Latently-infected CD4+ T cells are widely considered to be the major barrier to a cure for HIV. Much of our understanding of HIV latency comes from latency models and blood cells, but most HIV-infected cells reside in lymphoid tissues such as the gut. We hypothesized that tissue-specific environments may impact the mechanisms that govern HIV expression. To assess the degree to which different mechanisms inhibit HIV transcription in the gut and blood, we quantified HIV transcripts suggestive of transcriptional interference (U3-U5; "Read-through"), initiation (TAR), 5' elongation (R-U5-pre-Gag; "Long LTR"), distal transcription (Nef), completion (U3-polyA; "PolyA"), and multiple splicing (Tat-Rev) in matched peripheral blood mononuclear cells (PBMCs) and rectal biopsies, and matched FACS-sorted CD4+ T cells from blood and rectum, from two cohorts of ART-suppressed individuals. Like the PBMCs, rectal biopsies showed low levels of read-through transcripts (median = 23 copies/106 cells) and a gradient of total (679)>elongated(75)>Nef(16)>polyadenylated (11)>multiply-spliced HIV RNAs(<1) [p<0.05 for all], demonstrating blocks to HIV transcriptional elongation, completion, and splicing. Rectal CD4+ T cells showed a similar gradient of total>polyadenylated>multiply-spliced transcripts, but the ratio of total to elongated transcripts was 6-fold lower than in blood CD4+ T cells (P = 0.016), suggesting less of a block to HIV transcriptional elongation in rectal CD4+ T cells. Levels of total transcripts per provirus were significantly lower in rectal biopsies compared to PBMCs (median 3.5 vs. 15.4; P = 0.008) and in sorted CD4+ T cells from rectum compared to blood (median 2.7 vs. 31.8; P = 0.016). The lower levels of HIV transcriptional initiation and of most HIV transcripts per provirus in the rectum suggest that this site may be enriched for latently-infected cells, cells in which latency is maintained by different mechanisms, or cells in a "deeper" state of latency. These are important considerations for designing therapies that aim to disrupt HIV latency in all tissue compartments.

Plasma human immunodeficiency virus type 1 (HIV-1) RNA levels in women are lower early in untreated HIV-1 infection compared with those in men, but women have higher T-cell activation and faster disease progression when adjusted for viral load. It is not known whether these sex differences persist during effective antiretroviral therapy (ART), or whether they would be relevant for the evaluation and implementation of HIV-1 cure strategies. We prospectively enrolled a cohort of reproductive-aged women and matched men on suppressive ART and measured markers of HIV-1 persistence, residual virus activity, and immune activation. The frequency of CD4+ T cells harboring HIV-1 DNA was comparable between the sexes, but there was higher cell-associated HIV-1 RNA, higher plasma HIV-1 (single copy assay), and higher T-cell activation and PD-1 expression in men compared with women. These sex-related differences in immune phenotype and HIV-1 persistence on ART have significant implications for the design and measurement of curative interventions.

IL-10 producing T cells inhibit Ag-specific CD8+ T cell responses and may play a role in the immune dysregulation observed in HIV infection. We have previously observed the presence of HIV-specific IL-10-positive CD8+ T cells in advanced HIV disease. In this study, we examined the suppressive function of the Gag-specific IL-10-positive CD8+ T cells. Removal of these IL-10-positive CD8+ T cells resulted in increased cytolysis and IL-2, but not IFN-γ, production by both HIV- and human CMV-specific CD8+ T cells. In addition, these IL-10-positive CD8+ T cells mediated suppression through direct cell-cell contact, and had a distinct immunophenotypic profile compared with other regulatory T cells. We describe a new suppressor CD8+ T cell population in advanced HIV infection that may contribute to the immune dysfunction observed in HIV infection.

SummaryChronic HIV infection is associated with T cell abnormalities and altered effector function. Regulatory T cells (Treg) are CD4 + T cells that play a critical role in regulating the immune system. The impact of regulatory T cells on HIV infection and disease progression may be highly significant. We hypothesize that chronic antigenic stimulation from a persistent, high viraemic state may promote a population of Treg that contributes to HIV-associated immune dysfunction. We evaluated the pattern of Treg in chronically infected, HIVpositive individuals over a course of 6 months. Treg are depleted at a distinct rate from that of absolute CD4 cells and loss of Treg is slower in the presence of viral suppression. In vitro depletion of CD25 + CD4 + cells resulted in increased Gag-specific CD4 and CD8 responses. A significant correlation between ex vivo measurement of Treg and Gag-specific CD4 T cell responses was observed (r = -0·41, P = 0·018) with a trend observed with Gag-specific CD8 T cell responses (P = 0·07). The impact of HIV infection on the Treg population directly complicates the measured effect of Treg on the immune dysfunction although our data support the important role of Treg on modulating the effector T cell response in chronic infection.

