Interferon-signaling pathways are upregulated in people with HIV with abnormal pulmonary diffusing capacity (DL CO ).

OBJECTIVE: People with HIV (PWH) are at greater risk of developing lung diseases even when they are antiretroviral therapy (ART)-adherent and virally suppressed. The most common pulmonary function abnormality in PWH is that of impaired diffusing capacity of the lungs for carbon monoxide (DL CO ), which is an independent risk factor for increased mortality in PWH. Earlier work has identified several plasma biomarkers of inflammation and immune activation to be associated with decreased DL CO . However, the underpinning molecular mechanisms of HIV-associated impaired DL CO are largely unknown. DESIGN: Cross-sectional pilot study with PWH with normal DL CO (values greater than or equal to the lower limit of normal, DL CO  ≥ LLN, N = 9) or abnormal DL CO (DL CO  < LLN, N = 9). METHODS: We compared the gene expression levels of over 900 inflammation and immune exhaustion genes in PBMCs from PWH with normal vs. abnormal DL CO using the NanoString technology. RESULTS: We found that 26 genes were differentially expressed in the impaired DL CO group. These genes belong to 4 categories: 1. Nine genes in inflammation and immune activation pathways, 2. seven upregulated genes that are direct targets of the interferon signaling pathway, 3. seven B-cell specific genes that are downregulated, and 4. three miscellaneous genes. These results were corroborated using the bioinformatics tools DAVID (Database for Annotation, Visualization and Integrated Discovery) and GSEA (Gene Sets Enrichment Analysis). CONCLUSION: The data provides preliminary evidence for the involvement of sustained interferon signaling as a molecular mechanism for impaired DL CO in PWH.

Sex-Based Differences in Human Immunodeficiency Virus Type 1 Reservoir Activity and Residual Immune Activation.

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.

Human Immunodeficiency Virus Persistence and T-Cell Activation in Blood, Rectal, and Lymph Node Tissue in Human Immunodeficiency Virus-Infected Individuals Receiving Suppressive Antiretroviral Therapy.

Background: Immune activation and inflammation remain elevated in human immunodeficiency virus (HIV)-infected individuals receiving antiretroviral therapy (ART) and may contribute to HIV persistence. Methods: Using flow cytometry expression of CD38, HLA-DR and PD-1 were measured in blood (n = 48), lymph node (LN; n = 9), and rectal tissue (n = 17) from virally suppressed individuals. Total and integrated HIV DNA, 2-LTR circles, and cell-associated unspliced HIV RNA were quantified. Results: CD4+ T cells from rectal tissue had a higher frequency of integrated HIV DNA compared with blood (4.26 fold-change in DNA; 95% confidence interval [CI] = 2.61-7.00; P < .001) and LN (2.32 fold-change in DNA; 95% CI = 1.22-4.41; P = .01). In rectal tissue, there were positive associations between integrated HIV DNA with PD-1+ CD4+ T-cells (1.44 fold-change in integrated HIV DNA per 10-unit increase in PD-1+ CD4+ T cells; 95% CI = 1.01-2.05; P = .045) and CD38+HLA-DR+ CD8+ T cells (1.40 fold-change in integrated HIV DNA per 1-unit increase in CD38+HLA-DR+ CD8+ T cells; 95% CI = 1.05-1.86; P = .02). Both associations were independent of current and nadir CD4+ T-cell counts. Conclusions: During ART, rectal tissue is an important reservoir for HIV persistence with a high frequency of activated CD4+ and CD8+ T cells. PD-1 may represent a marker of HIV persistence in rectal tissue.

Site-specific differences in T cell frequencies and phenotypes in the blood and gut of HIV-uninfected and ART-treated HIV+ adults.

Gastrointestinal T lymphocytes are critical for mucosal immunity and HIV pathogenesis, yet little is known about normal T cell numbers and phenotypes in different regions of the gut, or the degree to which ART can restore levels to those of HIV-uninfected individuals. To investigate these questions, we measured T cell frequencies and markers of memory, activation, anergy, and homing in the blood, ileum, and rectum of HIV- and ART-suppressed HIV+ adults. In HIV- individuals, T cell frequencies and phenotypes differed significantly between sites. Compared to HIV- adults, HIV+ adults had lower absolute CD4+T cell counts in the ileal lamina propria and lower relative CD4+T cell counts in the blood and ileum. In the gut, HIV+ adults had a higher proportion of CD38+ CD4+T cells, a lower proportion of terminally-differentiated effector cells, and, in the rectum, a higher proportion of CTLA-4+ CD4+T cells. In HIV+ individuals, relative CD4+T cell numbers in the ileum correlated with the proportion of CTLA-4+ CD4+T cells, whereas in the rectum, they tended to correlate with the proportion of circulating CD4+T cells expressing α4ß7 or CCR6. Mechanisms of T cell reconstitution may differ throughout the gut, with homing contributing more in the rectum while ileal reconstitution is associated with mucosal CD4+T cell anergy.

