BackgroundIdentifying the immune correlates of reduced susceptibility to HIV remains a key goal for the HIV vaccine field, and individuals who are HIV-exposed, seronegative (HESN) may offer important clues. Reduced systemic immune activation has been described in HESN individuals. Conversely, pro-inflammatory T cell subsets, particularly CD4+ T cells producing the cytokine IL17 (Th17 cells), may represent a highly susceptible target for HIV infection after sexual exposure. Therefore, we characterized the cellular pro-inflammatory and IL17/IL22 cytokine immune milieu in the genital mucosa and blood of HESN female sex workers (FSWs).Methods and ResultsBlinded lab personnel characterized basal and mitogen-induced gene and cytokine immune responses in the cervix and blood of HESN FSWs (n = 116) and non-FSW controls (n = 17) using qPCR and ELISA. IL17 and IL22 production was significantly reduced in both the cervix and blood of HESNs, both in resting cells and after mitogen stimulation. In addition, HESN participants demonstrated blunted production of both pro-inflammatory cytokines and β-chemokines.Discussion and ConclusionsWe conclude that HIV exposure without infection was associated with blunted IL17/IL22 and pro-inflammatory responses, both systemically and at the site of mucosal HIV exposure. It will be important for further studies to examine the causal nature of the association and to define the cell subsets responsible for these differences.

CD1d-dependent NKT cells have been extensively studied; however, the function of CD8+NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8+NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8+NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8+NKT-like cell development is normal in CD1d−/− mice, which suggests that CD8+NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8+NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8+NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8+NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract.

The graft-versus-leukemia effect reminds us to observe the allogeneic cell elicited anti-tumor immune responses. Here we immunized recipient B6 mice with different types of allogenic leukocytes and found that vaccination with allogenic dendritic cells (alloDC) elicited the most efficient protection against broad-spectrum tumors. The recipient lymphocytes were analyzed and the data showed that CD8 T cells increased significantly after immunization and expressed effector memory T cell marker KLRG1. Functional evaluation demonstrated that these KLRG1+CD8 T cells could kill tumor cells in vitro and in vivo in Granzyme B- and Fas/FasL-dependent manners with no tumor antigen specificity, and tend to migrate into tumor sites by high expression of heparanase. Adoptive transfer of these cells could provide antitumor protection against tumors. AlloDC could also treat mice with residual tumors and combination of anti-PD1 antibody could enhance this effects. Together, our study showed that alloDC-immunization could induce potent antitumor effect through the expansion of KLRG1+CD8 T cells, which can work as both preventive and therapeutic tumor vaccines.