Mechanisms leading to the observed immune dysregulation in HIV-1 infection are not well understood. HIV-specific IL-10-positive CD8+ T cells are increased in advanced HIV disease. We have previously reported that Gag-specific IL-10-positive CD8+ T cells suppressed cytolysis. In this study we describe the suppressive effect of Nef-specific IL-10-positive CD8+ T cells. Interestingly, simultaneous removal of both Gag- and Nef-specific IL-10-positive CD8+ T cells led to higher HIV-specific cytolysis compared with the removal of Nef-specific IL-10-positive CD8+ T cells alone. We also examined the level of programmed cell death-1 (PD-1) as a measure of immune dysfunction in association with IL-10-positive suppressor CD8+ T cells. The level of PD-1 expression on CD107-positive effector CD8+ T cells was significantly increased when IL-10-positive suppressor CD8+ T cells were present (p &lt; 0.05). Our results suggest that IL-10-positive suppressor CD8+ T cells contribute to the immune dysfunction observed in advanced HIV infection and that the concomitant presence of multiple IL-10-positive CD8+ T cell populations may have an additive suppressive effect.

BackgroundUnderstanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.Methods and FindingsTo find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10–15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.ConclusionsLeukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

T cell activation is an important mechanism in HIV-associated immune depletion. We have previously demonstrated an association between the hyperactivation of CD4(+) and CD8(+) T cells and low CD4 status in HIV-infected Ugandan children. In this study, we explore differences in activation between naive and memory T cells in HIV-infected Ugandan children. A significant correlation between CD4- and CD8-mediated immune activation and CD4 status was observed only in the memory T cells. Antiretroviral (ART) untreated and treated HIV-positive and HIV-negative children displayed similar profiles of activation and distribution within the CD4(+) naive T cells. In contrast, significantly higher immune activation of the memory CD4(+) T cell subset was seen in ART-untreated children when compared to ART-treated or HIV-negative children. ART-mediated viral suppression led to the correction of CD4(+) immune activation to levels seen in uninfected children but did not increase the size of the memory CD4(+) T cell population. High levels of CD8(+) immune activation were also found in both naive and memory cell subsets. Antiretroviral treatment led to the normalization of CD8(+) T cell activation but did not correct the distribution of naive CD8(+) T cells. We also assessed PD-1 expression on CD8(+) T cells as a measure of immune dysfunction. Upregulation of PD-1 was highest in untreated children but persisted in ART-treated children compared to uninfected children. The mechanisms of immunopathogenesis in pediatric HIV infection likely involve distinct contributions from individual naive and memory T cells subsets.

Human immunodeficiency virus (HIV) or AIDS is currently the leading cause of death in Uganda, with at least three HIV clades (subtypes) accounting for most new infections. Whether an effective vaccine formulated on viruses from a single clade will be able to protect against infection from other local clades remains unresolved. We examined the T-cell immune responses from a cohort of HIV-seropositive individuals in Uganda with predominantly clade A and D infections. Surprisingly, we observed similar frequencies of cross-clade T-cell responses to the gag, env, and nef regions. Our data suggest that the level of viral sequence variability between distinct HIV strains does not predict the degree of cross-clade responses. High sequence homologies were also observed between consensus peptides and sequences from viral isolates, supporting the use of consensus amino acid sequences to identify immunogenic regions in studies of large populations.Human immunodeficiency virus (HIV) or AIDS continues to be the leading cause of death in sub-Saharan Africa. Although there is a critical need for an effective HIV vaccine, the undefined correlates of protective immunity in HIV type 1 (HIV-1) infection remain an important obstacle. Nevertheless, a wealth of evidence suggests that a robust cellular immune response may curtail viral replication; therefore, HIV vaccine designs have, to date, focused on eliciting strong cellular immune responses.T-cell epitope recognition is dependent on the individual class I human leukocyte antigen (HLA) alleles. The pattern of antigen recognition and immunodominance may be driven by the prevalence of these alleles in the studied population (19). The identification of highly immunogenic and conserved regions is also complicated by HIV sequence diversity (8). T-cell responses against HIV can, in turn, influence the HIV populations in generating immune escape viral variants (33).There is comprehensive data on T-cell epitopes for HIVinfected individuals of Caucasian descent. However, there is a relative paucity of information for the geographic regions where the HIV epidemic is spreading the fastest at this time, particularly sub-Saharan Africa (1,2,7,34,35). In Uganda, clades A and D (as well as A/D recombinant strains) are responsible for approximately 95% of HIV-1 infections (3,11,22,23). Previous studies of HIV-1-specific cellular immune responses in small cohorts of HIV-infected individuals and vaccine recipients in Uganda have shown cross-clade immune recognition (5, 6, 31). Nevertheless, the issue of whether an effective vaccine formulated on viruses from a single clade could protect equally well against viruses from other local clades remains unresolved.We evaluated the pattern of T-cell antigen recognition in the context of the HLA alleles and multiple circulating HIV subtypes in a cross-sectional study of HIV-infected Ugandan adults. We investigated factors that determine cross-clade Tcell recognition in the context of vaccine design for Uganda and sub-Saharan Africa. MATERIALS AND METHODS St...