CD32+ and PD-1+ Lymph Node CD4 T Cells Support Persistent HIV-1 Transcription in Treated Aviremic Individuals

Abstract

A recent study conducted in blood has proposed CD32 as the marker identifying the "elusive" HIV reservoir. We have investigated the distribution of CD32⁺ CD4 T cells in blood and lymph nodes (LNs) of HIV-1-uninfected subjects and viremic untreated and long-term-treated HIV-1-infected individuals and their relationship with PD-1⁺ CD4 T cells. The frequency of CD32⁺ CD4 T cells was increased in viremic compared to treated individuals in LNs, and a large proportion (up to 50%) of CD32⁺ cells coexpressed PD-1 and were enriched within T follicular helper (Tfh) cells. We next investigated the role of LN CD32⁺ CD4 T cells in the HIV reservoir. Total HIV DNA was enriched in CD32⁺ and PD-1⁺ CD4 T cells compared to CD32^- and PD-1^- cells in both viremic and treated individuals, but there was no difference between CD32⁺ and PD-1⁺ cells. There was no enrichment of latently infected cells with inducible HIV-1 in CD32⁺ versus PD-1⁺ cells in antiretroviral therapy (ART)-treated individuals. HIV-1 transcription was then analyzed in LN memory CD4 T cell populations sorted on the basis of CD32 and PD-1 expression. CD32⁺ PD-1⁺ CD4 T cells were significantly enriched in cell-associated HIV RNA compared to CD32^- PD-1^- (averages of 5.2-fold in treated individuals and 86.6-fold in viremics), CD32⁺ PD-1^- (2.2-fold in treated individuals and 4.3-fold in viremics), and CD32^- PD-1⁺ (2.2-fold in ART-treated individuals and 4.6-fold in viremics) cell populations. Similar levels of HIV-1 transcription were found in CD32⁺ PD-1^- and CD32^- PD-1⁺ CD4 T cells. Interestingly, the proportion of CD32⁺ and PD-1⁺ CD4 T cells negatively correlated with CD4 T cell counts and length of therapy. Therefore, the expression of CD32 identifies, independently of PD-1, a CD4 T cell population with persistent HIV-1 transcription and coexpression of CD32 and PD-1, the CD4 T cell population with the highest levels of HIV-1 transcription in both viremic and treated individuals.IMPORTANCE The existence of long-lived latently infected resting memory CD4 T cells represents a major obstacle to the eradication of HIV infection. Identifying cell markers defining latently infected cells containing replication-competent virus is important in order to determine the mechanisms of HIV persistence and to develop novel therapeutic strategies to cure HIV infection. We provide evidence that PD-1 and CD32 may have a complementary role in better defining CD4 T cell populations infected with HIV-1. Furthermore, CD4 T cells coexpressing CD32 and PD-1 identify a CD4 T cell population with high levels of persistent HIV-1 transcription.

Keywords: CD32; PD-1; Tfh cells; human immunodeficiency virus; lymph node.

FIG 1

CD32 expression on CD4 T cells in LNs and blood of HIV-1-infected and uninfected individuals. LN and PB mononuclear cells were isolated from the same HIV-uninfected (n = 9), HIV-1-infected ART-treated (n = 19), and HIV-1-infected viremic (n = 9) individuals. Cells were stained with anti-CD3, anti-CD4, anti-CD45RA, and anti-CD32 antibodies. (A) Representative flow cytometry profiles of blood and LN memory (CD45RA⁻) CD4 T cell populations expressing CD32 in representative HIV-1-uninfected, ART-treated, and viremic subjects. (B) Cumulative data on the frequencies of CD32⁺ memory CD4 T cells in blood and LN mononuclear cells of HIV-1-uninfected, ART-treated, and viremic individuals. P values were obtained by a Mann-Whitney test to compare the three groups and a Wilcoxon signed-rank test to compare frequencies between blood and LNs. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars denote means ± standard errors of the means (SEM).

FIG 2

CD32⁺ CD4 T cells from LNs are enriched in PD-1⁺ and Tfh cells. (A) Representative flow cytometry profiles of CD32 expression in LN memory CD4 T cell populations defined by PD-1 and CXCR5 expression in HIV-1-uninfected and HIV-1-infected ART-treated and viremic individuals. (B) Cumulative data on the frequencies of CD32⁺ memory CD4 T cells within PD-1⁻ CXCR5⁻, PD-1⁻ CXCR5⁺, PD-1⁺ CXCR5⁻, and Tfh (PD-1⁺ CXCR5⁺) cell populations isolated from LNs of 9 HIV-1-uninfected, 19 ART-treated, and 9 viremic individuals. (C) Percentages of PD-1⁺ cells in gated CD32⁻ and CD32⁺ memory (CD45RA⁻) CD4 T cell populations in the three groups. (D) Distribution of Tfh cells within CD32⁻ and CD32⁺ memory CD4 T cell populations. (E) Distribution of Tfh cells within CD32⁻, CD32⁺, and PD-1⁺ CD4 T cell populations from lymph nodes. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (values were obtained by a Wilcoxon signed-rank test, and error bars denote means ± SEM).

