Brian O. Porter

Elevated frequencies of highly activated CD4+ T cells in HIV+ patients developing immune reconstitution inflammatory syndrome

Immune reconstitution inflammatory syndrome (IRIS) is a considerable problem in the treatment of HIV-infected patients. To identify immunologic correlates of IRIS, we characterized T-cell phenotypic markers and serum cytokine levels in HIV patients with a range of different AIDS-defining illnesses, before and at regular time points after initiation of antiretroviral therapy. Patients developing IRIS episodes displayed higher frequencies of effector memory, PD-1(+), HLA-DR(+), and Ki67(+) CD4(+) T cells than patients without IRIS. Moreover, PD-1(+) CD4(+) T cells in IRIS patients expressed increased levels of LAG-3, CTLA-4, and ICOS and had a Th1/Th17 skewed cytokine profile upon polyclonal stimulation. Elevated PD-1 and Ki67 expression was also seen in regulatory T cells of IRIS patients. Furthermore, IRIS patients displayed higher serum interferon-γ, compared with non-IRIS patients, near the time of their IRIS events and higher serum interleukin-7 levels, suggesting that the T-cell populations are also exposed to augmented homeostatic signals. In conclusion, our findings indicate that IRIS appears to be a predominantly CD4-mediated phenomenon with reconstituting effector and regulatory T cells showing evidence of increased activation from antigenic exposure. These studies are registered online at http://clinicaltrials.gov as NCT00557570 and NCT00286767.

Mycobacterial Antigen Driven Activation of CD14++CD16- Monocytes Is a Predictor of Tuberculosis-Associated Immune Reconstitution Inflammatory Syndrome.

Paradoxical tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) is an aberrant inflammatory response occurring in a subset of TB-HIV co-infected patients initiating anti-retroviral therapy (ART). Here, we examined monocyte activation by prospectively quantitating pro-inflammatory plasma markers and monocyte subsets in TB-HIV co-infected patients from a South Indian cohort at baseline and following ART initiation at the time of IRIS, or at equivalent time points in non-IRIS controls. Pro-inflammatory biomarkers of innate and myeloid cell activation were increased in plasma of IRIS patients pre-ART and at the time of IRIS; this association was confirmed in a second cohort in South Africa. Increased expression of these markers correlated with elevated antigen load as measured by higher sputum culture grade and shorter duration of anti-TB therapy. Phenotypic analysis revealed the frequency of CD14++CD16− monocytes was an independent predictor of TB-IRIS, and was closely associated with plasma levels of CRP, TNF, IL-6 and tissue factor during IRIS. In addition, production of inflammatory cytokines by monocytes was higher in IRIS patients compared to controls pre-ART. These data point to a major role of mycobacterial antigen load and myeloid cell hyperactivation in the pathogenesis of TB-IRIS, and implicate monocytes and monocyte-derived cytokines as potential targets for TB-IRIS prevention or treatment.

Paradoxical Tuberculosis Immune Reconstitution Inflammatory Syndrome (TB-IRIS) in HIV Patients with Culture Confirmed Pulmonary Tuberculosis in India and the Potential Role of IL-6 in Prediction

Background The incidence, manifestations, outcome and clinical predictors of paradoxical TB-IRIS in patients with HIV and culture confirmed pulmonary tuberculosis (PTB) in India have not been studied prospectively. Methods HIV+ patients with culture confirmed PTB started on anti-tuberculosis therapy (ATT) were followed prospectively after anti-retroviral therapy (ART) initiation. Established criteria for IRIS diagnosis were used including decline in plasma HIV RNA at IRIS event. Pre-ART plasma levels of interleukin (IL)-6 and C-reactive protein (CRP) were measured. Univariate and multivariate logistic regression models were used to evaluate associations between baseline variables and IRIS. Results Of 57 patients enrolled, 48 had complete follow up data. Median ATT-ART interval was 28 days (interquartile range, IQR 14–47). IRIS events occurred in 26 patients (54.2%) at a median of 11 days (IQR: 7–16) after ART initiation. Corticosteroids were required for treatment of most IRIS events that resolved within a median of 13 days (IQR: 9–23). Two patients died due to CNS TB-IRIS. Lower CD4+ T-cell counts, higher plasma HIV RNA levels, lower CD4/CD8 ratio, lower hemoglobin, shorter ATT to ART interval, extra-pulmonary or miliary TB and higher plasma IL-6 and CRP levels at baseline were associated with paradoxical TB-IRIS in the univariate analysis. Shorter ATT to ART interval, lower hemoglobin and higher IL-6 and CRP levels remained significant in the multivariate analysis. Conclusion Paradoxical TB–IRIS frequently complicates HIV-TB therapy in India. IL-6 and CRP may assist in predicting IRIS events and serve as potential targets for immune interventions.

