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BackgroundOvarian hormones are known to modulate the immune system in the female genital tract (FGT). We sought to define the impact of the menstrual cycle on the mucosal HIV target cell levels, and tissue-resident CD4 T cells.Materials and methodsHere, we characterized the distribution, phenotype, and function of CD4 T cells with special emphasis on HIV target cells (CCR5+ and α4β7+) as well as tissue-resident memory (TRM; CD69+ and CD103+) CD4 T cells in FGT of cycling women. Peripheral blood and Endocervical cells (EC-collected from cytobrush) were collected from 105 healthy women and performed multicolor flow cytometry to characterize the various subsets of CD4 T cells. Cervicovaginal lavage (CVL) were collected for cytokine analysis and plasma were collected for hormonal analysis. All parameters were compared between follicular and luteal phase of menstrual cycle.ResultsOur findings revealed no significant difference in the blood CD4 T cell subsets between the follicular and luteal phase. However, in EC, the proportion of several cell types was higher in the follicular phase compared to the luteal phase of menstrual cycle, including CCR5+α4β7-cells (p=0.01), CD69+CD103+ TRM (p=0.02), CCR5+CD69+CD103+ TRM (p=0.001) and FoxP3+ CD4 T cells (p=0.0005). In contrast, α4β7+ CCR5- cells were higher in the luteal phase (p=0.0004) compared to the follicular phase. In addition, we also found that hormonal levels (P4/E2 ratio) and cytokines (IL-5 and IL-6) were correlated with CCR5+ CD4 T cells subsets during the follicular phase of the menstrual cycleConclusionOverall, these findings suggest the difference in the expression of CCR5 and α4β7 in TRM CD4 T cell subsets in endocervix of HIV seronegative women between the follicular and luteal phase. Increase in the CCR5+ expression on TRM subsets could increase susceptibility to HIV infection during follicular phase of the menstrual cycle.

Measurement(s) parasitemia • blood Chemistry • reticulocyte count • platelet activation status • neutrophil and monocyte activation after stimulation • T-cell phenotyping • B-cell phenotyping • monocyte phenotyping • dendritic cell phenotyping • lymphocyte phenotyping • multiplexed cytokine assay • erythropoietin measurement • total IgG concentration • transcriptome • metabolomics • lipidomics • proteomics Technology Type(s) brightfield microscopy • iSTAT machine • immunological flow cytometry • chemokine receptor markers • intracellular staining • ELISA • LC/MS Factor Type(s) Parasitemia Sample Characteristic - Organism Macaca mulatta • Plasmodium cynomolgi B/M strain • Plasmodium cynomolgi Ceylon Sample Characteristic - Environment venous blood • capillary blood

Plasmodium knowlesi poses a health threat throughout Southeast Asian communities and currently causes most cases of malaria in Malaysia. This zoonotic parasite species has been studied in Macaca mulatta (rhesus monkeys) as a model for severe malarial infections, chronicity, and antigenic variation. The phenomenon of Plasmodium antigenic variation was first recognized during rhesus monkey infections. Plasmodium-encoded variant proteins were first discovered in this species and found to be expressed at the surface of infected erythrocytes, and then named the Schizont-Infected Cell Agglutination (SICA) antigens. SICA expression was shown to be spleen dependent, as SICA expression is lost after P. knowlesi is passaged in splenectomized rhesus. Here we present data from longitudinal P. knowlesi infections in rhesus with the most comprehensive analysis to date of clinical parameters and infected red blood cell sequestration in the vasculature of tissues from 22 organs. Based on the histopathological analysis of 22 tissue types from 11 rhesus monkeys, we show a comparative distribution of parasitized erythrocytes and the degree of margination of the infected erythrocytes with the endothelium. Interestingly, there was a significantly higher burden of parasites in the gastrointestinal tissues, and extensive margination of the parasites along the endothelium, which may help explain gastrointestinal symptoms frequently reported by patients with P. knowlesi malarial infections. Moreover, this margination was not observed in splenectomized rhesus that were infected with parasites not expressing the SICA proteins. This work provides data that directly supports the view that a subpopulation of P. knowlesi parasites cytoadheres and sequesters, likely via SICA variant antigens acting as ligands. This process is akin to the cytoadhesive function of the related variant antigen proteins, namely Erythrocyte Membrane Protein-1, expressed by Plasmodium falciparum.

The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to an HIV/SIV cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCFs) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of NK cells in macaque lymph nodes that expressed BCF-homing receptor CXCR5 and accumulated within BCFs during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g., perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular Th cells that comprise a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared with the CXCR5– subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, and this may have important implications for HIV cure strategies.

Mucosa-associated invariant T (MAIT) cells are recently characterized as a novel subset of innate-like T cells that recognize microbial metabolites as presented by the MHC-1b-related protein MR1. The significance of MAIT cells in anti-bacterial defense is well-understood but not clear in viral infections such as SIV/HIV infection. Here we studied the phenotype, distribution, and function of MAIT cells and their association with plasma viral levels during chronic SHIV infection in rhesus macaques (RM). Two groups of healthy and chronic SHIV-infected macaques were characterized for MAIT cells in blood and mucosal tissues. Similar to human, we found a significant fraction of macaque T cells co-expressing MAIT cell markers CD161 and TCRVα-7.2 that correlated directly with macaque MR1 tetramer. These cells displayed memory phenotype and expressed high levels of IL-18R, CCR6, CD28, and CD95. During chronic infection, the frequency of MAIT cells are enriched in the blood but unaltered in the rectum; both blood and rectal MAIT cells displayed higher proliferative and cytotoxic phenotype post-SHIV infection. The frequency of MAIT cells in blood and rectum correlated inversely with plasma viral RNA levels and correlated directly with total CD4 T cells. MAIT cells respond to microbial products during chronic SHIV infection and correlated positively with serum immunoreactivity to flagellin levels. Tissue distribution analysis of MAIT cells during chronic infection showed significant enrichment in the non-lymphoid tissues (lung, rectum, and liver) compared to lymphoid tissues (spleen and LN), with higher levels of tissue-resident markers CD69 and CD103. Exogenous in vitro cytokine treatments during chronic SHIV infection revealed that IL-7 is important for the proliferation of MAIT cells, but IL-12 and IL-18 are important for their cytolytic function. Overall our results demonstrated that MAIT cells are enriched in blood but unaltered in the rectum during chronic SHIV infection, which displayed proliferative and functional phenotype that inversely correlated with SHIV plasma viral RNA levels. Treatment such as combined cytokine treatments could be beneficial for enhancing functional MAIT cells during chronic HIV infection in vivo.

Background: Tularemia is a potential biological weapon due to its high infectivity and ease of dissemination. This study aimed to characterize the innate and adaptive responses induced by two different lots of a live attenuated tularemia vaccine and compare them to other well-characterized viral vaccine immune responses. Methods: Microarray analyses were performed on human peripheral blood mononuclear cells (PBMCs) to determine changes in transcriptional activity that correlated with changes detected by cellular phenotyping, cytokine signaling, and serological assays. Transcriptional profiles after tularemia vaccination were compared with yellow fever [YF-17D], inactivated [TIV], and live attenuated [LAIV] influenza. Results: Tularemia vaccine lots produced strong innate immune responses by Day 2 after vaccination, with an increase in monocytes, NK cells, and cytokine signaling. T cell responses peaked at Day 14. Changes in gene expression, including upregulation of STAT1, GBP1, and IFIT2, predicted tularemia-specific antibody responses. Changes in CCL20 expression positively correlated with peak CD8+ T cell responses, but negatively correlated with peak CD4+ T cell activation. Tularemia vaccines elicited gene expression signatures similar to other replicating vaccines, inducing early upregulation of interferon-inducible genes. Conclusions: A systems vaccinology approach identified that tularemia vaccines induce a strong innate immune response early after vaccination, similar to the response seen after well-studied viral vaccines, and produce unique transcriptional signatures that are strongly correlated to the induction of T cell and antibody responses.

T follicular helper CD4 cells (Tfh) are essential for the development and maintenance of germinal center (GC) reactions, a critical process that promotes the generation of long-lived high affinity humoral immunity. It is becoming increasingly evident that GC-Tfh cells are heterogeneous in nature with some cellular characteristics associated with a Th1, Th2, and Th17 phenotype. Emerging studies suggest that GC-Tfh cells are directed to differentiate into distinct phenotypes during chronic HIV/SIV infection and these changes in GC-Tfh cells can greatly impact the B cell response and subclass of antibodies generated. Studies in HIV-infected humans have shown that certain Tfh phenotypes are associated with the generation of broadly neutralizing antibody responses. Moreover, the susceptibility of particular GC-Tfh subsets to HIV infection within the secondary lymphoid sites can also impact GC-Tfh/B cell interactions. In this review, we discuss the recent advances that show Tfh heterogeneity during chronic HIV/SIV infection. In particular, we will discuss the dynamics of GC-Tfh cells, their altered differentiation state and function, and their impact on B cell responses during HIV/SIV infection. In addition, we will also discuss the potential role of a recently described novel subset of follicular homing CXCR5+ CD8 T cells (Tfc) and their importance in contributing to control of chronic HIV/SIV infection. A better understanding of the mechanistic role of follicular homing CD4 and CD8 T cells during HIV/SIV infection will aid in the design of vaccines and therapeutic strategies to prevent and treat HIV/AIDS.

Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient "Hit and Run" infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.