Dynamics of Immune Reconstitution and Activation Markers in HIV+ Treatment-Naïve Patients Treated with Raltegravir, Tenofovir Disoproxil Fumarate and Emtricitabine

Background: The dynamics of CD4+ T cell reconstitution and changes in immune activation and inflammation in HIV-1 disease following initiation of antiretroviral therapy (ART) are incompletely defined and their underlying mechanisms poorly understood. Methods: Thirty-nine treatment-naïve patients were treated with raltegravir, tenofovir DF and emtricitabine. Immunologic and inflammatory indices were examined in persons with sustained virologic control during 48 weeks of therapy. Results: Initiation of ART increased CD4+ T cell numbers and decreased activation and cell cycle entry among CD4+ and CD8+ T cell subsets, and attenuated markers of coagulation (D-dimer levels) and inflammation (IL-6 and TNFr1). These indices decayed at different rates and almost all remained elevated above levels measured in HIV-seronegatives through 48 weeks of viral control. Greater first and second phase CD4+ T cell restoration was related to lower T cell activation and cell cycling at baseline, to their decay with treatment, and to baseline levels of selected inflammatory indices, but less so to their changes on therapy. Conclusions: ART initiation results in dynamic changes in viral replication, T cell restoration, and indices of immune activation, inflammation, and coagulation. These findings suggest that determinants of T cell activation/cycling and inflammation/coagulation may have distinguishable impact on immune homeostasis. Trial Registration Clinicaltrials.gov NCT0066097

Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions

Methotrexate Inhibits T Cell Proliferation but Not Inflammatory Cytokine Expression to Modulate Immunity in People Living With HIV.

Inflammation associated with increased risk of comorbidities persists in people living with HIV (PWH) on combination antiretroviral therapy (ART). A recent placebo-controlled trial of low-dose methotrexate (MTX) in PWH found that numbers of total CD4 and CD8 T cells decreased in the low-dose MTX arm. In this report we analyzed T cell phenotypes and additional plasma inflammatory indices in samples from the trial. We found that cycling (Ki67+) T cells lacking Bcl-2 were reduced by MTX but plasma inflammatory cytokines were largely unaffected. In a series of in vitro experiments to further investigate the mechanisms of MTX activity, we found that MTX did not inhibit effector cytokine production but inhibited T cell proliferation downstream of mTOR activation, mitochondrial function, and cell cycle entry. This inhibitory effect was reversible with folinic acid, suggesting low-dose MTX exerts anti-inflammatory effects in vivo in PWH largely by blocking T cell proliferation via dihydrofolate reductase inhibition, yet daily administration of folic acid did not rescue this effect in trial participants. Our findings identify the main mechanism of action of this widely used anti-inflammatory medicine in PWH and may provide insight into how MTX works in the setting of other inflammatory conditions

Gut Epithelial Barrier Dysfunction and Innate Immune Activation Predict Mortality in Treated HIV Infection

Background. While inflammation predicts mortality in treated human immunodeficiency virus (HIV) infection, the prognostic significance of gut barrier dysfunction and phenotypic T-cell markers remains unclear. Methods. We assessed immunologic predictors of mortality in a case-control study within the Longitudinal Study of the Ocular Complications of AIDS (LSOCA), using conditional logistic regression. Sixty-four case patients who died within 12 months of treatment-mediated viral suppression were each matched to 2 control individuals (total number of controls, 128) by duration of antiretroviral therapy–mediated viral suppression, nadir CD4(+) T-cell count, age, sex, and prior cytomegalovirus (CMV) retinitis. A similar secondary analysis was conducted in the SCOPE cohort, which had participants with less advanced immunodeficiency. Results. Plasma gut epithelial barrier integrity markers (intestinal fatty acid binding protein and zonulin-1 levels), soluble CD14 level, kynurenine/tryptophan ratio, soluble tumor necrosis factor receptor 1 level, high-sensitivity C-reactive protein level, and D-dimer level all strongly predicted mortality, even after adjustment for proximal CD4(+) T-cell count (all P ≤ .001). A higher percentage of CD38(+)HLA-DR(+) cells in the CD8(+) T-cell population was a predictor of mortality before (P = .031) but not after (P = .10) adjustment for proximal CD4(+) T-cell count. Frequencies of senescent (defined as CD28(−)CD57(+) cells), exhausted (defined as PD1(+) cells), naive, and CMV-specific T cells did not predict mortality. Conclusions. Gut epithelial barrier dysfunction, innate immune activation, inflammation, and coagulation—but not T-cell activation, senescence, and exhaustion—independently predict mortality in individuals with treated HIV infection with a history of AIDS and are viable targets for interventions

Proportions of activated (CD38+HLA-DR+) and cycling (Ki67+) CD4+ and CD8+ T cells decreased following initiation of ART.

<div><p>Among both A) CD4+ and B) CD8+ T cell populations, initiation of raltegravir plus emtricitabine/tenofovir resulted in a significant decrease from baseline in the proportion of activated cells by 2 days and 7 days (p= 0.05 and 0.015, respectively). By week 48, the proportions of activated CD4+ and CD8+ T cells in these patients did not approach the proportions measured in healthy controls. Also among both C) CD4+ and D) CD8+ T cell populations, initiation of ART significantly decreased the proportions of Ki67+ cells by week 8 and by day 7 respectively (p<0.001 and p=0.028). E) Naïve CD4+ T cells (CD4+,CD45RA+, CCR7+) and F) CM CD4+ T cells (CD4+,CD45RA-, CCR7+) that express Ki67 were significantly reduced from baseline by week 8 (p=0.005 and p=0.001, respectively). In all four T cell subsets, proportions of Ki67+ cells did not approach the proportions seen in healthy controls by week 48 of the study. Symbols used in the figure:</p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div

Markers of inflammation and coagulation are reduced following initiation of ART.

<div><p>Plasma samples were thawed and levels of A) interleukin-6 (IL-6) B) tumor necrosis factor receptor type 1 (TNFr1) C) D-dimers D) Lipopolysaccharide (LPS) and E) CD14 (sCD14) were measured. Initiation of ART resulted in significant decreases from baseline in plasma levels of IL-6 and TNFr1 by week 4 (p=0.002 and p=0.038) D-dimer levels were significantly reduced by day 7 (p=0.031). Levels of sCD14 were significantly reduced by day 2 following initiation of therapy (p<0.001). LPS levels were significantly reduced from baseline 24 weeks after initiation of ART (p<0.001). None of these markers, except for IL-6, consistently reached the levels seen in healthy controls by the end of the study. Symbols used in the figure: </p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div

Initiation of antiretroviral therapy (ART) results in an increase in the number of CD4+T cells and a decrease in the number of CD8+ T cells.

<div><p>Absolute CD4+ and CD8+ T cell counts were obtained in real time on fresh whole blood samples. Lymphocytes were identified by flow cytometry based on size and granularity; T cell subsets were identified by positive expression of CD4 or CD8. The numbers of circulating A) CD4+ and B) CD8+ T cells within this HIV-1 infected patient population changed significantly from baseline by day 2 (p<0.001 and p<0.002, respectively), but did not reach levels seen in healthy controls within 48 weeks of ART treatment. C) naïve (CD45RA+ CCR7+) and D) central memory (CM, CD45RA-CCR7+) CD4+ T cell numbers increased significantly by day 2 (p<0.001) and day 7 (p=0.001) respectively. Symbols used in the figure:</p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div

Circulating human CD4 and CD8 T cells do not have large intracellular pools of CCR5

CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells