Modulating Tolerance of Tumor Specific T cells in a Murine Model of Prostate Cancer

The development of genetically modified murine models of cancer has profoundly aided in understanding T cell responses to tumor antigens. To study T cell tolerance and tumor immunity, we utilize the TRAMP model, an autochthonous model of prostate cancer that highly resembles the pathogenesis and progression of prostate cancer in humans. TRAMP mice carry the -426/+28 fragment of the androgen-driven, prostate-specific rat Probasin (PB) regulatory element fused to the SV40 T/t antigen gene (TAg) (Greenberg et al. 1995). The TRAMP model system has been proven by us and others to be a valid model to study immunological tolerance and potential immunotherapeutics aimed at overcoming T cell tolerance to tumors (Granziero et al. 1999; Hurwitz et al. 2000; Tourkova et al. 2004).

Traditionally, generating an effective antitumor immune response has primarily focused on priming CD8+ T cells to tumor antigens. CD8+ T cells have been strategically targeted because the majority of tumors express MHC I, not MHC II, and upon recognition, these cytotoxic T cells may be able to kill these MHC I+ tumor cells. We recently characterized the fate of naïve tumor-specific CD8+ T cells in TRAMP mice (Table 1). We demonstrated that naïve, tumor-specific CD8+ T cells (TcR-I) adoptively transferred into TRAMP mice were inefficiently primed and underwent abortive proliferation. Addition of an ex vivo matured, peptide-pulsed DC vaccine resulted in effective TcR-I priming and protection of these cells from initial tolerization (Anderson et al. 2007). A slowing of prostate tumors in mice treated with the DC vaccine and tumor-specific TcR-I cells was also noted. However, these effects are not durable and T cells were eventually tolerized due to the immunosup-pressive environment of the transgene-driven tumor that appears to continually exert suppressive effects on the infiltrating T cells (Anderson et al. 2007).

Table 1. Relative Infiltration and Responsiveness of TcR cells in the Prostate of Tumor-bearing Mice.

TcR Transfer1

Infiltration into Prostate2

T cell Responsiveness {tumor-infiltrating cells)-' D5 D10 D20

TcR-1 only TcR-I + DC Vaccine TcR-II only TcR-I + TcR-Il

'Twelve-week-old male TRAMP nticc were transferred with 3D x 106 TcR-I (CDS-0 andor TcR-11 (CtM+>T cells or given 9 si peptide pulsed IX' vaccine.

M.'sins magnetic heads, TcR T cells were isolated from prostate tissue on day 5 CDS), day 10 iDIO). atid day 20 (D2II) alter transfer and the number of TcR T cells were enumerated- Data is presented as the day of peak TcR T cell infiltration into the tumor and the relative number with respect to type of transfer.

3l'sing magnetic beads. TcR T cells were isolated from prostate tissue on day 5 (D51. day 10 (D10). and day 20 (D20) after transfer.

Isolated TcR-I cells were assayed for functional capacit using IFN-y and Granzyme B KLISI'OT assays. TcR-11 cells were assayed for functional capacity using an interleukin-2 and [bN-f KLISltJT assays. Data is presented relative activity in die functional assays with respect to type of and day after transfer.

The signals CD8+ T cells receive during their initial differentiation and maintenance of their immune response are critical for shaping their antitumor functions (Gattinoni et al. 2005). CD4+ T lymphocytes play a pivotal role in activating DCs and other APCs to efficiently prime CD8 T cells. Activation of APCs by CD4+ can be mediated through CD40/CD40L interactions that in turn upregulate the expression of costimulatory molecules (e.g., CD80, CD86, and ICAM) and cytokines (e.g., IL-12) on the APCs needed to generate CD8+ effector and memory cells (Bennett et al. 1998; Schoenberger et al. 1998). This has been referred to as APC licensing. Additionally, CD4+ T cells are a source of cytokines that are important for CTL differentiation, expansion, and survival (Kalams and Walker 1998; Moroz et al. 2004). With respect to tumor immunity, CD4+ T cells may be important to maintain tumor-specific CD8+ T cell numbers, to enhance their infiltration into the tumor microenvironment, and to sustain their effector functions once in the tumor microenvironment (Marzo et al. 2000). Taken together, these data suggest that CD4+ T cells provide important and critical provisions for the generation of CD8+ T cells with antitumor functions.

Given these findings, we are currently using the TRAMP model to test whether tumor-specific CD4+ (TcR-II) T cells can affect tolerization of TcR-I cells. We first characterized TcR-II cell trafficking, activation, and functional kinetics in tumor-bearing mice. Naïve TcR-II T adoptively transferred into TRAMP mice encounter their cognate antigen in lymph nodes, undergo several rounds of proliferation, express activation markers (CD25 and CD44), and traffic to the prostate. Over time, TcR-II cells become functionally tolerant in the prostate, as measured by their inability to secrete IL-2 and IFN-y in response to their cognate antigen (Table 1). Therefore, like CD8+ TcR-I cells, some naïve TcR-II T cells can resist deletion and persist in the prostate in a tolerant state.

We have also tested the effect of CD4+ TcR-II cells on the fate and function of TcR-I cells. Co-transfer of TcR-II cells enhanced the frequency, activation, survival, and function (IFN-y and GrB secretion) of TcR-I cells in TRAMP mice. However, this protection was not durable, as TcR-I cell tolerance was noted 3 weeks after transfer into TRAMP mice. These data suggest that TcR-II cells facilitate efficient priming and differentiation of TcR-I cells, but CD4-mediated help is not sufficient for the maintenance of TcR-I antitumor effector functions in the tumor microenvironment (Table 1). Our current studies are testing whether CD4+ cells can reverse CD8+ T cell tolerance. By understanding the role of CD4+ T helper cells in enhancing or rescuing tumor-specific cytotoxic T cells' responsiveness, we hope to facilitate the design of novel approaches to stimulate an immune response that would be therapeutically effective at controlling and eradicating tumors.

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