Phenotypic analysis of prostate-infiltrating lymphocytes reveals TH17 and Treg skewing

Abstract

Purpose: Pathologic examination of prostate glands removed from patients with prostate cancer commonly reveals infiltrating CD4+ and CD8+ T cells. Little is known about the phenotype of these cells, despite accumulating evidence suggesting a potential role for chronic inflammation in the etiology of prostate cancer.

Experimental design: We developed a technique that samples the majority of the peripheral prostate through serial needle aspirates. CD4+ prostate-infiltrating lymphocytes (PIL) were isolated using magnetic beads and analyzed for subset skewing using both flow cytometry and quantitative reverse transcription-PCR. The transcriptional profile of fluorescence-activated cell sorted prostate-infiltrating regulatory T cells (CD4+, CD25+, GITR+) was compared with naïve, peripheral blood T cells using microarray analysis.

Results: CD4+ PIL showed a paucity of TH2 (interleukin-4-secreting) cells, a surprising finding given the generally accepted association of these cells with chronic, smoldering inflammation. Instead, CD4+ PIL seemed to be skewed towards a regulatory Treg phenotype (FoxP3+) as well as towards the TH17 phenotype (interleukin-17+). We also found that a preponderance of TH17-mediated inflammation was associated with a lower pathologic Gleason score. These protein level data were reflected at the message level, as analyzed by quantitative reverse transcription-PCR. Microarray analysis of pooled prostate-infiltrating T(reg) revealed expected Treg-associated transcripts (FoxP3, CTLA-4, GITR, LAG-3) as well as a number of unique cell surface markers that may serve as additional Treg markers.

Conclusion: Taken together, these data suggest that TH17 and/or Treg CD4+ T cells (rather than TH2 T cells) may be involved in the development or progression of prostate cancer.

Fig. 1

Frequency of T_helper subsets in peripheral blood and prostate tissue of patients with prostate cancer. Positively isolated CD4⁺ T cells were stimulated for 4 h in the presence of phorbol 12-myristate13-acetate and ionomycin and analyzed by flow cytometry. Representative fluorescence-activated cell sorting plots of CD4⁺IFN-γ⁺ (T_H1), CD4⁺IL-4⁺ (T_H2), CD4⁺IL-17⁺ (T_H17), and CD4⁺FoxP3⁺ (T_reg) T cells in peripheral blood and prostate tissue of prostate cancer patients with representative positive controls for each cellular marker from independent in vitro skewing experiments as well as a summary of the data. Percentage of CD4⁺ T cells positive for IFN-γ, IL-4, IL-17, and FoxP3. P values were calculated using two-sided Student’s t test.

Fig. 2

Prostate-infiltrating T cells are not skewed toward a T_H2 phenotype. A, ratio of prostate-infiltrating T cells positive for each of the markers shown/peripheral blood T cells for that marker. B, correlative data for FoxP3 vs. IL-17 and IFN-γ vs. IL-17 (patients were compared by ratio of prostate/peripheral blood for each marker). R² value determined by linear least-squares regression.

Fig. 3

T_H17 skewing of prostate-infiltrating CD4⁺ T cells inversely correlates with Gleason grade. Y-axis, ratio of prostate-infiltrating CD4⁺ T cells positive for a given cytokine or marker/peripheral blood CD4⁺ T cells positive for that cytokine or marker. Bar, median; P values were calculated by two-sided Student’s t test.

Fig. 4

Prostate-infiltrating CD4⁺ T cells have heterogeneous cytokine gene expression profiles. A, qRT-PCR results normalized to CD4 message levels. B, ratio of message levels in prostate versus peripheral blood. IL-4 and IL-12 expression were not shown because message levels were undetectable in all patients. ND, not detectable. The Gleason scores for patients1 to 4 were as follows: 3 + 3 = 6; 3 + 4 = 7; 3 + 4 = 7; 4 + 3 = 7 (tertiary 5).