Human prostate-infiltrating CD8+ T lymphocytes are oligoclonal and PD-1+

Abstract

Background: Prostate-infiltrating CD8(+) T lymphocytes (CD8(+) PIL) are prevalent in men with prostate cancer (PCa), however, it is unclear whether the presence of such cells reflects a non-specific immune infiltrate or an oligoclonal, antigen-driven adaptive immune response.

Methods: We investigated the complexity of the T-cell receptor (TCR) repertoire in the prostate gland by examining the diversity of CD8(+) TCR beta chain variable region (Vbeta) gene sequences in both the peripheral blood and prostates of cancer patients. Vbeta repertoire analysis was performed by family-specific Vbeta spectratyping and flow cytometry, as well as direct sequence analysis (5' RACE and cloning). Programmed cell death 1 (PD-1 or PDCD1) expression on peripheral blood CD8(+) T cells and CD8(+) PIL was analyzed by flow cytometry.

Results: CD8(+) PIL isolated from cancer patients exhibited restricted TCR Vbeta gene usage, and identical clones were identified in multiple sites within the prostate. Furthermore, CD8(+) PIL express high levels of the inhibitory receptor PD-1, a cell surface protein associated with an "exhausted" CD8(+) T-cell phenotype.

Conclusions: CD8(+) PIL appear to have undergone clonal expansion in response to an as yet unidentified antigen; however, due to the high expression of PD-1, these cells are likely incapable of mounting an effective immune response. The results provide an important basis for further efforts aimed at the identification of specific antigens involved in prostatic inflammation, and suggest that PD-1 blockade may be useful in immunotherapy for PCa.

Figure 1

TCR Vβ skewing of select Vβ families in peripheral blood and prostate of PCa patients. A) Examples of skewing observed by TCR Vβ CDR3 spectratyping. Skewed CDR3 sizes in CD8⁺ PIL are observed in both the right and left lobes of the prostate, but not in the peripheral blood. B-E) Positively isolated CD8⁺ T cells were stained directly ex vivo and analyzed by flow cytometry. Representative FACS plots of CD3+CD8+Vβ2+, CD3+CD8+Vβ8+, CD3+CD8+Vβ13.6+, and CD3+CD8+Vβ14+ T cells in peripheral blood and prostate tissue of PCa patients are shown as well as a summary of the data. Several individual patients demonstrate a relative up-regulation of a particular Vβ family within the prostate gland. P values were calculated by paired Student’s t test, two sided.

Figure 2

Variation in clonality by patient and by Vβ family. Flow cytometry data (Fig. 1b-e) are shown as difference between % of prostate CD8⁺ T cells positive for given Vβ family - % of peripheral blood CD8⁺ T cells positive for given Vβ family. Positive values indicate higher expression on prostate-infiltrating CD8⁺ T cells and negative values higher expression on peripheral blood CD8⁺ T cells. * = Significant difference as defined by % difference > +/−2 standard deviations (S.D.) of expression of Vβ family in peripheral blood of all patients. ND = Not Determined.

Figure 3

Clonality of prostate-infiltrating CD8⁺ T cells as evidenced by TCR Vβ CDR3 repertoire sequencing. A) RNA from positively isolated peripheral blood and prostate CD8⁺ T cells was subject to 5′ RACE analysis using a TCR Cβ constant region primer. PCR products were cloned and sequenced. Identical sequences found either five or more times in one anatomic site (blue) or ≥ 5 times in both right and left peripheral prostate (red) are shown. Identical sequences (< 5) were also identified in both right and left peripheral zone of the prostate of Patients 1,2,3,4 and 6. PB = peripheral blood, RP = right peripheral prostate, LP = left peripheral prostate. Number in parentheses is number of clones sequenced. B) Amino acid sequence of junctional regions observed from all sequences belonging to Vβ family 13.6 derived from Patient 1 and Vβ family 2.1 derived from Patient 3. * = Identical sequences observed in both right and left peripheral prostate. C) The same RNA samples used for Patient 1 5′ RACE and sequence analysis were reverse transcribed using an oligo d(T) primer. cDNA was then amplified using a Vβ13.6 family-specific forward primer and a 6-FAM labeled TCR Cβ constant region reverse primer. Spectratyping analysis of the PCR products reveals marked skewing towards one CDR3 region length for both right and left peripheral prostate, indicative of local clonal expansion. These results are in concordance with the nucleotide sequence analyses.

Figure 4

Prostate-infiltrating CD8⁺ T cells exhibit an exhausted phenotype. A) FACS analysis of prostate-infiltrating CD8⁺ T cells. Plots are gated on CD8⁺ T cells and gates set using appropriate isotype controls. Far right, mean fluorescence intensity (MFI) comparison of peripheral blood CD8⁺PD-1⁺ T cells (black histogram) and prostate CD8⁺PD-1⁺ T cells (gray histogram). B) Summary of data above. % CD8⁺ T cells positive for PD-1 are shown. Bar = mean. C) Summary of data above. PD-1 MFI is shown. Bar = mean. P values were calculated for control peripheral blood (P. blood) versus PCa P. blood by unpaired Student’s t test, two sided and for PCa P. blood versus prostate by paired Student’s t test, two sided. D) Correlative analysis for PD-1 MFI for PCa P. blood versus prostate for low grade (Gleason 6) versus high grade (Gleason 7-9) disease.