Recovery of zeta-chain expression and changes in spontaneous IL-10 production after PSA-based vaccines in patients with prostate cancer

Abstract

Circulating T lymphocytes of patients with prostate cancer have been reported to have functional deficits, including low or absent zeta-chain expression. To determine whether these functional impairments could be reversed by prostate specific antigen-based vaccination therapy, 10 patients treated with recombinant human prostate specific antigen plus GM-CSF and eight others receiving prostate specific antigen plus oil emulsion in two pilot clinical trials were evaluated prior to and after vaccination for several immunologic end points, including zeta-chain expression and cytokine production by circulating T cells as well as the frequency of T cells able to respond to prostate specific antigen in ELISPOT assays. The flow cytometry assay for zeta-chain expression was standardized to allow for a reliable comparison of pre- vs post-vaccination samples. Prior to therapy, the patients were found to have significantly lower zeta-chain expression in circulating CD3(+) cells and a higher percentage of zeta-chain negative CD3(+) and CD4(+) cells than normal donors. The patients' peripheral blood mononuclear cells spontaneously produced more IL-10 ex vivo than those of normal controls. After vaccination, recovery of zeta-chain expression was observed in 50% of patients in both clinical trials. Also, spontaneous IL-10 secretion by peripheral blood mononuclear cells decreased following immunotherapy in patients treated with prostate specific antigen and GM-CSF. The frequency of prostate specific antigen-reactive T cells was detectable in 7 out of 18 patients vs 4 out of 18 patients prior to vaccination. Only one of 18 patients was a clinical responder. The vaccine had stimulatory effects on the patients' immune system, but post-vaccine immune recovery could not be correlated to progression-free survival in this small cohort of patients with prostate cancer.

Figure 1

Intra-individual variability of MFI for ζ-chain expression and the per cent of ζ-chain-negative cells in CD3⁺, CD4⁺ and CD8⁺ T cell subsets obtained from six normal donors (C1–6). The samples were drawn at three different time points 1 month apart. PBMC were cryopreserved and tested in the same assay to establish the biologic variation within each individual evaluated over a 3 month period. The solid line connects the median values. Of note, the percentages of ζ-chain-negative T cells are very low in normal controls, except for CD8⁺ T cells in C5.

Figure 2

Comparisons of the ζ-chain expression in T cells and T-cell subsets between patients and healthy controls are presented as box- whisker plots. The white bar represents the median; the box represents the interquartile range; the whiskers represent 1½ times the interquartile range; and the single lines show the outliers. Nineteen healthy controls were compared with 18 patients with prostate cancer. (A), MFI for ζ-chain expression and (B), percentages of ζ-chain-negative cells in patients and healthy controls are shown. The P values were calculated by the exact Wilcoxon test.

Figure 3

Flow cytometry dot plots for ζ-chain expression in two representative patients with prostate carcinoma. The gate was set on CD3⁺ T cells. In patient no. 401, ζ-chain-positive and ζ-chain-negative populations of T cells are present. In patient no. 411 practically all T cells express ζ-chain. After vaccination, nearly all T cells of patient no. 401 express ζ-chain, and MFI for ζ is increased in T cells of both patients.

Figure 4

Treatment-related changes in ζ-chain expression in T cells of all the patients. The upper charts summarize changes in MFI and the lower charts, changes in the percentage of ζ-negative T cells. The diagonal dotted lines indicate no change in zeta expression from pre- to post-therapy.

Figure 5

ζ-chain expression prior to and after therapy with PSA-based vaccines. (A) The data for the patients in the 200 series (PSA+GM-CSF). (B) The data for the patients in the 400 series (PSA emulsified in mineral oil). The numbers identify patients with most pronounced pre- to post-therapy changes in ζ-expression. Note that the changes in the 400 series are generally more pronounced. Comparison between pre and post treatment by 2-tailed signed rank test showed significant changes (P<0.05) only for the patients in the 400 series. MFI of ζ-chain expression increased for CD3⁺, CD4⁺ and CD8⁺ cells. The comparison of percentage ζ-chain-negative cells showed a significant decrease (P<0.05) only in the CD3⁺ cells.

Figure 6

Cytokine secretion by the patients' PBMC. Either spontaneous or PHA-stimulated secretion of IL-10 or IL-2 by PBMC, were determined by ELISA in the supernatants after 48 h culture. Note that patients nos. 401 and 402 have high pre-vaccination levels of IL-10, which decreased after treatment. Only changes in IL-10 spontaneous release for patients in the 200 series are statistically significant at P<0.01.