Yeast Hsp7O genes constitute a multigene family in which at least five of the nine members are heat inducible. Hsp7O RNA levels also vary dramatically during stationary arrest and sporulatien. During growth to stationary phase, SSBI-SSB2 and SSCI RNAs decreased in abundance as cell density increased. In contrast, SSAI-SSA2 RNA levels increased before the diauxic shift and then decreased as cells approach stationary phase. SSA3 RNA was detected only after the diauxic shift and accumulated to high levels as cells entered stationary phase. This accumulation was reversed by addition of glucose. Studies with cyri mutants indicated that SSA3 RNA accumulation is stimulated by decreasing intracellular cyclic AMP concentrations. When cells were incubated in sporulation medium, most Hsp7O RNAs, with the exception of SSAI-SSA2 RNA, decreased in abundance. This finding contrasted with the SSAI-SSA2 pattern observed during growth to stationary phase. SSA3 RNA was not detected during growth in acetate-based medium but accumulated after several hours. SSA3 RNA accumulation was higher in sporulating cells than in nonsporulating cells and was reversed by addition of glucose.

SummaryProstatic acid phosphatase (PAP) is a tumor antigen in prostate cancer and the target of several anti-tumor vaccines in earlier clinical trials. Ultimately, the goal of anti-tumor vaccines is to elicit a sustainable immune response, able to eradicate a tumor, or at least restrain its growth. We have investigated plasmid DNA vaccines and have previously conducted a phase 1 trial in which patients with recurrent prostate cancer were vaccinated with a DNA vaccine encoding PAP. In this study, we investigated the immunologic efficacy of subsequent booster immunizations, and conducted more detailed longitudinal immune analysis, to answer several questions aimed at guiding optimal schedules of vaccine administration for future clinical trials. We report that antigen-specific cytolytic T-cell responses were amplified after immunization in 7 of 12 human leukocyte antigen-A2-expressing individuals, and that multiple immunizations seemed necessary to elicit PAP-specific interferon-γ-secreting immune responses detectable by enzyme-linked immunosorbent spot assay. Moreover, among individuals who experienced a ≥200% increase in prostate-specific antigen doubling time, long-term PAP-specific interferon-γ-secreting T-cell responses were detectable in 6 of 8, but in only 1 of 14 individuals without an observed change in prostate-specific antigen doubling time (P=0.001). Finally, we identified that immune responses elicited could be further amplified by subsequent booster immunizations. These results suggest that future trials using this DNA vaccine, and potentially other anti-tumor DNA vaccines, could investigate ongoing schedules of administration with periodic booster immunizations. Moreover, these results suggest that DNA vaccines targeting PAP could potentially be combined in heterologous immunization strategies with other vaccines to further augment PAP-specific T-cell immunity. Despite the significant effort in developing and evaluating anti-tumor vaccines, there is little consensus as to the "best" antigens to target and the optimal means of targeting these antigens. The prioritization of anti-tumor vaccine antigens, in fact, has been the focus of recent efforts led by the National Cancer Institute. 12 Most vaccine approaches targeting individual antigens have focused on eliciting CD8 + T cells, as these are part of the adaptive arm of the immune system with direct cytolytic activity. Consequently, to specifically elicit CD8 + T cells, most approaches have used antigen-loaded antigen-presenting cellular vaccines [13][14][15] or genetic vaccines using viral vaccines or naked DNA plasmids. [3][4][5]8,10 Of these particular approaches, DNA vaccines are generally believed to be a "weaker" immunization strategy given the absence of a concurrent inflammatory antiviral response, and the low level of in-vivo transfection of antigen-presenting cells that occurs after direct administration. However, we have been particularly interested in DNA vaccines as a simpler means of antigen-specific immunization given our experience i...

Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4+CD25+ Tregs and myeloid-derived suppressor cells, tumor antigen-specific regulatory T cells interfere with the detection of anti-tumor immunity following immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses following immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8+CTLA-4+, and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-β. Moreover, antigen-specific CD8+ regulatory T cells were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8+CTLA-4+ suppressor T cells expressed IL-35, which was decreased following blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8+CTLA-4+ T cells also suppressed T-cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8+CTLA-4+ IL-35-secreting tumor antigen-specific regulatory T cells arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of antigen-specific effector responses by an IL-35-dependent mechanism.