A comparison of methods for measuring rectal HIV levels suggests that HIV DNA resides in cells other than CD4+ T cells, including myeloid cells.

We compared different techniques for measuring gut HIV reservoirs and assessed for HIV in non-CD4 T cells. HIV DNA levels were similar when measured from rectal biopsies and isolated rectal cells, while HIV RNA tended to be higher in rectal cells. HIV DNA levels in total rectal cells were greater than those predicted from levels in sorted CD4 T cells, suggesting a reservoir in non-CD4 T cells, and HIV DNA was detected in sorted myeloid cells (7/7 subjects).

The distribution of HIV DNA and RNA in cell subsets differs in gut and blood of HIV-positive patients on ART: implications for viral persistence.

Even with optimal antiretroviral therapy, human immunodeficiency virus (HIV) persists in plasma, blood cells, and tissues. To develop new therapies, it is essential to know what cell types harbor residual HIV. We measured levels of HIV DNA, RNA, and RNA/DNA ratios in sorted subsets of CD4+ T cells (CCR7+, transitional memory, and effector memory) and non-CD4+ T leukocytes from blood, ileum, and rectum of 8 ART-suppressed HIV-positive subjects. Levels of HIV DNA/million cells in CCR7+ and effector memory cells were higher in the ileum than blood. When normalized by cell frequencies, most HIV DNA and RNA in the blood were found in CCR7+ cells, whereas in both gut sites, most HIV DNA and RNA were found in effector memory cells. HIV DNA and RNA were observed in non-CD4+ T leukocytes at low levels, particularly in gut tissues. Compared to the blood, the ileum had higher levels of HIV DNA and RNA in both CD4+ T cells and non-CD4+ T leukocytes, whereas the rectum had higher HIV DNA levels in both cell types but lower RNA levels in CD4+ T cells. Future studies should determine whether different mechanisms allow HIV to persist in these distinct reservoirs, and the degree to which different therapies can affect each reservoir.

Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy.

BACKGROUND: The gut is a major reservoir for human immunodeficiency virus (HIV) in patients receiving antiretroviral therapy (ART). We hypothesized that distinct immune environments within the gut may support varying levels of HIV. METHODS: In 8 HIV-1-positive adults who were receiving ART and had CD4(+) T cell counts of >200 cells/µL and plasma viral loads of <40 copies/mL, levels of HIV and T cell activation were measured in blood samples and endoscopic biopsy specimens from the duodenum, ileum, ascending colon, and rectum. RESULTS: HIV DNA and RNA levels per CD4(+) T cell were higher in all 4 gut sites compared with those in the blood. HIV DNA levels increased from the duodenum to the rectum, whereas the median HIV RNA level peaked in the ileum. HIV DNA levels correlated positively with T cell activation markers in peripheral blood mononuclear cells (PBMCs) but negatively with T cell activation markers in the gut. Multiply spliced RNA was infrequently detected in gut, and ratios of unspliced RNA to DNA were lower in the colon and rectum than in PBMCs, which reflects paradoxically low HIV transcription, given the higher level of T cell activation in the gut. CONCLUSIONS: HIV DNA and RNA are both concentrated in the gut, but the inverse relationship between HIV DNA levels and T cell activation in the gut and the paradoxically low levels of HIV expression in the large bowel suggest that different processes drive HIV persistence in the blood and gut. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00884793 (PLUS1).

Poor predictive value of cytomegalovirus (CMV)-specific T cell assays for the development of CMV retinitis in patients with AIDS.

