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Objective: To assess whether weakly recognized epitope variants induce anergy in HIV-1-specific CD8 T lymphocyte (CTL) clones as a mechanism of dysfunction., Design: HIV-1-specific CTL clones were exposed to suboptimally recognized epitope variants, and screened for anergy and other T-cell dysfunction markers, and subsequent capability to kill target cells bearing index epitope., Methods: In addition to the optimally recognized index epitope, two suboptimally recognized epitope variants were selected based on titration curves for killing of peptide-labeled target cells by three HIV-1-specific CTL clones targeting the epitopes SLYNTVATL (Gag 77-85, A02-restricted), RPAEPVPLQL (Rev 66-75, B07-restricted), and KRWIIMGLNK (Gag 263-272, B27-restricted). Consequences of suboptimal stimulation were assessed by cytokine secretion, gene expression, and capacity to kill index epitope-labeled target cells upon rechallenge., Results: Suboptimal recognition of epitope variants reduced cytokine production by CTL similarly to reduction in killing of target cells. Gene expression profiles after suboptimal stimulation demonstrated no patterns consistent with T-cell dysfunction due to anergy, exhaustion, or apoptosis. Preexposure of CTL to epitope variants had no discernable impact on their subsequent capacity to kill index epitope-bearing target cells., Conclusion: Our data explore the hypothesis that poorly recognized epitope variants not only facilitate HIV-1 evasion of CTL recognition, but also induce CTL dysfunction through suboptimal signaling causing anergy. However, the results do not suggest that suboptimal signaling induces anergy (or exhaustion or apoptosis), indicating that the major role of CTL epitope variation is likely viral escape.

Objectives: HIV-1-infected persons spontaneously controlling viremia without treatment (SCV) are rare. Sex and race effects on prevalence and outcome are poorly defined, and it is unclear whether SCV qualitatively or quantitatively differs from typical infection. These issues are examined in this article., Design: Medical records of 46 524 persons receiving outpatient care for HIV-1 infection were reviewed. Of these, 29 811 had adequate viremia testing for SCV screening., Methods: SCV was defined as at least three consecutive plasma viremia measurements <50 RNA copies/ml spanning at least 1 year without treatment. SCV loss was defined as at least three consecutive viremia measurements ≥50 RNA copies/ml or one ≥1000 RNA copies/ml. Demographics of persons with SCV were compared with the total population. Viremia and blood CD4 T-cell levels during SCV were compared between demographic subgroups and persons who maintained or lost SCV during observation., Results: In total, 53 persons (0.18%) met SCV criteria. Prevalence was higher for women versus men and blacks versus whites; these appeared independent. Loss of SCV was observed at 1.22% per year, and significantly associated with viremia 'blips'. Blip magnitudes fit log-normal distributions with means below 50 RNA copies/ml., Conclusion: Our novel observation of higher SCV prevalence in women and blacks is consistent with prior studies of typical chronic infection. Viremia blips correspond to greater risk of loss of SCV, likely reflecting higher set-point viremia under the limit of detection. Our findings suggest that SCV represents an extreme along a continuum of HIV-1 infection, and not qualitative difference.

Objective: The pathogenesis of immune dysfunction in chronic HIV-1 infection is unclear, and a potential role for oxidized lipids has been suggested. We hypothesize that both oxidized HDL and LDL (HDLox and LDLox) contribute to HIV-1-related immune dysfunction., Study: In the AIDS Clinical Trials Group A5260, 234 HIV-infected antiretroviral therapy (ART)-naive participants were randomized to receive tenofovir-emtricitabine and protease inhibitors or raltegravir and had HIV-1 RNA less than 50 copies/ml by week 24 and thereafter., Methods: Associations between biomarkers of inflammation (IL-6, high-sensitivity C-reactive protein and D-dimer), immune activation (sCD163, sCD14, soluble IL-2 receptor, CD38 and HLA-DR), inflammatory monocytes (CD14CD16), T-cell senescence (CD28 and CD57) and exhaustion (PD1), and HDLox, LDLox were assessed at entry and after ART (week 96) with Spearman (partial) correlations., Results: HDLox declined and LDLox increased over 96 weeks of ART. Positive associations were observed at baseline and over time between HDLox (but not consistently for LDLox) and most markers of inflammation and immune activation (but not senescence/exhaustion), even after adjustment for multiple comparisons, demographics, entry CD4 cell count and HIV-1 RNA. HDLox was positively associated with IL-6 (r = 0.19 - 0.29, P < 0.01) and sCD163 (r = 0.14 - 0.41, P ≤ 0.04) at all time points., Conclusion: These prospective longitudinal data suggest that oxidized lipoproteins may contribute to persistent immune activation on ART., Competing Interests: Potential conflicts of interest: All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Objectives: AIDS is caused by CD4 T-cell depletion. Although combination antiretroviral therapy can restore blood T-cell numbers, the clonal diversity of the reconstituting cells, critical for immunocompetence, is not well defined., Methods: We performed an extensive analysis of parameters of thymic function in perinatally HIV-1-infected (n = 39) and control (n = 28) participants ranging from 13 to 23 years of age. CD4 T cells including naive (CD27 CD45RA) and recent thymic emigrant (RTE) (CD31/CD45RA) cells, were quantified by flow cytometry. Deep sequencing was used to examine T-cell receptor (TCR) sequence diversity in sorted RTE CD4 T cells., Results: Infected participants had reduced CD4 T-cell levels with predominant depletion of the memory subset and preservation of naive cells. RTE CD4 T-cell levels were normal in most infected individuals, and enhanced thymopoiesis was indicated by higher proportions of CD4 T cells containing TCR recombination excision circles. Memory CD4 T-cell depletion was highly associated with CD8 T-cell activation in HIV-1-infected persons and plasma interlekin-7 levels were correlated with naive CD4 T cells, suggesting activation-driven loss and compensatory enhancement of thymopoiesis. Deep sequencing of CD4 T-cell receptor sequences in well compensated infected persons demonstrated supranormal diversity, providing additional evidence of enhanced thymic output., Conclusion: Despite up to two decades of infection, many individuals have remarkable thymic reserve to compensate for ongoing CD4 T-cell loss, although there is ongoing viral replication and immune activation despite combination antiretroviral therapy. The longer term sustainability of this physiology remains to be determined.

