Dendritic cells program non-immunogenic prostate-specific T cell responses beginning at early stages of prostate tumorigenesis

Abstract

Background: Prostate cancer promotes the development of T cell tolerance towards prostatic antigens, potentially limiting the efficacy of prostate cancer vaccines targeting these antigens. Here, we sought to determine the stage of disease progression when T cell tolerance develops, as well as the role of steady state dendritic cells (DC) and CD4(+)CD25(+) T regulatory cells (Tregs) in programming tolerance.

Methods: The response of naïve HA-specific CD4(+) T cells were analyzed following adoptive transfer into Pro-HA x TRAMP transgenic mice harboring variably-staged HA-expressing prostate tumors on two genetic backgrounds that display different patterns and kinetics of tumorigenesis. The role of DC and Tregs in programming HA-specific CD4 cell responses were assessed via depletion.

Results: HA-specific CD4 cells underwent non-immunogenic responses at all stages of tumorigenesis in both genetic backgrounds. These responses were completely dependent on DC, but not appreciably influenced by Tregs.

Conclusions: These results suggest that tolerogenicity is an early and general property of prostate tumors.

Fig. 1

The course of disease progression in Pro-HA × TRAMP mice on the B10.D2 (B10) versus F₁ B10.D2 × FVB (B10 × FVB) genetic backgrounds. Scatter plots showing total prostate weights (A) and pathological scores of prostate tumors (B) for individual mice, with horizontal lines designating mean values. Note that the prostate weights of 8 week-old double transgenic Pro-HA × TRAMP mice are similar to that of single transgenic Pro-HA mice (data not shown).

Fig. 2

Time course of T cell prostatic antigen recognitionin B10.D2 Pro-HA × TRAMP mice. A: Representative CFSE-dilution histograms of adoptively transferred clonotypic CD4 cells recovered 5 days post-transfer from the prostate-draining periaortic and prostate-non-draining cervical LN of B10.D2 Pro-HA × TRAMP mice aged 8,16, 20, or 28 weeks. A dashed line separates divided from undivided clonotypic CD4 cells, and the percentage of clonotypic CD4 cells with diluted CFSE is indicated. B: Scatter plot showing the percentage of clonotypic CD4 cells with diluted CFSE for individual mice, with horizontal lines designating mean values.

Fig. 3

Time course of T cell prostatic antigen recognition in F₁ FVB × B10.D2 Pro-HA × TRAMP mice. Representative CFSE-dilution histograms (A) and scatter plot showing the percentage of clonotypic CD4 cells with diluted CFSE for individual mice (B) are presented as in Figure 2.

Fig. 4

IFN-γ expression potential of CFSE-diluted clonotypic CD4 cells recovered from F₁ FVB × B10.D2 Pro-HA × TRAMP mice. Clonotypic CD4 cells recovered from F₁ Pro-HA × TRAMP mice described in Figure 3 were restimulated in vitro with HA peptide-pulsed APCs prior to fixation, permeabilization, and intracellular staining for IFN-γ. Representative histogram plots (from two to four mice per group) of IFN-γ expression gated on CFSE-diluted clonotypic CD4 cells are shown with a dashed line indicating positive IFN-γ expression. Restimulated clonotypic CD4 cells recovered from the spleen of a vacc-HA-infected F₁ mouse is shown as a positive control for IFN-γ expression.

Fig. 5

DC are essential for presenting prostatic antigen during tumorigenesis. Pro-HA single transgenic and Pro-HA × TRAMP double transgenic mice on the B10.D2 background were lethally irradiated and reconstituted with bone marrow from either control NT or CD11c-DTR (DTR) donors. At least 8 week slater, chimeras were treated with DT(4ηg/gbody weight)or PBS on Days −4, −1, and +2, and received adoptive transfers of naœve CFSE-labeled clonotypic CD4 cells on Day 0 which were subsequently recovered for analysis 5 days post-transfer from the prostate-draining LN. Chimeric recipients were between 3 and 4 months of age at the time of adoptive transfer. Representative CFSE-dilution histograms (A) and scatter plot showing the percentage of clonotypic CD4 cells with diluted CFSE for individual mice(B)are presented as in previous figures.

Fig. 6

CD4⁺CD25⁺ T regulatory cells do not appreciably influence the naœve CD4 cell response to prostatic antigen during prostate tumorigenesis. C3-HA, vacc-HA-infected NT (vacc-HA), and Pro-HA × TRAMP recipients on the B10.D2 background were treated with either anti-CD25 (PC61) or control (rat Ig) antibody 4 days prior to receiving CFSE-labeled naœve clonotypic CD4 cells, which were subsequently recovered for analysis 5 days later from the prostate-draining LN. A: Representative FACS plots showing the frequency of CD4⁺CD25⁺cells within the total peripheral blood CD4⁺ population both before (Day 0) and 4 days following treatment with either PC61 or rat Ig.CD25 staining was performed using the 7D4 mAb, which does not cross-react with PC61. B: Representative CFSE-dilution histograms and graph showing frequencies of clonotypic CD4 cells. C: Representative FACS plots of IFN-γ versus TNF-α expression following in vitro restimulation with HA peptide-pulsed APCs, fixation/permeabilization, and intracellular staining, gated on CFSE-diluted clonotypic CD4 cells. D: Quantitative analysis of IFN-γ and TNF-α expression. Total cytokine expression is expressed in arbitrary units (calculated as the product of the % of cytokine positive clonotypic CD4 cells multiplied by the mean fluorescence intensity of cytokine staining per positively expressing cell) as previously described [11]. All quantitative data is expressed as the mean ± SEM, and n = 3 for each group.