FIG 3

Frequencies of blood memory CD4 T cell populations on the basis of CD32 and PD-1. (A) Representative flow cytometry profiles of blood memory (CD45RA⁻) CD4 T cell populations expressing CD32 and/or PD-1 from representative HIV-1-uninfected, ART-treated, and viremic individuals. (B) Cumulative data on the frequencies of CD32⁻ PD-1⁻, CD32⁺ PD-1⁻, CD32⁻ PD-1⁺, and CD32⁺ PD-1⁺ CD4 T cell populations in blood mononuclear cells from HIV-1-uninfected (n = 9) and HIV-1-infected ART-treated (n = 19) and viremic (n = 9) individuals. **, P < 0.01; ***, P < 0.001 (values were obtained by a Mann-Whitney test, and error bars denote means ± SEM).

FIG 4

Mass cytometry analysis of LN memory CD4 T cells defined by CD32 and/or PD-1 expression. Mass cytometry staining was performed on LN mononuclear cells isolated from 9 HIV-1-uninfected, 16 HIV-1-infected ART-treated, and 9 viremic individuals. Cells were stained with a panel of 30 cell surface markers (Table 2). (A and B) Heat maps of mean marker expression levels in memory CD4 T cells defined on the basis of CD32 (A) and CD32 and PD-1 (B) expression. Columns (i.e., markers) are scaled to facilitate comparisons of expression values color-coded from blue (low) to yellow (high). (C) Venn diagram summarizing common trends and the direction of changes for significant markers (as in panel B) in CD45RA⁻ CD4 T cells defined on the basis of PD-1 and CD32 expression. Black arrows indicate trends that are common to all study groups (HIV-uninfected, ART-treated, and viremic individuals). Green, red, and blue arrows indicate trends that are specific to each study group.

FIG 5

Levels of total HIV DNA and cell-associated HIV RNA in CD32⁺ and PD-1⁺ CD4 T cell populations. (A) Gating strategy for CD32⁺ and CD32⁻ and PD-1⁺ and PD-1⁻ memory (CD45RA⁻) CD4 T cell populations. (B) Quantification of total HIV DNA (copies per million cells) of sorted memory (CD45RA⁻) CD4 T cells expressing or not expressing CD32 and PD-1 from 7 aviremic ART-treated (round circles) and 7 viremic (squares) individuals. (C) Frequencies of cells with inducible replication-competent HIV in sorted CD32⁻, PD-1⁻, and total memory CD4 T cells estimated using conventional limiting-dilution methods by extreme limiting-dilution analysis (http://bioinf.wehi.edu.au/software/elda/) in 3 aviremic ART-treated individuals. (D) Gating strategy for CD32⁻ PD-1⁻, CD32⁺ PD-1⁻, CD32⁻ PD-1⁺, and CD32⁺ PD-1⁺ memory (CD45RA⁻) CD4 T cell populations. (E) Levels of cell-associated unspliced HIV RNA (copies/million cells) in sorted CD32⁻ PD-1⁻, CD32⁺ PD-1⁻, CD32⁻ PD-1⁺, and CD32⁺ PD-1⁺ memory CD4 T cell populations isolated from 10 aviremic ART-treated and 6 viremic individuals. *, P < 0.05; **, P < 0.01; ns, not significant (values were obtained by a Wilcoxon signed-rank test, and error bars denote means ± SEM).

FIG 6

Correlations between memory CD4 T cell populations defined by CD32 and PD-1 CD4 counts. Shown are correlations between the percentages of CD32⁺ and PD-1⁺ memory CD4 T cells and CD4 T cell counts. A Spearman rank test was used for correlations.

FIG 7

Correlation between memory CD4 T cell populations defined by CD32 and/or PD-1 and years of suppressive ART. (A) Correlation between the percentages of CD32⁺ and PD-1⁺ memory CD4 T cells and years of suppressive ART. (B) Correlation between the percentages of CD32⁻ PD-1⁻, CD32⁺ PD-1⁻, CD32⁻ PD-1⁺, and CD32⁺ PD-1⁺ populations and years of suppressive ART. A Spearman rank test was used for correlations.