Objective: T-cell receptor (TCR) gene therapy is an approach being considered for HIV-1, but epitope mutation is a significant barrier. We assessed whether HIV-specific TCR can be modified to have broader coverage of epitope variants by recombining polymorphisms between public clonotype TCR sequences., Design: Public clonotype TCRs recognizing the same epitope often differ by polymorphisms in their third complementarity determining regions (CDR3). We assessed whether novel combinations of such polymorphisms could improve TCR recognition of epitope variation., Methods: A TCR recognizing the HLA A*0201-restricted epitope SLYNTVATL (Gag 77-85, SL9) was engineered to have combinations of four polymorphisms in the CDR3 regions compared to another SL9-specific TCR. These novel TCRs were screened for functional avidities against SL9 epitope variants and abilities to mediate cytotoxic T-lymphocyte suppression of HIV-1 containing the same epitope variants., Results: The TCRs varied modestly in functional avidities for SL9 variants, due to alterations in affinity. This translated to differences in antiviral activities against HIV-1 when functional avidity changes crossed the previously defined threshold required for efficient recognition of HIV-1-infected cells. Higher avidity TCR mutants had generally broader recognition of SL9 variants., Conclusion: These results indicate that rationally targeted increases in functional avidities can be utilized to maximize the antiviral breadth of transgenic TCRs. In contrast to previously reported random mutagenesis to markedly increase functional avidities, tuning through recombining naturally occurring polymorphisms may offer a more physiologic approach that minimizes the risk of deleterious TCR reactivities.

As interferon-gamma (IFN-gamma) is produced at lower levels by neonatal than adult T cells, we determined whether IFN-gamma influences HIV-1 replication in thymocytes. IFN-gamma significantly decreased replication of R5 but not X4 viruses, and reduced depletion of CD3(hi)CD27 (mature) thymocytes, the preferential targets for R5 HIV-1. Thus infection and depletion of functionally mature thymocytes that can produce endogenous IFN-gamma may mutually contribute to HIV-1 replication in the thymus and to reduced T-cell output.

Objective: Although the epitopic breadth of HIV-1-specific CD8 T lymphocyte (CTL) responses has been described, the T cell receptor (TCR) diversity of virus-specific cells remains poorly defined., Design and Methods: To address this issue, we applied a novel technique for subtractive analysis of the HIV-1-specific CTL repertoire, combining specific deletion of peptide-specific cells by 5-fluorouracil with TCR spectratyping to identify clonal breadth of CTL recognizing individual peptides., Results: Comprehensive analysis of an infected individual reveals that nine identified HIV-1-specific responses are comprised of at least 38 distinct T-cell clones (ranging from two to 10 distinct clones per epitope)., Conclusion: Given the potentially crucial role of T-cell receptor breadth for viral recognition and avoidance of escape, this subepitopic analysis of CTL may offer an important measure of cellular immunity for pathogenesis and vaccine studies.

Objective: We examined the association between immunogenic exposure and T-cell receptor (TCR) diversity to more clearly assess the impact of HIV-1 infection on the T-cell repertoire., Methods: : To estimate the extent of T-cell clonality attributable to HIV-1 infection, we evaluated T-cell repertoires in low-risk and at-risk seronegative men and HIV-1 seropositive men by assessment of T-cell receptor beta-chain (TCR beta) complimentary determining region 3 (CDR3) lengths., Results: The frequency of T-cell clonality in both HIV-1 infected and at-risk uninfected men was elevated in comparison to low-risk uninfected men. Among low-risk and at-risk seronegative, and HIV-1 seropositive men, clonal expansions were present in 3, 8, and 10% of CD4+ CDR3 lengths, and 18, 22, and 28% of CD8+ CDR3 lengths respectively. In addition, the longitudinal conservation of clonal expansions was observed in at-risk seronegative men. Based on comparisons to at-risk seronegative men, we estimate that at-risk seropositive men with chronic HIV-1 infection exhibit a 27% increase in the number of expanded CD8+ CDR3 lengths., Conclusion: These findings provide an approximation of the magnitude of the T-cell response in individuals undergoing chronic HIV-1 infection and demonstrate a significant association between the history of immunogenic challenge and the magnitude of clonality within the T-cell repertoire. In addition, these findings underscore the necessity of selecting controls with similar antigenic exposure histories when investigating T-cell dynamics in HIV-infected individuals.