Markers of endothelial dysfunction, coagulation and tissue fibrosis independently predict venous thromboembolism in Hiv

Objective:HIV infection is associated with coagulation abnormalities and significantly increased risk of venous thrombosis. It has been shown that higher plasma levels of coagulation and inflammatory biomarkers predicted mortality in HIV. We investigated the relationship between venous thrombosis and HIV-related characteristics, traditional risk factors of hypercoagulability, and pre-event levels of biomarkers. Design:A retrospective case–control study of 23 HIV-infected individuals who experienced an incident venous thromboembolic event while enrolled in National Institutes of Health studies from 1995 to 2010 and 69 age-matched and sex-matched HIV-infected individuals without known venous thromboembolism (VTE). Methods:Biomarkers of inflammation, endothelial dysfunction, coagulation, tissue fibrosis, and cytomegalovirus (CMV) reactivation were assessed by ELISA-based assays and PCR using plasma obtained prior to the event. Results:VTE events were related to nadir CD4 cell count, lifetime history of multiple opportunistic infections, CMV disease, CMV viremia, immunological AIDS, active infection, and provocation (i.e., recent hospitalization, surgery, or trauma). VTE events were independently associated with increased plasma levels of P-selectin (P = 0.002), D-dimer (P = 0.01), and hyaluronic acid (P = 0.009) in a multivariate analysis. No significant differences in antiretroviral or interleukin-2 exposures, plasma HIV viremia, or other traditional risk factors were observed. Conclusion:Severe immunodeficiency, active infection, and provocation are associated with venous thromboembolic disease in HIV. Biomarkers of endothelial dysfunction, coagulation, and tissue fibrosis may help identify HIV-infected patients at elevated risk of VTE.

d-Dimer and CRP levels are elevated prior to antiretroviral treatment in patients who develop IRIS.

Biomarkers could be useful in evaluating immune reconstitution inflammatory syndrome (IRIS). A cohort of 45 HIV-1-infected, antiretroviral treatment (ART)-naive patients with baseline CD4 T cell counts <or=100 cells/microL who were started on ART, suppressed HIV-RNA to <50 copies/mL, and seen every 1-3 months for 1 year were retrospectively evaluated for suspected or confirmed IRIS. d-Dimer, C-reactive protein (CRP), and selected autoantibodies were analyzed at baseline, 1 and 3 months post-ART in cryopreserved plasma. Median differences between cases and controls were compared with Mann-Whitney and Fishers exact tests. Sixteen patients (35.6%) developed IRIS (median of 35 days post-ART initiation): unmasking=8, paradoxical=7, autoimmune=1. Pre-ART d-dimer and CRP were higher in IRIS cases versus controls (d-dimer: 0.89 mg/L versus 0.66 mg /L, p=0.037; CRP: 0.74 mg/L versus 0.39 mg/L, p=0.022), while d-dimer was higher in unmasking cases at IRIS onset (2.04 mg/L versus 0.36 mg /L, p=0.05). These biomarkers may be useful in identifying patients at risk for IRIS.

Decreases in IL-7 levels during antiretroviral treatment of HIV infection suggest a primary mechanism of receptor-mediated clearance.

