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The bacterial pathogen Mycobacterium tuberculosis (Mtb) successfully evades host innate immune responses to cause tuberculosis (TB) disease. Recent studies showed that myeloid-derived suppressor cells (MDSCs) are recruited to the lung after Mtb infection and harbor Mtb. While MDSC-mediated negative regulation of tumor micro-environments in cancer has been extensively studied, in-depth analyses of the phenotype, functions and transcriptional signatures of MDSCs in TB are lacking. In this study we sought to characterize MDSCs in the lungs of Mtb-infected mice and develop tools for depleting MDSCs as a strategy for host-directed therapy. We determined MDSCs in the aerosol mouse model of TB and in human blood using flow cytometry. MDSCs are recruited into the lung as early as 2 weeks post-infection and expresses immunomodulatory mediators associated with immune suppression such as iNOS, IDO and arginase. We recently reported the development of novel synthetic nanoparticle antibodies (SNAbs) that specifically target and deplete MDSCs via antibody-like mechanisms. We used SNAbs to deplete Mtb-induced MDSCs in vitro and in human PBMCs ex vivo. Interestingly, we found that SNAbs efficiently depleted MDSCs in both models. To test the efficacy of SNAb-mediated depletion of MDSCs in mouse lungs in vivo, we first used a murine lung cancer model and found that intra-tracheal delivery of SNAbs successfully depleted MDSCs in vivo. Ongoing studies include evaluating the ability of SNAbs to deplete lung MDSCs following Mtb infection of mice and to assess the impact on lung T cell functions, pathology and Mtb burden. These studies will establish a platform to investigate MDSCs as a target for host-directed therapies that improve TB treatment efficacy. Supported by grant from NIH (1R01AI155023-01A1)

Female sex hormones are known to regulate the immune functions of the female genital tract (FGT). While the majority of immune cells found in the FGT are T cells and Natural Killer cells (NK cells), very few studies have focused on NK cells in the FGT in contrast to numerous studies focusing on T-cells. Here, we characterized the distribution, phenotype and function of NK cells in FGT of HIV seronegative women using different mucosal sampling methods; cervicovaginal lavage (CVL), endocervical cytobrush (CB), and cervicovaginal biopsy and compared them to blood. We isolated cells from blood and FGT and performed multi-color flow cytometry to identify cellular phenotypes. First, we looked at the distribution of CD56brightand CD56dimpopulations of NK cells across the samples and found that CD56brightNK cells were lower in the FGT tissues compared to blood. Between the CD56brightand CD56dimpopulations, the CD56dimpopulation was significantly reduced in the follicular phase compared to the luteal phase in tissues. The NK cells within the FGT samples express higher levels of tissue resident markers CD69 and CD103. The co-expression of CD69+CD103+ markers seem to be significantly higher in follicular phase compared to luteal phase in tissues. These cells have high proliferative capacity with high levels of HLADR expression, suggesting that these cells are highly functional and activated. The gut tissue homing marker alpha4beta7 expression significantly reduced in the follicular phase, however other homing markers CCR5 and CCR7 expression were elevated in the follicular phase. Altogether, these data demonstrate that FGT is enriched with tissue resident NK cells with high activation and proliferation profile with distinct homing potential.

OBJECTIVE To characterize the correlates of protection from systemic infection in a vaccinated rhesus macaque, RAt-9, which had been challenged sequentially with two related clade C simian/human immunodeficiency viruses (SHIV-Cs) yet remained aviremic for more than 5 years despite indirect evidence of cryptic infection. DESIGN To measure long-term anti-SHIV-C immunity, host genetics and gene-expression patterns for protective correlates. METHODS Long-term immune reactivity was evaluated and identification of virus in RAt-9 was attempted by RT-PCR analysis of concentrated plasma and blood transfer to CD8(+) cell-depleted infant macaques. Full MHC genotyping of RAt-9, TRIM5α and KIR3DL allelic expression analysis of PBMC, and microarray gene expression analysis were performed. RESULTS All attempts to detect/isolate virus, including blood transfer to CD8(+) cell-depleted infant rhesus macaques, were negative, and the animal maintained normal levels of memory CD4(+) T cells in both peripheral blood and gut tissues. However, RAt-9 maintained high levels of anti-SHIV-C humoral and cellular immunity, including reactivity to nonvaccine neoantigens (Nef and Rev), up to 63 months postinitial challenge, suggesting chronic sub-threshold infection. RAt-9 expressed the Mamu A*001 allele negative for B*008 and B*017, had a B13 serotype, and had increased expression of killer-cell immunoglobulin-like receptors (KIRs) previously linked to favorable outcomes of lentiviral infection. Elements of the gene expression profiling coincided with genotyping results. RAt-9 also displayed CD8 cell noncytotoxic antiviral response (CNAR) activity. CONCLUSION Monkey RAt-9 is the first example of a virus-exposed, persistently aviremic animal that has maintained long-term, high-level cellular and humoral antiviral immunity in the absence of an identifiable cryptic reservoir.

