Restoration of CMV-Specific-CD4 T Cells with ART Occurs Early and Is Greater in Those with More Advanced Immunodeficiency

<div><p>Objectives</p><p>Restoration of Cytomegalovirus-specific-CD4 T cell (CMV-Sp-CD4) responses partly accounts for the reduction of CMV-disease with antiretroviral-therapy (ART), but CMV-Sp-CD4 may also drive immune activation and immunosenescence. This study characterized the dynamics of CMV-Sp-CD4 after ART initiation and explored associations with CD4 T cell recovery as well as frequency of naïve CD4 T cells at week 96.</p> <p>Methods</p><p>Fifty HIV-infected, ART-naïve Thai adults with CD4 T cell count ≤350cells/µL and starting ART were evaluated over 96 weeks (<a href="http://clinicaltrials.gov" target="_blank">ClinicalTrials.gov</a> identifier NCT01296373). CMV-Sp-CD4 was detected by co-expression of CD25/CD134 by flow cytometry after CMV-antigen stimulation.</p> <p>Results</p><p>All subjects were CMV sero-positive, 4 had quantifiable CMV-DNA (range 2.3-3.9 log₁₀ copies/mL) at baseline but none had clinically apparent CMV-disease. Baseline CMV-Sp-CD4 response was positive in 40 subjects. Those with CD4 T cell count <100cells/µL were less likely to have positive baseline CMV-Sp-CD4 response (P=0.003). Positive baseline CMV-Sp-CD4 response was associated with reduced odds of quantifiable CMV-DNA (P=0.022). Mean CD4 T cell increase at week 96 was 213 cells/µL. This was associated positively with baseline HIV-VL (P=0.001) and negatively with age (P=0.003). The frequency of CMV-Sp-CD4 increased at week 4 (P=0.008), then declined. Those with lower baseline CMV-Sp-CD4 (P=0.009) or CDC category C (P<0.001) had greater increases in CMV-Sp-CD4 at week 4. At week 96, CD4 T cell count was positively (P<0.001) and the frequency of CMV-Sp-CD4 was negatively (P=0.001) associated with the percentage of naïve CD4 T cells.</p> <p>Conclusions</p><p>Increases in CMV-Sp-CD4 with ART occurred early and were greater in those with more advanced immunodeficiency. The frequency of CMV-Sp-CD4 was associated with reduced naïve CD4 T cells, a marker associated with immunosenescence.</p> </div

Changes in cerebral function parameters over 3 years.

<p><i>SD = standard deviation, NAA/Cr = N-acetyl-aspartate/Creatine, Cho/Cr = Choline/Cr, mI/Cr = myo-Inositol/Cr. P</i>-values <0.05 shown in bold.</p><p>Changes in cerebral function parameters over 3 years.</p

Dynamics of changes in cerebral metabolite ratios in the Frontal White matter over 3 years.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118608#pone.0118608.g001" target="_blank">Fig. 1A</a>: Mean changes in N-acetyl-aspartate/Creatine ratio; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118608#pone.0118608.g001" target="_blank">Fig. 1B</a>: Mean changes in Choline/Creatine ratio and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118608#pone.0118608.g001" target="_blank">Fig. 1C</a>: Mean changes in myo-Inositol/Creatine ratio (error bars represent 1 standard error).</p

Gating strategies for CMV-SP-CD4 T cells.

<p>All flow cytometry plots are from a single subject of the RESTORE study. Lymphocytes were identified using forward and side scatter (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077479#pone-0077479-g001" target="_blank">Figure 1A</a>), followed by gating on CD4+ T cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077479#pone-0077479-g001" target="_blank">Figure 1B</a>). Gates for CD25+ and CD134+ cells were placed based on comparison with negative control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077479#pone-0077479-g001" target="_blank">Figure 1C</a>) and PHA positive control (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077479#pone-0077479-g001" target="_blank">Figure 1D</a>) to include cells highly co-expressing CD25 and CD134. A representative example of the dynamics of responses to CMV antigens over 96 weeks of follow-up in a RESTORE subject (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077479#pone-0077479-g001" target="_blank">Figure 1E-K</a>).</p

Baseline positive and persistent positive biomarkers predicting incident anal histologic HSIL at subsequent visits among those who were free of disease at baseline.

<p>HSIL, high-grade squamous intraepithelial lesion; HPV, human papillomavirus; CI, confidence interval.</p><p>HSIL included anal intraepithelial neoplasia (AIN) 2 or AIN 3 on histology.</p>a<p>High-risk HPV DNA included HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68.</p>b<p>E6/E7 mRNA positivity was defined as greater than or equal to 2% of cells which had E6/E7 mRNA over-expression in the sample.</p>c<p>p16 immunocytochemistry was considered positive if there was a presence of cells with cytoplasmic and/or nuclear staining.</p>d<p>Multivariate model adjusted for HIV status and low-grade squamous intraepithelial lesion at baseline.</p

Virologic response at Week 96, by randomised arm, study population and screening strata.

<p>RAL = lopinavir/ritonavir+raltegravir Control = lopinavir/ritonavir+2/3 nucleoside/nucleotide reverse transcriptase inhibitors. *grey circle = screening plasma viral load strata, P interaction = 0.81, bars are 95%confidence intervals (CI).</p

Clinical outcomes by randomised arm.

<p>*Grade 3/4 adverse events summarised by MedDRA System Organ Class which were reported by 5 or more people</p><p>py: person years</p><p>Clinical outcomes by randomised arm.</p

Baseline positivity of anal cytology and biomarkers by histologic anal diagnosis.

<p>P values correspond to a comparison of the proportion of subjects with no SIL or LSIL versus those with HSIL, who were identified by each biomarker.</p><p>SIL, squamous intraepithelial lesion; LSIL, low-grade squamous intraepithelial lesion; HSIL, high-grade squamous intraepithelial lesion; HPV, human papillomavirus.</p><p>LSIL group included MSM with anal intraepithelial neoplasia (AIN) 1 on histology. HSIL group included MSM with AIN 2 or AIN 3 on histology. No SIL group included MSM without LSIL or HSIL.</p

Mean change from baseline in CD4+ T cells/mm³ over 96 weeks by randomised arm.

<p>RAL = lopinavir/ritonavir+raltegravir Control = lopinavir/ritonavir+2/3 nucleoside/nucleotide reverse transcriptase inhibitors. (Control- - - RAL ---, bars indicate standard deviation).</p

Absolute mean cholesterol fractions and ratio over 96 weeks by randomised arm.

<p>(A) total cholesterol, (B) high density lipoprotein (HDL) cholesterol, (C) low density lipoprotein (LDL) cholesterol, (D) Total:HDL cholesterol ratio. RAL = lopinavir/ritonavir+raltegravir Control = lopinavir/ritonavir+2/3 nucleoside/nucleotide reverse transcriptase inhibitors. (Control - - - RAL ---, bars indicate standard deviation).</p