IL-7 is essential for T-cell homeostasis. Elevated serum IL-7 levels in lymphopenic states, including HIV infection, are thought to be due to increased production by homeostatic feedback, decreased receptor-mediated clearance, or both. The goal of this study was to understand how immune reconstitution through antiretroviral therapy (ART) in HIV(+) patients affects IL-7 serum levels, expression of the IL-7 receptor (CD127), and T-cell cycling. Immunophenotypic analysis of T cells from 29 HIV(-) controls and 43 untreated HIV(+) patients (30 of whom were followed longitudinally for ≤ 24 months on ART) was performed. Restoration of both CD4(+) and CD8(+) T cells was driven by increases in CD127(+) naive and central memory T cells. CD4(+) T-cell subsets were not fully restored after 2 years of ART, whereas serum IL-7 levels normalized by 1 year of ART. Mathematical modeling indicated that changes in serum IL-7 levels could be accounted for by changes in the receptor concentration. These data suggest that T-cell restoration after ART in HIV infection is driven predominantly by CD127(+) cells and that decreases of serum IL-7 can be largely explained by improved CD127-mediated clearance.

Graves’ disease as immune reconstitution disease in Hiv-positive patients is associated with naive and primary thymic emigrant Cd4+ T-cell recovery

Objective:Immune restoration disease (IRD) can develop in HIV-infected patients following antiretroviral therapy (ART) initiation as unmasking or paradoxical worsening of opportunistic infections and, rarely, autoimmune phenomena. Although IRD usually occurs in the first months of ART during memory CD4+ T-cell recovery, Graves’ disease occurs as a distinctive late-onset IRD and its pathogenesis is unclear. Design:Seven patients who developed Graves’ disease following ART initiation from the primary HIV care clinic at the National Institutes of Health were retrospectively identified and each was matched with two HIV-infected controls based on age, sex, and baseline CD4+ T-cell count. Laboratory evaluations on stored cryopreserved samples were performed. Methods:Immunophenotyping of peripheral blood mononuclear cells (PBMCs), T-cell receptor excision circle (TREC) analysis in PBMCs, measurement of serum cytokines, and luciferase immunoprecipitation systems (LIPS) analysis for autoimmune antibodies were performed on stored samples for cases and controls at baseline and longitudinally following ART initiation. TSH/thyrotropin receptor (TSH-R) antibody testing was performed on serum from cases. Data were analyzed using nonparametric testing. Results:In comparison with controls, the proportion of naive CD4+ T cells increased significantly (P = 0.0027) in the Graves’ disease-IRD patients. TREC/106 PBMCs also increased significantly following ART in Graves’ disease-IRD patients compared with controls (P = 0.0071). Similarly, LIPS analysis demonstrated increases in nonthyroid-related autoantibody titers over time following ART in cases compared with controls. Conclusion:Our data suggest that Graves’ disease-IRD, in contrast to early-onset IRD, is associated with naive and primary thymic emigrant CD4+ T-cell recovery and inappropriate autoantibody production.

Interleukin-2 cycling causes transient increases in high-sensitivity C-reactive protein and D-dimer that are not associated with plasma HIV-RNA levels.

Objective:To determine the effects of interleukin (IL)-2 treatment on inflammatory and thrombotic biomarkers in chronically HIV-infected adults receiving antiretroviral therapy. Methods:Cryopreserved plasma was evaluated retrospectively for C-reactive protein (CRP) and D-dimer at baseline, end of an IL-2 cycle, and long-term follow up from two randomized, controlled trials: 57 IL-2-naive adults receiving either three to six cycles of IL-2 as well as antiretroviral therapy (nucleoside analogues) or antiretroviral therapy alone for 12 months, and 40 IL-2-experienced adults on highly active antiretroviral therapy who either interrupted or continued therapy for 6 months after a baseline IL-2 cycle. High-sensitivity CRP (hsCRP) was measured by immunonephelometry (detection limit 0.175 mg/l) and D-dimer by latex agglutination (detection limit 0.20 mg/l). Median within-group differences and pre and post-IL-2 changes between groups were assessed via nonparametric Wilcoxon signed-rank and Mann–Whitney U-tests. Spearmans rank test was used to assess correlations between changes in hsCRP, D-dimer, and HIV-RNA viral load. Results:Significant increases in hsCRP (study 1: 138.6 mg/l; study 2: 58.9 mg/l) and D-dimer (study 1: 3.1 mg/l; study 2: 0.4 mg/l, all P < 0.0001) occurred by the end of the initial IL-2 cycle, returning to baseline by the end of study. No correlations were seen between changes in hsCRP or D-dimer and HIV-RNA, CD4 T-cell count, or proliferation (Ki67 expression). No thrombotic or cardiovascular serious adverse events occurred during these study periods. Conclusion:IL-2 dosing caused transient increases in plasma hsCRP and D-dimer levels, regardless of HIV-RNA viral load, suggesting the possibility of increased risk for thrombotic events.

