Sample data were acquired in an LSR Fortessa II (BD Biosciences) circulation cytometer and subsequently analyzed using FlowJo software (Tree Star). For a more complex visualization of the flow cytometry data for memory and naive T cell populations, we used value of <0.05 was considered significant. ACKNOWLEDGMENTS This study was supported by NIH, National Institute of Allergy and Infectious Diseases, grants R21-AI131763 and U01-AI35041. We thank Lisa Borghesi for her help in using the tSNE analysis software in FlowJo. CD8+ T cells into effector T cells expressing high levels of T-box transcription factor (T-bethi) and eomesodermin (Eomes+). In contrast, PD-1 blockade enhanced the overall magnitude of memory HIV-specific CTL responses and reversed the worn out memory phenotype from a T-betlow/Eomes+ to a T-bethi/Eomes+ phenotype. These results indicate that this PD-L1/PD-1 signaling pathway has a previously unappreciated dual role in the induction and regulation P005672 HCl (Sarecycline HCl) of HIV-1-specific CTL immunity, which is usually greatly determined by the context and differentiation stage of the responsive CD8+ T cells. IMPORTANCE Targeting the PD-1/PD-L1 immune checkpoint axis with signaling inhibitors has proven to be a powerful immunotherapeutic strategy to enhance the functional quality P005672 HCl (Sarecycline HCl) and survival of existing antigen-specific effector T cells. However, our study demonstrates that this context and timing of PD-1 signaling in T cells greatly impact the outcome of the effector response. In particular, we show that PD-1 activation plays a positive role during the DC-mediated initiation stage of the primary T cell response, while it serves as an Mouse monoclonal to BNP inhibitory mechanism during the effector phase of the response. Therefore, caution should be taken in the design of therapies that include targeting of the PD-1/PD-L1 signaling pathway in order to avoid potential unfavorable impacts around the induction of T cell responses. (18, 19) and in the nonhuman primate simian immunodeficiency computer virus model (24). Although PD-1/PD-L1 signaling inhibition appears to have beneficial effects in reversing T cell exhaustion in several contexts of malignancy and chronic infections, PD-1/PD-L1 signaling is also required for proper development of main Th1 responses against intracellular bacteria (25,C28). Interestingly, we demonstrated that this PD-1 blockade experienced opposing effects on CTL function when implemented during main versus secondary activation in the setting of human papillomavirus (29). However, whether PD-1 has any role in the priming and differentiation of naive T cells into effector CD8+ T cells or whether PD-1 blockade has a differential impact on naive versus memory CD8+ T cell responses remains unclear. Recent findings from our group spotlight the use of antigen-presenting dendritic cells (DC) to induce main CD8+ CTL responses from naive T cell precursors, rather than merely recalling memory T cells, to effectively target P005672 HCl (Sarecycline HCl) and kill HIV-1-infected cells during chronic HIV-1 contamination (30). Therefore, in this study we evaluated the role of the PD-1 pathway in DC-induced main and memory T cell responses in chronic HIV-1 contamination. RESULTS Type 1 polarized DC (MDC1) stimulated with CD40L primary naive CD8+ T cell responses to natural HIV-1 Gag 9-mers. MDC1 are known to be effective drivers of Th1-skewed cell-mediated T cell responses in part because of their ability to secrete copious amounts of IL-12p70 upon CD40L activation (31, 32). This unique house of MDC1 supports their potential as an immunotherapy for HIV-1 contamination (33, 34). To demonstrate the importance of this T helper transmission, we evaluated the ability of MDC1 to induce main HIV-1 Gag-specific T cell responses in the presence or absence CD40L. HIV-1 peptide-loaded MDC1 were generated from HLA-A2+ HIV-1-seronegative donors, harvested, and cocultured with autologous CD8+ T cells in the presence or absence of gamma-irradiated CD40L-expressing J558 cells (J558-CD40L) (35). It is important to note that this parental murine cell collection J558 does not produce factors that activate human DC production of cytokines or activate T cells (36). Because of this, these CD40L transfected cells have been routinely used as a standard surrogate for Th cell CD40L help in numerous DC-mediated T cell activation studies (31, 32, 35) and as a quality assurance monitoring tool for DC clinical trials (37). After 12?days of stimulation, CD8+ T cells were then restimulated with gamma-irradiated, Gag peptide-loaded, HLA-A2+ T2 cells. At day 21 postpriming, the T cells were tested for production of IFN- in response to the relevant peptide antigens by IFN- enzyme-linked immunospot (ELISPOT) assay. We observed that MDC1 were unable to generate strong HIV-1 Gag-specific IFN- responses to any of the five peptides utilized for priming (Fig. 1A, top wells, and Fig. 1B, black symbols) when cultures were initiated in the absence of J558-CD40L. In contrast, when J558-CD40L was present at the initiation of the MDC1-T cell coculture, long-term HIV-1 Gag-specific IFN- responses were generated against 2 out of 5 peptides in the first P005672 HCl (Sarecycline HCl) donor tested.