BACKGROUND: We examined the potential clinical utility of using a cytomegalovirus (CMV)-specific T cell immunoassay to determine the risk of developing new-onset CMV retinitis (CMVR) in patients with acquired immunodeficiency syndrome (AIDS). METHODS: CMV-specific T cell assays were performed by multiparameter flow cytometry using stored peripheral blood mononuclear cells that had been obtained in an observational study 2-6 months before new-onset CMVR was diagnosed in case patients (at a study visit during which a dilated ophthalmologic examination revealed no evidence of CMVR) and at the same study visit in control subjects (matched by absolute CD4(+) T cell count at entry) who did not subsequently develop retinitis during 1-6 years of study follow-up. RESULTS: There were no significant differences in CMV-specific CD4(+) or CD8(+) T cell interferon-gamma or interleukin-2 expression in peripheral blood mononuclear cells from case patients and control subjects. Although there were trends toward lower percentages and absolute numbers of CMV-specific, cytokine-expressing CD8(+) T cells with a "late memory" phenotype (CD27(-)CD28(-)) as well as with an "early memory" phenotype (CD27(+)CD28(+)CD45RA(+)) in case patients than in control subjects, these differences were not statistically significant. CONCLUSIONS: Many studies have reported that CMV-specific CD4(+) and CD8(+) T cell responses distinguish patients with active CMVR (i.e., who lack CMV-protective immunity) from those with inactive CMVR after immune restoration by antiretroviral treatment (i.e., who have CMV-protective immunity). However, the multiple CMV-specific immune responses we measured do not appear to have clinical utility for predicting the risk for patients with AIDS of developing new-onset CMVR with sufficient accuracy to be used in guiding therapeutic management.

Protective immunity to cytomegalovirus (CMV) retinitis in AIDS is associated with CMV-specific T cells that express interferon- gamma and interleukin-2 and have a CD8+ cell early maturational phenotype.

To determine potential correlates of immune recovery from AIDS-related cytomegalovirus retinitis (CMVR), multiparameter flow cytometry was used to characterize CMV-specific T cells from subjects with CMVR. Individuals with active retinitis were compared with those who had been clinically immunorestored by antiretroviral therapy and had > or =2 years of ophthalmologic follow-up without anti-CMV therapy or retinitis reactivation or progression. In comparison with patients with active retinitis, immunorestored patients had higher circulating CD4(+) and CD8(+) T cells expressing interleukin-2 and interferon- gamma in response to combined CMV pp65 and IE1 peptide pool stimulation. CD4(+) T cell responses were predominantly to pp65, whereas CD8(+) T cell responses were predominantly to IE. Immunorestored patients, compared with patients with active retinitis, had increased levels of circulating CMV-specific CD8(+) T cells with "early" (CD27(+)CD28(+)CD45RA(+), CD27(+)CD28(+)CD45RA(-)) and "intermediate" (CD27(-)CD28(+)CD45RA(-)) phenotypes. Recovery from AIDS-related CMVR after the initiation of antiretroviral therapy may be mediated by CMV-specific CD4(+) and CD8(+) T cells capable of promoting antigen-specific CD8(+) T cell proliferation.

Safety and immunogenicity of Towne cytomegalovirus vaccine with or without adjuvant recombinant interleukin-12.

The Towne, human cytomegalovirus (CMV) vaccine is safe and immunogenic but has not prevented infection at doses tested to date. We administered 3000 pfu Towne CMV vaccine, with or without adjuvant recombinant interleukin-12 (rhIL-12), to CMV-seronegative healthy volunteers and then measured CMV gB-specific IgG titers and CMV-specific CD4+ and CD8+ T cell proliferation and IFNgamma expression after stimulation with whole viral lysate and immunodominant peptide CMV antigens. Adjuvant rhIL-12 at doses up to 2 microg were well-tolerated and associated with (1) dose-related increases in peak anti-CMV gB IgG titers (though not in sustained titers), (2) dose-related increases in the weak CMV viral lysate-specific CD4+ T cell proliferation responses induced by vaccine alone after 360 days of follow-up, and (3) decreases in the very robust CMV IE-specific peak CD4+ T cell and Day 360 CD8+ T cell proliferation responses induced by the vaccine alone. Also, qualitative CD8+ T cell IFNgamma responses to stimulation with the immunodominant CMV antigen, pp65, tended to occur more frequently in vaccinees who received 0.5-2.0 microg rhIL-12 compared to lower dose or no rhIL-12. Thus, adjuvant IL-12 may be a promising strategy for improving antibody and T cell immune responses to a CMV vaccine.