Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo - PubMed (original) (raw)

Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo

Mei-Ling Chen et al. Proc Natl Acad Sci U S A. 2005.

Abstract

Cancer patients can harbor significant numbers of CD8 and CD4 T cells with specificities to tumor antigens (Ags). Yet, in most cases, such T cells fail to eradicate the tumor in vivo. Here, we investigated the interference of Ag-specific CD4(+)CD25(+) regulatory T cells (Treg) with the tumor-specific CD8 T cell immune response in vivo, by monitoring the homing, expansion, and effector function of both subsets in draining and nondraining lymph nodes. The results show that CD8 cells expand to the same extent and produce similar levels of IFN-gamma in the presence or absence of Ag-specific Treg. Nevertheless, these Treg abrogate CD8 T cell-mediated tumor rejection by specifically suppressing the cytotoxicity of expanded CD8 cells. The molecular mechanism of suppression involves TGF-beta because expression of a dominant-negative TGF-beta receptor by tumor-specific CD8 cells renders them resistant to suppression and is associated with tumor rejection and unimpaired cytotoxicity.

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Figures

Fig. 1.

Fig. 1.

HA-specific CD8 T cells selectively reject HA-expressing tumors in the absence of HA-specific CD4 Treg. On day -1, BALB/c mice were not treated (a) or were adoptively transferred with different combinations of HA-specific T cells as follows: 105 naïve CD8 T cells (b), 105 naïve CD8 T cells and 105 CD4 Treg (c), or 105 naïve CD8 and 105 naïve CD4 T cells (d). On day 0, two colon carcinoma cell lines, namely CT26 (HA-) and CT44 (HA+), were s.c. inoculated in the right and left footpads, respectively. Tumor size was measured over a period of 2 wk. Results show mean and SD values of nine or more mice from at least three independent experiments.

Fig. 2.

Fig. 2.

HA-specific CD4 Treg neither impair proliferation nor accelerate apoptosis of HA-specific CD8 T cells. Thy1.1 BALB/c mice were adoptively transferred on day -1 with different combinations of CFSE-labeled Thy1.2 HA-specific T cells and challenged with CT26 (HA-) and CT44 (HA+) tumors on day 0 as described in Fig. 1. (a) CT44 draining LN from mice that received either naïve HA-specific CD8 T cells (○) or naïve HA-specific CD8 T cells and HA-specific CD4 Treg (•) were collected at different time points, and frequency of the transferred CD8 T cells (defined as CD8+Thy1.2+) was quantified by flow cytometry. (b) CD8 T cells in CT44 draining LN were assessed on day 6 for cell division (CFSE distribution) and apoptosis (binding to annexin V). (c) Similar analysis was performed with cells retrieved in CT26 tumor draining LN at the same time point. Similar results were obtained on days 4 and 8 (data not shown). Results show mean and SD values of six or more mice from at least three independent experiments.

Fig. 3.

Fig. 3.

HA-specific CD4 Treg do not control the production of inflammatory cytokines by HA-specific CD8 T cells. Thy1.1 BALB/c mice were adoptively transferred on day -1 with different combinations of Thy1.2 HA-specific T cells and challenged with CT44 (HA+) tumors on day 0 as described in Fig. 1, from which CT44 draining LN cells were collected on day 6. HA-specific CD8 T cells were analyzed for IFN-γ (a) and TNF-α (b) production after 4-h restimulation with exogenously added HA512-520 and HA107-119 peptides or PMA/ionomycin. (c) HA-specific CD8 T cells were analyzed for IFN-γ production after 4-h restimulation with DCs previously pulsed with only HA512-520 or both HA512-520 and HA107-119 peptides, or PMA/ionomycin. Results show mean and SD values of nine or more mice from at least three independent experiments.

Fig. 4.

Fig. 4.

HA-specific CD4 Treg suppress the cytotoxic activity of HA-specific CD8 T cells in vivo. BALB/c mice were adoptively transferred on day -1 with different combinations of HA-specific T cells and challenged with CT44 (HA+) tumors on day 0 as described in Fig. 1. Mice were injected intravenously with a 1:1 mixture of syngeneic splenocytes previously labeled with 10 μM CFSE and pulsed with HA512-520 peptide and of cells previously labeled with 1 μM CFSE but not pulsed with peptide. CT26 and CT44 draining LN were collected 16 h later, and Ag-specific killing of HA512-520 positive targets was measured by flow cytometry. (a) Representative examples for in vivo killing of HA+ targets. (b) Summary of in vivo killing of HA+ targets. n.s., not significant; *, P < 0.0001. Results show mean and SD values of nine or more mice from at least three independent experiments.

Fig. 5.

Fig. 5.

Suppression of HA-specific CD8 T cell cytotoxic activity by HA-specific CD4 Treg requires TGF-β receptor signaling. On day -1, 105 naïve HA-specific CD8 T cells expressing or not expressing dnTGFβR were adoptively transferred into BALB/c mice either alone or with 105 HA-specific CD4 Treg. Mice were challenged with CT44 (HA+) tumors on day 0. (a) HA-specific CD8 T cells in CT44 draining LN were quantified on day 6. (b) In vivo cytotoxic activity in CT44 draining LN was measured on day 6, as described in Fig. 4. n.s., not significant; *, P < 0.0001. (c) Tumor size was measured over a period of 2 wk in mice not receiving (Left) or receiving (Right) Treg. Results show mean and SD values of nine or more mice from at least three independent experiments.

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