Inferiority of Il-2 alone versus Il-2 with Haart in maintaining Cd4 T cell counts during Haart interruption: a randomized controlled trial

Objective:To evaluate whether interleukin (IL)-2 in patients with chronic HIV infection can maintain CD4 T cell counts during 6 months of HAART interruption. Design:Prospective, randomized, controlled, open-label phase II noninferiority trial comparing IL-2 with HAART interruption or continuous HAART. Methods:Forty-one IL-2-experienced (three or more prior cycles) HIV-1-infected adults with CD4 cell count at least 500 cells/μl were randomized in the ratio 2: 1 to interrupted (I = 27) or continuous (C = 14) HAART for 6 months following an initial IL-2 cycle. Subsequent IL-2 cycles were triggered by CD4 T cell counts less than 90% of baseline. Immune, metabolic, and quality of life indices were compared (Mann–Whitney and Fishers exact tests), defining noninferiority as a percentage difference (C– I) in treatment success (CD4 T cells ≥90% of baseline at 6 months) with a 95% confidence interval (CI) lower limit greater than −20%. Results:Demographic and immune parameters were similar between the groups at baseline. Median CD4 T cell count, HIV viral load, and treatment success differed significantly at 6 months (I: 866 cells/μl, 39 389 copies/ml, 48.1%; C: 1246 cells/μl, <50 copies/ml, 92.3%; P ≤ 0.001). Group I was inferior to C (% difference = −44.2%; 95% CI: −64.2%, −11.2%; P = 0.013). Minor statistically significant differences in HgbA1c and energy level occurred at 6 months (I > C). Following HAART interruption, single cases of acute retroviral syndrome, secondary syphilis, non-Hodgkins lymphoma, and Kaposis sarcoma recurrence were observed. Conclusion:IL-2 alone was inferior to IL-2 with HAART in maintaining baseline CD4 T cell counts. HAART interruption had a small impact on metabolic parameters and quality of life.

Administration of interleukin-7 increases CD4 T cells in idiopathic CD4 lymphocytopenia.

Idiopathic CD4 lymphopenia (ICL) is a rare syndrome defined by low CD4 T-cell counts (<300/µL) without evidence of HIV infection or other known cause of immunodeficiency. ICL confers an increased risk of opportunistic infections and has no established treatment. Interleukin-7 (IL-7) is fundamental for thymopoiesis, T-cell homeostasis, and survival of mature T cells, which provides a rationale for its potential use as an immunotherapeutic agent for ICL. We performed an open-label phase 1/2A dose-escalation trial of 3 subcutaneous doses of recombinant human IL-7 (rhIL-7) per week in patients with ICL who were at risk of disease progression. The primary objectives of the study were to assess safety and the immunomodulatory effects of rhIL-7 in ICL patients. Injection site reactions were the most frequently reported adverse events. One patient experienced a hypersensitivity reaction and developed non-neutralizing anti-IL-7 antibodies. Patients with autoimmune diseases that required systemic therapy at screening were excluded from the study; however, 1 participant developed systemic lupus erythematosus while on study and was excluded from further rhIL-7 dosing. Quantitatively, rhIL-7 led to an increase in the number of circulating CD4 and CD8 T cells and tissue-resident CD3 T cells in the gut mucosa and bone marrow. Functionally, these T cells were capable of producing cytokines after mitogenic stimulation. rhIL-7 was well tolerated at biologically active doses and may represent a promising therapeutic intervention in ICL. This trial was registered at www.clinicaltrials.gov as #NCT00839436.