Immunity to murine prostatic tumors: continuous provision of T-cell help prevents CD8 T-cell tolerance and activates tumor-infiltrating dendritic cells

Abstract

We reported previously that tumor-specific CD8(+) T cells (TcR-I) become tolerant in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. In this study, we show that CD4(+) TcR transgenic (TcR-II) T cells transferred into TRAMP mice became activated in lymph nodes, trafficked to the prostate, and initially functioned as T(H)1 cells. Although a single cotransfer of TcR-II cells delayed TcR-I cell tolerization, repeated transfer of TcR-II cells was required to prevent TcR-I cell tolerization and significantly slowed progression of TRAMP prostate tumors. After transfer of TcR-II cells, dendritic cells within the tumor expressed higher levels of costimulatory molecules and displayed an enhanced ability to stimulate proliferation of naive T cells. Blockade of CD40-CD40L interactions during TcR-II transfer resulted in a profound reduction in dendritic cell stimulatory capacity and a partial loss of TcR-I effector functions and tumor immunity. These data show that sustained provision of activated tumor-specific CD4(+) T cells alters the immunosuppressive tumor microenvironment, ultimately leading to the control of tumor growth. These findings will assist in the design of more effective immunotherapeutic approaches for cancer.

Figure 1. Progressive loss of TcR-II cell proliferation and cytokine secretion

A, TcR-II T cells isolated from TRAMP or WT mice 3 days after transfer were stimulated with increasing concentrations of antigen (TAg_362−384) and tested for IL-2 (left) and IFN-γ (right). Data is expressed as mean ± SEM and is representative of results from 3 separate experiments. B,C The frequency of TcR-II T cells isolated from TRAMP prostates or WT mice that secrete IL-2 (B) or IFN-γ (C) in response to stimulation with TAg_362−384 was tested at the indicated time after transfer. (B): WT vs TRAMP: P < 0.01 (day 5) and P < 0.001 (days 10 and 20). (C): WT vs. TRAMP mice: P < 0.001 on days 10 and 20. P values represent significant differences between experimental groups for data across all antigen concentrations ≥ 0.002 μg/ml. Data is expressed as mean ± SEM and is representative of 3 separate experiments.

Figure 2. TcR-II cells enhance TcR-I cell priming and trafficking to the tumor

The percentage (A) and total number (B) of TcR-I cells present in nDLN, pDLN, or prostates of TRAMP mice at various times after transfer of the indicated cell populations. The percentage of TcR-I cells was determined by gating on the Thy1.1⁺, CD8⁺ population. Data is presented as representative of 3 (A) or 5 (B) separate experiments and is expressed as mean ± SEM (*P<0.05, ***P < 0.001).

Figure 3. TcR-II cell enhancement of anti-tumor effector functions is not sustained in the tumor microenvironment

The frequency of IFN-γ-secreting (A) and GrB-secreting (B) TcR-I cells from TRAMP mice receiving TcR-I cells alone or as co-transfer (TcR-I + TcR-II) was determined by ELISPOT assay. The number of IFN-γ-secreting TcR-I cells from the co-transfer group is significantly greater at day 5 and 10 after transfer (P < 0.001 for data across all antigen concentrations ≥ 0.001 μg/ml). The number of GrB-secreting TcR-I cells from the co-transfer group is significantly greater than TcR-I alone at all time points (P < 0.001 for data across all antigen concentrations ± 0.01 μg/ml). Data is expressed as mean ± SEM and is representative of 5 separate experiments.

Figure 4. Continuous provision of TcR-II cell prevents the induction of tolerance of TcR-I cells

A, The frequency of IFN-γ (A) or GrB (B) secreting TcR-I cells isolated from TRAMP mice was determined by ELISPOT assay. Mice receiving TcR-I with multiple TcR-II cell transfers (TcR-I + Multi TcR-II) versus single co-transfer (TcR-I + TcR-II) or TcR-I alone produced significantly greater IFN-γ (P < 0.05, all time points) and GrB (P < 0.001, day 5; (P < 0.05, day 18 and 30). Data are expressed as means ± SEM and is representative of 3 separate experiments. C, To estimate tumor burden, the prostatic complex was dissected and weighed. The mean wet weight of WT prostate is indicated by a dashed line. In non-transferred TRAMP mice, the average prostatic weight was 0.25 ± 0.02g. Each dot represents an individual mouse and horizontal bars represent the group mean (*P < 0.05, **P < 0.01 and ***P < 0.001).

Figure 5. APCs from prostate tumors have an increased stimulatory ability after TcR-II cell transfer

APCs were isolated from WT or TRAMP prostates at the indicated time after transfer of TcR cells. A, Phenotype of isolated APCs day 20 after T cell transfer (similar results were observed on days 5 and 10 after transfer). B,C, APCs isolated from non-transferred or mice receiving single transfers of TcR-I or TcR-II cells were analyzed for the ability to stimulate naïve CD8⁺ T cell proliferation in vitro. Data is expressed as mean ± SEM (**P = 0.0017, B) and is representative of at least 3 separate experiments. D, Mice were treated with FTY720 prior to TcR cell transfer and APCs were isolated 7 days after transfer of TcR cells and analyzed for the ability to stimulate naïve CD8⁺ T cell proliferation in vitro. Data is expressed as average CPM ± SEM (**P < 0.01).

Figure 6. Blockade of CD40:CD154 reduced APC activation but only partially reduced TcR-I effector functions

APCs were isolated from the indicated treatment groups and used in stimulation assays as described in Figure 5 (A) or cultured overnight and the supernatants tested for IL-12 expression (B). Mice were treated with anti-CD154 prior to T cell transfer. T cells were purified from the indicated treatment groups 6 days after transfer and tested for the frequency of IFN-γ-expressing (top) and GrB-expressing (bottom) cells. Prostate weights were determined 12 days after T cell transfers. Data is expressed as average CPM ± SEM (**P < 0.05. ***P<0.001).