Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia

Abstract

Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

Figures

Figure 1

EmTh-1 cells impair tumor-induced iTreg generation. CD4+CD25−CD62L+ naive T cells were isolated from mouse spleens using magnetic activated cell sorting. The cells were activated using T-cell expander beads (cell:bead ratio 1:1), resuspended in the culture medium from different tumor cell lines (12B1, B16, 4T1), and treated with emTh-1 supernatant. (A) FoxP3 expression was determined by flow cytometry. (B) Pooled data from 3 independent experiments are depicted. Student t tests were used to analyze the diagrams. *P < .001, a significant difference compared with the corresponding group without emTh-1 supernatant.

Figure 2

EmTh-1 supernatant impairs TGF-β–induced iTreg generation and promotes Tbet+ T-lymphocyte differentiation. CD4+CD25−CD62L+ naive T cells were cultured for 72 hours with T-cell expander beads (cell:bead ratio 1:1) with or without TGF-β1 (5 ng/mL) in the presence or absence of the emTh-1 supernatant (emTh-1 sup). Cells were then analyzed by flow cytometry. (A-B) Representative dot plots or histogram plots from 10 independent experiments. (C) Percentage of CD4+CD25+FoxP3−- activated T cells in total CD4+ T lymphocytes. *P < .01, a significant difference compared with cells cultured without emTh-1 supernatant. (D) The expression of the transcription factors FoxP3, Tbet, and GATA-3 was determined in CD4+CD25−CD62L+ T cells cultured for 72 hours with T-cell expander beads with or without TGF-β1 treated or not with emTh-1 supernatant. Results are representative of 3 independent experiments.

Figure 3

EmTh-1 cell–mediated inhibition of TGF-β–induced iTreg generation depends on IFN-γ. CD4+CD25−CD62L+ naive T cells were cultured for 72 hours with T-cell expander beads with or without TGF-β1 in the presence or absence of emTh-1 supernatant and with or without blocking antibodies against (A) mouse IFN-γ (*P < .05, a significant difference compared with the TGF-β + emTh-1 group) or (B) mouse TNF-α. (C-D) CD4+CD25−CD62L+ naive T lymphocytes were isolated from mouse spleens (IFN-γR−/−) and cultured for 72 hours with T-cell expander beads with or without TGF-β1 and in the presence or absence of emTh-1 supernatant. Percentage of FoxP3-expressing cells was determined by flow cytometry. Results of 3 independent experiments have been combined. *P < .01, a significant difference compared with cells from wild-type (IFN-γR+/+) mice cultured in the same conditions.

Figure 4

EmTh-1 supernatant inhibits nTreg immunosuppressive function. (A) CD4+CD25+ nTregs were isolated from BALB/c mouse lymphoid tissues and cultured for the indicated periods of time with plate-bound anti-CD3 (5 ng/mL), soluble anti-CD28 (5 ng/mL), and IL-2 (20 IU/mL) with or without emTh-1 supernatant. FoxP3 expression was then determined using flow cytometry. (B) CD4+CD25+ nTregs were cultured for 48 hours with plate-bound anti-CD3, soluble anti-CD28, and IL-2 with or without emTh-1 supernatant. Cells were then washed extensively with complete medium. Responder CD4+CD25− T lymphocytes were stimulated with anti-CD3/anti-CD28 T-cell expander beads in the absence (CD25−) or presence of untreated nTregs (CD25− + untreated nTreg) or in the presence of emTh-1 supernatant–treated nTregs (CD25− + [nTreg]emTh-1 sup). Responder CD4+CD25− T-lymphocyte proliferation was determined after 48 hours using BrdU incorporation assays. NS, nonsignificant; *P < .001, a significant difference compared with responder CD25− T cells cultured with untreated Tregs. (C) CD4+CD25− T lymphocytes were first treated ([CD25−]emTh-1 sup) or not (untreated CD25−) for 48 hours with emTh-1 supernatant, washed extensively with complete medium, and cocultured for 48 hours with freshly isolated CD4+CD25+ nTregs (+ nTreg). Proliferation of responder CD25− T cells was then determined using BrdU incorporation assays. *P < .001. (D) IFN-γ concentration was assessed in the cocultures as described for panel C. *P < .001; **P < .0001.

