to IL-12 plus IL-7 or IL-21 for 3 days followed by culture without IL-12 for another 3–5 days. This cytokine cocktail reproducibly yielded TCR gene–engineered CD8 T cells displaying CD62L^highCD28^high CD127^highCD27^highCCR7^high phenotype associated with less-differentiated cells. Moreover, these cells with a less-differentiated phenotype engrafted significantly better than the cells grown exclusively in IL-2 following ACT in NOD/SCID/γc−/− mice.

We chose to adopt the culture conditions where IL-12 was removed at day 7 as these conditions in combination with IL-7 or IL-21 yielded more CD8 + T cells with markers CD27 and CD127 (Fig. 1c). In TIL used for ACT of patients with metastatic melanoma, the factors associated with objective response included longer telomeres of the infused cells, increased numbers of CD8+/CD27+ cells, and the persistence of the infused cells in the circulation at 1 month [5]. In addition, we previously reported that following administration of tumor antigen-specific T cells, a rapid up-regulation and continued expression of CD127 was an important factor in intermediate and long-term T-cell survival [39]. Moreover, two months after transfer of T cells with a late effector cell phenotype (CD27lowCD28lowCD62L-CCR7-), there was a transition to the development of long-term, melanoma-reactive memory CD8+ T cells in vivo showing a phenotype of CD27+CD28+ cells [39]. Recently several groups have reported that IL-21 can have profound effects on tumor antigen-specific T cells, including increases in CD62L, CD28, CD27, and CD127 [13, 20, 21]. Our observations that genetically engineered CD8+ T cells can be similarly programmed by culture conditions including cytokines IL-7 or IL-21 could have immediate impact on ACT trials using TCR- or CAR-engineered T cells.

One of the unexpected observations in this report was the finding that highly differentiated TIL can undergo phenotypic change by cytokines (Figs. 5, ​,6).6). When we cultured TIL with a terminally differentiated cell-like phenotype using the recipe of combined cytokines for 6 days, we observed a significant up-regulation of CD62L, and CD127 and a decrease in CD70 and CD57. This observation along with the induction of transcripts associated with stem cells and a distinct morphological change suggests that TIL can be re-programmed in vitro. Using the strategy pioneered by Yamanaka, multiple investigators have reported that mature T cells can be re-programmed to induced pluripotent stem cells (iPS) while retaining their rearranged T-cell receptor [41–43]. It is thus conceivable that these iPS could then be differentiated to functional naïve T cells (Tn) or T stem cell memory cells (Tscm) that possess enhanced anti-tumor reactivity[34, 44].

Our study suggests that cytokine combinations can be used to mediate the differentiation of in vitro-cultured human anti-tumor CD8+ T cells. In addition, the recipe of cytokines we proposed for generating less-differentiated CD8 + T cells in vitro was also capable of re-programming the late effector cells from TIL (although these experiments do not rule out the possibility that culture selectively enriched for less-differentiated TIL). As more and more cytokines become commercially available and are provided as GMP reagents, the optimized utilization of cytokines, such as we propose here, may lead to the design of versatile protocols for better ACT of cancer.

Acknowledgments

References