Mucosa-associated invariant T-cells (MAIT) are abundant in humans and recognize bacterial ligands. Here, we have studied the distribution and function of MAIT cells during chronic SHIV infection using rhesus macaques (RM). Two groups of RM, healthy and SHIV infected were studied. lymphocytes from various tissues were analysed for MAIT (TCR7.2++ CD161++) cells using multicolor flow cytometry and characterized for their distribution, phenotype and function during chronic SHIV infection. Similar to human, we found significant fraction of (~2%) RM CD8 T cells co-express MAIT cell markers TCR7.2, CD161, IL-18R, CCR6 and display central memory (CCR7+CD45RA−) phenotype. The frequency of MAIT cells were decreased during chronic SHIV infection and tissue analysis showed a significant enrichment of MAIT cells in liver (~8%) and BAL (3%) than in blood (1%), spleen(0.5%), lymph node(0.13%) and gut (0.2%). Importantly, during chronic SHIV infection more than 80% of MAIT cells expressed T cell exhaustion marker PD-1 in blood and the level of expression was higher (>95%) in tissue resident CD69+MAIT cells. These cells lack proliferating capacity (Ki-67) and cytokine production (IFN- g and IL-17) in the blood, suggesting their functional impairment. The preserved blood and gut MAIT cells correlate inversely with plasma viral RNA levels and associate directly with gut CD4 T cell levels, suggesting that CD4 help maintains MAIT cell frequencies in the gut. These data demonstrate that MAIT cells are decreased in blood and are functionally exhausted during chronic SHIV infection. Future studies focused on blocking the PD-1 pathway to retrive MAIT cell exhaustion during chronic SHIV/HIV infection may yield therapeutic benefit to HIV/SHIV infection.

Follicular helper CD4T cells (Tfh) play a critical role in determining the magnitude and quality of antibody (Ab) responses. Chronic HIV infection is associated with hypergammaglobulinemia and germinal center Tfh (GC-Tfh) in lymphoid tissue may contribute to this process. However, the heterogeneous nature of GC-Tfh cells and the relative contribution of specific GC-Tfh subsets to viral persistence and Ab production is not understood in HIV/SIV infections. Here, we characterized the phenotype and function of GC-Tfh cells based on the expression of chemokine receptors associated with Th1 (CXCR3), Th2 (CCR4) and Th17 (CCR6) in lymph nodes following SIV251 infection in rhesus macaques (RMs). In SIV naïve RM, only a small fraction of GC-Tfh expressed CXCR3, CCR4 and CCR6. During chronic SIV infection, despite a global depletion of CD4 T cells, there was an increase in total GC-Tfh cells. Interestingly the majority of expanded GC-Tfh cells expressed CXCR3, while the proportion of CCR4+ cells did not change but CCR6+ cells decreased. CXCR3+ but not CXCR3−GC-Tfh produced IFN-g and IL-21 while both expressed CD40L following stimulation. IHC analysis demonstrated an accumulation of IFN-g+ T cells within the follicles during chronic infection. CXCR3+GC-Tfh also expressed higher levels of ICOS, CCR5 and a4b7, contained more copies of SIV DNA compared to CXCR3−GC-Tfh cells. Both CXCR3+ and CXCR3−GC-Tfh delivered help to B cells in vitro, but CXCR3+ GC-Tfh favored the production of IgG and IgG1. These data demonstrate that chronic SIV infection promotes expansion of Th1-biased GC-Tfh cells, which are phenotypically and functionally distinct from conventional GC-Tfh cells and contribute to IgG1 biased hypergammaglobulinemia and viral reservoirs

Background The identification and treatment of individuals with tuberculosis (TB) is a global public health priority. Accurate diagnosis of pulmonary active TB (ATB) disease remains challenging and relies on extensive medical evaluations and detection of Mycobacterium tuberculosis (Mtb) in the patients’ sputum. Further, the response to treatment is monitored by sputum culture conversion, which takes 3–6 weeks for results. Here, we sought to identify blood-based host biomarkers associated with ATB and hypothesized that frequencies of Mtb-specific CD4+IFN-g+ T cells expressing caspase-3 would decrease following anti-TB treatment. Caspase-3, a member of cysteine proteases’ family, is highly expressed in CD4 effector T cells and has been shown to regulate T cell activation, cell cycle entry, proliferation and apoptosis. Methods Using polychromatic flow cytometry, we evaluated the expression of the active form of caspase-3 on Mtb-specific CD4+ T cells from individuals with asymptomatic latent Mtb infection (LTBI) (n=22), ATB (n=18), and from ATB patients undergoing anti-TB treatment (n=7). Results Frequencies of Mtb-specific IFN-g+CD4+ T cells that expressed active caspase-3 were higher in individuals with ATB compared to those with LTBI suggesting that apoptotic pathways are operant during pulmonary ATB. This marker also distinguished individuals with untreated ATB from those who had successfully completed anti-TB treatment and correlated with decreasing mycobacterial loads during treatment. Conclusion We have identified caspase-3 on Mtb-specific CD4+ T cells as a marker that distinguishes ATB and LTBI and may provide a tool for monitoring treatment response and cure.

Two of the most common causes of end-stage liver disease (ESLD) are hepatitis C virus (HCV) infection and alcoholic liver disease (ALD); the only therapeutic option for liver failure is a liver transplant. Extrahepatic manifestations of ESLD include antibody-mediated autoimmunity; an indication of B cell and CD4+ T cell dysfunction. To investigate the contribution of CD4+ T cells during liver disease, we induced liver fibrosis in mice by a conventional weekly carbon-tetrachloride treatment regimen. Fibrotic animals show elevated serum IgG, positive ANA titers and spontaneous IgG production from hepatic B cells. However, this was not evident in CD4+ T cell depleted fibrotic animals despite comparable fibrosis by pathology. Despite significant expression of Foxp3 on fibrotic liver CD4+ T cells, concomitant expression of CD27, PD-1 and CD40L may account for their ability to promote B cell activation. To understand the implications during ESLD in humans, we performed parallel analysis from human liver explant samples. In accordance with our fibrotic mouse model, we saw an enrichment of Foxp3+ CD4+ T cells as well as spontaneous IgG production that was not evident in healthy liver or PBMC. Taken together, these data suggest that altered hepatic CD4+ T cells aberrantly instruct B cells to drive extrahepatic sequelae of ESLD.