CD32+CD4+ memory T cells are enriched for total HIV-1 DNA in tissues from humanized mice

Abstract

CD32 has raised conflicting results as a putative marker of the HIV-1 reservoir. We measured CD32 expression in tissues from viremic and virally suppressed humanized mice treated relatively early or late after HIV-1 infection with combined antiretroviral therapy. CD32 was expressed in a small fraction of the memory CD4⁺ T-cell subsets from different tissues in viremic and aviremic mice, regardless of treatment initiation time. CD32⁺ memory CD4⁺ T cells were enriched in cell-associated (CA) HIV-1 DNA but not in CA HIV-1 RNA as compared to the CD32^-CD4⁺ fraction. Using multidimensional reduction analysis, several memory CD4⁺CD32⁺ T-cell clusters were identified expressing HLA-DR, TIGIT, or PD-1. Importantly, although tissue-resident CD32⁺CD4⁺ memory cells were enriched with translation-competent reservoirs, most of it was detected in memory CD32^-CD4⁺ T cells. Our findings support that CD32 labels highly activated/exhausted memory CD4⁺ T-cell subsets that contain only a small proportion of the translation-competent reservoir.

Keywords: Cell Biology; Immunology; Molecular Biology; Virology.

Graphical abstract

Figure 1

Design of the Study One hundred twenty humanized NSG mice were infected with HIV-1 JRCSF. At week one post-infection, the cART regimen was initiated (n = 58) in the early treated group and sustained for two months. Late treatment was started at seven weeks post-infection (n = 42) and continued for two months. In each group, a number of animals were sacrificed at the end of treatment, corresponding to week nine for early treated (n = 43) and to week fifteen for late treated (n = 34) animals. The untreated group (n = 20) never received cART and was euthanized correspondingly to the sacrifices of both treatment groups (week nine for early treated and week fifteen for late treated mice). Viral rebound after treatment interruption was monitored for the remaining early (n = 15) and late treated (n = 8) animals for a duration of six weeks or longer if animals were not detectable for HIV-1 viral load.

Figure 2

Early Treatment Significantly Decreased Time to Viral Suppression (A and B) (A) Plasma viral load of early treated (n = 43), late treated (n = 34), and untreated (n = 20) mice and (B) percentage of CD4⁺ T cells were determined every two weeks in peripheral blood. Background coloring of graphs highlights treatment periods: light green for early treatment, violet for late treatment, dark green is obtained by a two-week overlap (week seven to week nine) of early and late treatment periods (figure A and B). (C and D) (C) Time to viral suppression (median two and four weeks, respectively) (p < 0.0001) and (D) viral rebound (p = 0.107) for the early and late group. Statistical analysis was executed using Mantel COX rank test. (∗∗∗p < 0.005; ns, nonsignificant test p > 0.05).

Figure 3

cART Significantly Decreased Total HIV-1 DNA in Peripheral Organs and HIV-1-Related Immune Exhaustion and Activation on Human T Cells (A) Total HIV-1 DNA quantified from the bone marrow, lymph nodes, and spleen is represented for early treated (n = 9), late treated (n = 11), and untreated mice (n = 6). Percentages of cells expressing the respective markers PD-1, TIGIT, and HLA-DR on CD4+ T cells (B) and CD8+ T cells (C) extracted from the spleen at experimental endpoint (median age of nine months, eight weeks total cART duration for both treatment groups) are depicted for early treated mice (n = 17), late treated mice (n = 23), and untreated mice (n = 16). Statistical testing was performed using non-parametric Kruskal-Wallis tests with multiple comparison Dunn's correction. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗p < 0.001, ns, nonsignificant test, p > 0.05).

Figure 4

CD32 Is Expressed on Cell Fragments Attached to CD4⁺ T Cells with No Signs of T-B Cell Trogocytosis To assess whether the expression of CD32 on CD4⁺ T cell comes from T cells or T-B cell conjugates, imaging flow cytometry was performed on sorted CD32⁺CD4⁺ T cells (A, C, E, and F) or sorted CD19⁺ B cells (B and D) isolated from the spleen of cART suppressed HIV-1-infected humanized mice (A, B, E) or peripheral blood mononuclear cells (PBMCs) from cART suppressed HIV-1-infected patients (C, D, and F). Flow sorted B cells from humanized mice or patients were stained, acquired, and represented with the same settings and experimental procedures and,hence, serve as reference population with regard to CD32, CD19 ,and CD20 signal on images (B, D). Overlay shots of CD4 and CD32 signals of splenocytes from a humanized mouse (E) and of PBMCs from one HIV-1-infected patient (F).

Figure 5

Total HIV-1 DNA Is Higher in CD32⁺CD4⁺ Memory T Cells in the Spleen and the Bone Marrow Total HIV-1 DNA from flow sorted CD3⁺CD4⁺CD45RA⁻ T cells according to CD32 expression in bone marrow (A) and spleen (B) in virally suppressed mice (mixed from early and late treatment conditions) (bone marrow n = 11, spleen n = 10) depicted as aligned dot plots of individual measurements (CD32- in black circles and CD32 + in red squares). The flow gating strategy is depicted in Figure S3. (C) HIV-1 CA RNA in flow sorted CD4⁺ memory T cells (CD32^- (n = 9) CD32⁺ (n = 5)) from virally suppressed mice (mixed from early and late treatment conditions). Statistical testing was done using non-parametric paired Wilcoxon rank tests (A and B) or non-paired tests (C). Exact p values for HIV-1 DNA: bone marrow, 0.0278; spleen, 0.0039, (∗p < 0.05. ∗∗p < 0.01).

Figure 6

CD32 Expression Was Associated with High Expression Levels of Activation/Exhaustion Markers and Did Not Correlate with HIV-1 DNA (A–C) Expression of HLA-DR (A), PD-1 (B), and TIGIT (C) in CD32-positive and negative CD4⁺ memory T cells from the spleen. (D) Expression level of three combined activation markers (HLA-DR, CD38, and CD69) in flow sorted CD4⁺ memory T cells according to their CD32 expression. Geometric mean fluorescence intensity (GMFI) of activation markers plotted against the GMFI of CD32 from virally suppressed mice (mixed early and late treatment conditions). (E) Spearman correlation with fitted linear regression line (p value: 0.0004, R value: 0.6872). Statistical testing by non-parametric Mann-Whitney or Kruskal-Wallis tests with multiple comparison Dunn's correction. Two-tailed spearman correlation (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001, ns, nonsignificant test, p > 0.05).

Figure 7

High-Dimensional Analyses Highlighted Different Activated/Exhausted Clusters Expressing CD32 in Memory CD4 T Cells from the Spleen of Early and Late Treated Mice with cART (A) FlowSOM hierarchical tree colored according to metaclusters. (B) Metaclusters were annotated according to the heatmap shown in. Two-way hierarchical clustering of the 49 clusters defined by FlowSOM and the median expression of the 9 markers indicated on the right of the heatmap. Median expression across the three groups of mice is shown. Three viremic untreated mice (untreated mice, UT)) were kept four weeks without treatment and four viremic mice were kept eight weeks; twenty-two mice were virally suppressed by receiving early cART treatment (n = 12, ET mice) or receiving late cART treatment (n = 10, LT). The clusters significantly different (p < 0.05) between four weeks and eight weeks of treatment are depicted on the bottom of the heatmap. A significant increase or decrease is indicated in red or cyan, respectively.

Figure 8

P24 Production after Latency Reversal Is Predominantly Found in CD32⁻CD4⁺ Memory T Cells and Not in the CD32⁺ Fraction (A) Representative fluorescence activated cell sorting (FACS) plots of p24 staining in an untreated viremic mouse after five weeks of HIV-1 infection and one ART-treated mouse, as well as a non-infected control mouse after PMA/ionomycin reactivation. The strict gating assured no false positive signal. (B) Intracellular p24 staining after PMA/ionomycin activation in ART-treated animals (n = 4) revealed that the highest proportion of productive cells is CD32^- and not CD32 ⁺. (C) The frequency of p24 producing cells is higher within CD32⁺CD4⁺ memory T cells than in CD32^- cells. (D) Expression of HLA-DR, CD38, and PD-1 at the surface of the reactivated cells producing p24 from graph B. Statistics were performed using non-parametric testing. (∗p < 0.05, ns, nonsignificant test, p > 0.05).