Figure 5

The combination of emTh-1 and CRCL vaccine improves the survival of mice with 12B1 leukemia. (A) Naive BALB/c mice were injected subcutaneously in the right groin with 5 × 103 12B1 cells on day 0. Animals (8 mice per group) were then administered via footpads on days 3, 7, and 14 with: PBS (Control), 12B1-derived CRCL (CRCL, 25 μg/mouse), emTh-1 lymphocytes (emTh-1, 1 × 105 cells/mouse), or a combination of CRCL plus emTh-1 (emTh-1 + CRCL). **P < .0001. (B) SCID mice were injected subcutaneously in the right groin with 5 × 103 12B1 cells on day 0 and were administered via the footpad on days 3, 7, and 14 with: PBS (control), emTh-1 cells (emTh-1), or the combination of emTh-1 cells plus CRCL (emTh-1 + CRCL). NS, nonsignificant. (C) Immunocompetent Balb/c mice were injected with tumor cells and treated with PBS or with the combination of emTh-1 plus CRCL (emTh-1 + CRCL) on days 3, 7, and 14. In some groups of mice, NK cells were depleted using anti-asialo GM1 antibodies (+anti-asialo GM1) intraperitoneally 25 μg/mouse on days −1, +3 and +5 as described in “Tumor growth in vivo and combination immunotherapy.” In all of the experiments, survival of mice was monitored every other day and is depicted using Kaplan-Meier analysis. NS, nonsignificant; **P < .0001.

Figure 6

EmTh-1 plus CRCL immunotherapy induces tumor-specific immunity. Naive BALB/c mice were injected with 5 × 103 12B1 cells (subcutaneously in the right groin) on day 0 and vaccinated as described in the legend to Figure 5. Surviving tumor-free mice were then rechallenged with 5 × 103 12B1 cells given subcutaneously in the right groin and 1 × 106 A20 cells given subcutaneously in the left groin on day 45. Tumor volume was determined every other day. (A) A20 tumor volume monitored in control mice; (B) A20 tumor volume monitored in emTh-1 plus CRCL-treated mice; (C) 12B1 volume monitored in control group; (D) 12B1 tumor volume monitored in emTh-1 plus CRCL-treated animals. Results are representative of 2 independent experiments.

Figure 7

Effects of emTh-1 cells on antitumoral T lymphocytes and Tregs in vivo. (A) EmTh-1 plus CRCL immunotherapy induces tumor-specific killer T lymphocytes. B16 tumor–bearing mice were injected with control PBS or were treated with B16-derived CRCL and allogeneic emTh-1 cells as indicated in “Tumor growth in vivo and combination immunotherapy.” Seven days after the last vaccination, splenocytes were harvested and incubated for 3 days with CRCL (25 μg/mL) and 50 U/mL IL-2. T lymphocytes were then purified on a nylon wool column and incubated for 36 hours with either B16 tumor cells or irrelevant 4T1 breast cancer cell targets as indicated (effector T cells to target tumor cells ratio = 20:1). Cytotoxicity was determined as previously reported., + Control T, Tumor cells cultured with T lymphocytes from mice injected with PBS; +T [emTh-1], tumor cells cultured with T lymphocytes from mice treated with CRCL plus emTh-1. *P < .005, a significant difference compared with control T cells cultured with B16 melanoma cells. (B) EmTh-1 cells skew the differentiation of CD4+CD25−FoxP3− naive T lymphocytes toward CD4+CD25+FoxP3− effector T cells rather than Tregs in vivo. CD4+CD25−FoxP3− naive T lymphocytes isolated from Thy1.2 FoxP3EGFP transgenic BALB/c mice (107 cells) were transferred to 12B1 tumor–bearing congenic Thy1.1 BALB/c mice. Animals were treated with emTh-1 cells (+emTh-1 cells) or with control PBS (No emTh-1 cells). Endogenous T cells of recipient Thy1.1 mice express the Thy1.1 but not the Thy1.2 antigen, which allows the specific tracking and identification of Thy1.2 T lymphocytes in Thy1.1 mice. Spleens were harvested and dissociated, and conversion of transferred naive Thy1.2 CD4+CD25−FoxP3− T cells into GFP+ (FoxP3-expressing) Treg in vivo was determined by evaluating the frequency of Thy1.2+GFP+ cells after gating on the CD4+ T-cell population using flow cytometry. (C) EmTh-1 cells impair Treg suppressive function in vivo. Responder CD4+CD25− T lymphocytes were stimulated with anti-CD3/anti-CD28 T-cell expander beads in the absence (CD25−) or presence of Tregs isolated from the draining lymph nodes of untreated tumor-bearing mice (CD25− + [Treg]untreated) or of tumor-bearing mice treated with emTh-1 (CD25− + [Treg]emTh-1). Responder CD4+CD25− T-lymphocyte proliferation was determined after 48 hours using BrdU incorporation assays. NS, nonsignificant; *P < .02; **P < .001.