Early antiretroviral therapy-treated perinatally HIV-infected seronegative children demonstrate distinct long-term persistence of HIV-specific T-cell and B-cell memory

Abstract

Objective: To investigate long-term persistence of HIV-specific lymphocyte immunity in perinatally HIV-infected children treated within the first year of life.

Design: Twenty perinatally HIV-infected children who received ART therapy within the first year of life (early treated) and with stable viral control (>5 years) were grouped according to their serological response to HIV.

Methods: Western blot analysis and ELISA defined 14 HIV-seropositive and six seronegative patients. Frequencies of gp140-specific T-cell and B-cell, and T-cell cytokine production were quantified by flow cytometry in both seronegatives and seropositives. Transcriptional signatures in purified gp140-specific B-cell subsets, in response to in-vitro stimulation with HIV peptides was evaluated by multiplex RT-PCR.

Results: Gp140-specific T cells and B cells persist at similar levels in both groups. A higher production of IL-21 in gp140-specific T cells was found in seropositives vs. seronegatives (P = 0.003). Gene expression in switched IgM-IgD- gp140-specific memory B cells after stimulation with HIV peptides in vitro demonstrated a differential expression of genes involved in signal transduction and activation after BCR/TLR triggering and B-cell activation. Genes relating to antibody production (PRDM1) and T-B cognate stimulation (CXCR4, IL21R) were differentially induced after in-vitro stimulation in seronegatives vs. seropositives suggesting a truncated process of B-cell maturation.

Conclusion: HIV-specific memory B and T cells persist in early treated regardless their serological status. Seronegatives and seropositives are distinguished by gp140-specific T-cell function and by distinct transcriptional signatures of gp140-specific B cells after in-vitro stimulation, presumably because of a different antigen exposure. Such qualitative insights may inform future immunotherapeutic interventions.

Figure 1.. Gp140-specific T cells.

Left side panel of (A) shows a representative gate used to identify gp140-specific T cell subsets according to the expression of CD40L after in vitro stimulation. Right panel of (A) depicts frequencies of gp140-specific T cell gated on live CD3+CD4+ T cells. (B) Representative gate of gp140-specific T cells distribution within maturational subsets according to the expression of CD27 and CD45RO. (C) Contingency plot representing the median values of gp140-specific T cells within every T cell subset. (D) Scatter dot plot shows gp140-specific peripheral T follicular cells. Mann-Whitney test was used for all comparisons. SEB Staphylococcal Enterotoxin B.

Figure 2.. Cytokine production in gp140-specific T cells.

(A) Scatter dot plot representing intracellular staining measurment for IFN-γ, IL-2, TNFα, IL-21 production after in-vitro stimulation in gp140-specific CD4+ T cells. (B) SPICE program was used for Boolean analysis looking at the production of IFN-γ, IL-2, IL-21 and TNFα in gp140-specific CD4+ T cells in SN and SP. The bar graph represents the median frequency of all the Boolean subsets. (C) Each color in the pie charts corresponds to a specific combination of markers indicated at the bottom of the bar graph in (B), while every arc indicates the presence of that specific cytokine. (D) Permutation test was performed through SPICE program. Mann-Whitney was used for comparisons. *indicates p value≤0.05

Figure 3.. Gp140-specific B cell distribution in total CD19+ cells and among maturational subsets.

Left side panel in (A) depicts representative gates of gp140-specific staining in B cells with negative controls. Scatter dot plot on the right side of panel (A) represents the percentage of gp140-specific cells among total CD19+ B cells. (B) On Left side, demonstrative gate used to identify the surface expression of CD21 and CD27 on CD19+ B cells. On the right of panel (B) the scatter dot plot shows the frequencies of gp140-specific B cells among CD27 and CD21 subsets. Statistical analyses between the subsets were determined by Mann-Whitney test. (C-D) Median values of gp140-specific B cell distribution among CD27 and CD21 subsets were used to build contingency plots showed in the pie charts. Unpaired t-test or Mann-Whitney were used to compare normally or not-normally distributed data respectively. Abbreviations: HIV+, HIV positive patient; HC, Healthy Control; FMO, Fluorescence Minus One; AM, activated memory; REM, resting memory; IM, intermediate memory; TLM, tissue-like memory. * indicates statistical differences among REM cells and the other B cell populations.

Figure 4.. Gene expression analysis in gp140-specific B cell subsets.

(A) Volcano plot representing DEGs between SP and SN, with (red dots) or without (blue dots) in-vitro stimulation in sorted IgM-gp140-specific (triangle dots) and IgM+gp140-specific B cells (square dots). Only DEGs with a p value< 0.05 are shown in the volcano plot. (B) Column heatmaps demonstrate differentially induced Genes (DIGs) between stimulated and unstimulated samples in IgM-gp140-specific B cells (left side panel) and IgM+gp140-specific B cells (right side panel). Values depicted in this panel were calculated by the difference in the averages in Et between the 2 experimental conditions for both groups and displayed in the heatmap. Only genes resulte d significantly induced with a p value<0,05 at paired analysis are showed in the heatmap. Paired Wilcoxon test for not-normally distributed data and paired t-test for normally distributed data were used for comparisons. (C) A correlation matrix is depicted with color heatmap showing r values derived from Spearman and Pearson correlation analysis, respectively used for normally and not-normally distributed data. A color was assigned only to r values resulting from significant correlations between data with a p value<0.05. An r value of 0 (white) was assigned to p value > 0.05.