Decreased PD-1 Expression on CD8 Lymphocyte Subsets and Increase in CD8 Tscm Cells in Children with HIV Receiving Raltegravir

Abstract

We investigated the effect of combination antiretroviral therapy (cART) on immune recovery, particularly on the percentages of PD-1-positive cells within the major leukocyte subsets. Cryopreserved peripheral blood mononuclear cells and plasma samples collected longitudinally from a subset of 13 children and adolescents (between 9.7 and 18.2 years old) who were enrolled in the International Maternal Pediatric Adolescent AIDS Clinical Trials (IMPAACT) P1066 were used for this study. Immunophenotyping by flow cytometry was performed to determine the effect of raltegravir-containing cART regimen on the distribution of leukocyte populations, on the expression of PD-1 on T cell subpopulations, and on the expression of well-established markers of T cell activation (CD38 and HLA-DR) on CD8 T cells. C reactive protein (CRP), lipopolysaccharide (LPS), IL-6, and soluble CD163 were assayed in plasma samples by an enzyme-linked immunosorbent assay. Plasma viral loads were decreased in all subjects (by an average of 2.9 log units). The cART regimen, including raltegravir, induced changes in CD8 T cell subsets, consistent with an effective antiretroviral outcome and improved immunologic status, including increased percentages of CD8 stem cell memory T cells (Tscm). The percentages of CD8 PD-1-positive cells decreased significantly as compared with baseline levels. Among the proinflammatory markers measured in plasma, sCD163 showed a decline that was associated with cART. cART therapy, including raltegravir, over 48 weeks in children is associated with immune restoration, consistent with effective antiretroviral therapy, namely decreased percentages of PD-1⁺ CD8⁺ T cells, an increase in CD8 Tscm cells, and decreased levels of sCD163.

Keywords: HIV; T cell immunity; antiretroviral therapy; clinical trials for antivirals; integrase inhibitors/drug discovery.

FIG. 1.

Gating strategy for identifying T cell subpopulations and the expression pf PD-1. PBMCs were separated from blood samples by centrifugation on Ficoll, and cells were cryopreserved in liquid nitrogen until flow cytometry analysis was performed. Viable T cells were identified as CD3⁺ and L/D Blue-dim cells (a), and subpopulations of CD4⁺ and CD8⁺ T cells were further divided into central memory (Tcm), effector memory (Tem), and effector (Te) T cells based on the expression of CCR7 (CD197) and CD45RO (b). Naïve and stem cell-like memory T cells (Tscm) were gated based on their high expression of CD27 and low SSC values (c). Tscm cells were distinguished from naïve (Tn) T cells based on higher levels of FasR (CD95) expression (d). Histograms were used for gating PD-1-positive cells in each of the T cell subpopulations described (e–h). PBMC, peripheral blood mononuclear cell.

FIG. 2.

Viral load and percentages of CD4⁺ and CD8⁺ T cells. Viral loads (a), CD4 (b) and CD8 (c) T cell percentages in fresh whole blood samples were measured as described under the Materials and Methods section. Individual data points connected by solid lines are shown for each subject; blue dotted lines connect median values. Based on Bonferroni correction for three familywise comparisons, the level of significance for p-values was set to .0167. Correlations between CD4⁺ (d) and CD8⁺ (e) T cell percentages measured in fresh versus cryopreserved samples were significant for both subpopulations of CD4⁺ and CD8⁺ T cells (p < 10⁻⁶). (*p < 0.0167; **p < 10⁻³; ***p < 10⁻⁶).

FIG. 3.

T cell subpopulations in cryopreserved PBMCs. Percentages of naïve (Tn), central memory (Tcm), effector memory (Tem), effector (Te), and stem cell-like memory (Tscm) cells were determined in CD4⁺ (a–e) and CD8+ (f–j) T cells in cryopreserved PBMCs. Individual data points connected by solid lines are shown for each subject; blue dotted lines connect median values. The level of significance for p-values was set to .0167 based on Bonferroni correction for three familywise comparisons.

FIG. 4.

Expression of PD-1 on T cells. Percentages of PD-1-positive T cells, total, naïve (Tn), central memory (Tcm), effector memory (Tem), effector (Te), and stem cell-like memory (Tscm) cells were determined in cryopreserved PBMCs for CD4⁺ (a–f) and CD8⁺ (g–l) T cells. Individual data points connected by solid lines are shown for each subject; blue dotted lines connect median values. Based on Bonferroni correction for three familywise comparisons, the level of significance for p-values was set to .0167.

FIG. 5.

Markers of immune activation on CD8 T cells and inflammation markers in plasma. After the initiation of cART, the percentages of CD8⁺-T cells positive for CD38 or dual positive for HLA-DR and CD38 significantly decreased at 8 weeks and remained lower than at the beginning of the study (a, b). Correlations between viral loads and CD8⁺ CD38⁺ (c) or CD8⁺ CD38⁺ HLA-DR⁺ (d) are shown. Individual data points connected by solid lines are shown for each patient; dotted lines in (a) and (b) connect median values. Plasma levels of CRP (e), IL-6 (f), LPS (g), and soluble CD163 (h) were measured as described under the Materials and Methods section. Individual data points connected by solid lines are shown for each subject; dotted lines connect median values. The level of significance for p-values was set to .0167. cART, combination antiretroviral therapy; CRP, C reactive protein; LPS, lipopolysaccharide.