Progressive HIV infection is characterized by dysregulation of the intestinal immune barrier, translocation of immunostimulatory microbial products, and chronic systemic inflammation that is thought to drive progression of disease to AIDS. Elements of this pathologic process persist despite viral suppression during highly active antiretroviral therapy (HAART) and drivers of these phenomena remain poorly understood. Disrupted intestinal immunity can precipitate dysbiosis that induces chronic inflammation in the mucosa and periphery of mice. However, putative microbial drivers of HIV-associated immunopathology versus recovery have not been identified in humans. Using high-resolution bacterial community profiling, we identified a dysbiotic mucosal-adherent community enriched in Proteobacteria and depleted of Bacteroidia members that was associated with markers of mucosal immune disruption, T cell activation, and chronic inflammation in HIV-infected subjects. Furthermore, this dysbiosis was evident among HIV-infected subjects undergoing HAART, and the extent of dysbiosis correlated with activity of the kynurenine pathway of tryptophan metabolism and plasma concentrations of the inflammatory cytokine interleukin-6 (IL-6), two established markers of disease progression. Gut-resident bacteria with capacity to metabolize tryptophan through the kynurenine pathway were found to be enriched in HIV-infected subjects, strongly correlated with kynurenine levels in HIV-infected subjects, and capable of kynurenine production in vitro. These observations demonstrate a link between mucosal-adherent colonic bacteria and immunopathogenesis during progressive HIV infection, which is apparent even in the setting of viral suppression during HAART. This link suggests that gut-resident microbial populations may influence intestinal homeostasis during HIV disease.

HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell frequencies measured by viral outgrowth versus PCR assays is an urgent priority in HIV-1 cure research.

• Antimicrobial CD8 ϩ MAIT cells are activated, exhausted, and progressively and persistently depleted during chronic HIV-1 infection.• This decline in MAIT cell level and function may seriously impair the ability to mount immune responses to bacterial and fungal pathogens. IntroductionHIV-1 infection is associated with a range of pathologic changes to the immune system, including systemic immune activation, CD4 T-cell loss and CD8 T-cell expansion. The state of broad and persistent immune activation develops early during infection, 1,2 contributes to the rapid aging of the immune system seen during chronic progressive HIV-1 disease, and persists despite effective long-term virologic suppression by combination antiretroviral therapy (cART; reviewed in by Deeks, 3 Appay et al, 4 and Desai and Landay 5 ). These pathologic processes lead to the progressive destruction of lymphoid organs and loss of CD4 helper T cells. 6,7 Already during primary infection, HIV-1 depletes intestinal CD4 T cells and disrupts the structure and function of the intestinal immune system. [8][9][10][11][12][13] One consequence of this is increased permeability of the intestinal epithelium with translocation of microbial products into the local tissue, the portal circulation, the liver and eventually into systemic circulation. 14 This process may continue despite effective long-term cART. 15,16 Disruption of immune homeostasis and barrier function at the mucosa is a considerable challenge for the host immune system because the microbial proteins, carbohydrates, and lipids form a range of antigens that will engage innate as well as adaptive immune mechanisms (reviewed by Brenchley and Douek 17 ). Despite considerable advances in the treatment and management of HIV-1 disease, certain infections still present a significant clinical challenge particularly among HIV-infected individuals who are diagnosed at advanced stages, those who lack access to antiretroviral therapy, and those who cannot maintain adherence to therapy and clinical care. [18][19][20] This includes an increased risk of developing bacterial pneumonia even in HIV-1-infected patients with relatively normal CD4 counts, 21 indicating that impaired CD4 T-cell independent control of certain infections still exists even in the context of treated HIV-1 disease. Mucosal-associated invariant T (MAIT) cells are a relatively recently discovered subset of unconventional, innate-like T cells that are highly abundant in mucosal tissues, liver, and peripheral blood. [22][23][24][25] Human MAIT cells express an invariant T-cell receptor (TCR) carrying the V␣7.2 ␣-chain segment, a restricted V␤ repertoire (V␤2 or V␤13), and recognize antigens in complex with the evolutionarily conserved MHC-Ib-related protein (MR1). 24,25 In addition to the V␣7.2 TCR segment, MAIT cells are defined by Submitted July 27, 2012; accepted November 26, 2012. Prepublished online as Blood First Edition paper, December 13, 2012; DOI 10.1182 DOI 10. /blood-2012 The online version of this article contains a data suppleme...

A low CD4/CD8 ratio in elderly HIV-uninfected adults is associated with increased morbidity and mortality. A subset of HIV-infected adults receiving effective antiretroviral therapy (ART) fails to normalize this ratio, even after they achieve normal CD4+ T cell counts. The immunologic and clinical characteristics of this clinical phenotype remain undefined. Using data from four distinct clinical cohorts and three clinical trials, we show that a low CD4/CD8 ratio in HIV-infected adults during otherwise effective ART (after CD4 count recovery above 500 cells/mm3) is associated with a number of immunological abnormalities, including a skewed T cell phenotype from naïve toward terminally differentiated CD8+ T cells, higher levels of CD8+ T cell activation (HLADR+CD38+) and senescence (CD28− and CD57+CD28−), and higher kynurenine/tryptophan ratio. Changes in the peripheral CD4/CD8 ratio are also reflective of changes in gut mucosa, but not in lymph nodes. In a longitudinal study, individuals who initiated ART within six months of infection had greater CD4/CD8 ratio increase compared to later initiators (>2 years). After controlling for age, gender, ART duration, nadir and CD4 count, the CD4/CD8 ratio predicted increased risk of morbidity and mortality. Hence, a persistently low CD4/CD8 ratio during otherwise effective ART is associated with increased innate and adaptive immune activation, an immunosenescent phenotype, and higher risk of morbidity/mortality. This ratio may prove useful in monitoring response to ART and could identify a unique subset of individuals needed of novel therapeutic interventions.