Lymph-Node-Based CD3+ CD20+ Cells Emerge from Membrane Exchange between T Follicular Helper Cells and B Cells and Increase Their Frequency following Simian Immunodeficiency Virus Infection

Abstract

CD4⁺ T follicular helper (T_FH) cells are key targets for human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) replication and contribute to the virus reservoir under antiretroviral therapy (ART). Here, we describe a novel CD3⁺ CD20⁺ double-positive (DP) lymphocyte subset, resident in secondary lymphoid organs of humans and rhesus macaques (RMs), that appear predominantly after membrane exchange between T_FH and B cells. DP lymphocytes are enriched in cells displaying a T_FH phenotype (CD4⁺ PD1^hi CXCR5^hi), function (interleukin 21 positive [IL-21⁺]), and gene expression profile. Importantly, expression of CD40L upon brief in vitro mitogen stimulation identifies, by specific gene-expression signatures, DP cells of T_FH-cell origin versus those of B-cell origin. Analysis of 56 RMs showed that DP cells (i) significantly increase following SIV infection, (ii) are reduced after 12 months of ART in comparison to pre-ART levels, and (iii) expand to a significantly higher frequency following ART interruption. Quantification of total SIV-gag DNA on sorted DP cells from chronically infected RMs showed that these cells are susceptible to SIV infection. These data reinforce earlier observations that CD20⁺ T cells are infected and expanded by HIV infection, while suggesting that these cells phenotypically overlap activated CD4⁺ T_FH cells that acquire CD20 expression via trogocytosis and can be targeted as part of therapeutic strategies aimed at HIV remission. IMPORTANCE The HIV reservoir is largely composed of latently infected memory CD4⁺ T cells that persist during antiretroviral therapy and constitute a major barrier toward HIV eradication. In particular, CD4⁺ T follicular helper cells have been demonstrated as key targets for viral replication and persistence under ART. In lymph nodes from HIV-infected humans and SIV-infected rhesus macaques, we show that CD3⁺ CD20⁺ lymphocytes emerge after membrane exchange between T cells and B cells and are enriched in phenotypic, functional, and gene expression profiles found in T follicular helper cells. Furthermore, in SIV-infected rhesus macaques, these cells expand following experimental infection and after interruption of ART and harbor SIV DNA at levels similar to those found in CD4⁺ T cells; thus, CD3⁺ CD20⁺ lymphocytes are susceptible to SIV infection and can contribute to SIV persistence.

Keywords: CD3+ CD20+ cells; HIV infection; SIV infection; TFH; lymphoid tissues; nonhuman primates.

FIG 1

CD3⁺ CD20⁺ cells are found in secondary lymphoid organs of rhesus macaques. (A) Representative flow cytometry plots showing live lymphocytes from two RMs in which mononuclear cells from LNs were stained with anti-CD3 and anti-CD20 antibodies. A distinct CD3⁺ CD20⁺ population (double positive [DP]), is found in LNs. (B) Frequency of DP population within various secondary lymphoid organs of RMs (n = 9 for inguinal LNs (ILNs) and axillary LNs (ALNs) spleen, and PBMCs; n = 8 for mesenteric LNs [MLNs]) showing higher percentages in LNs and spleen. Statistical differences were assessed by ANOVA with the Tukey adjustment for multiple comparisons. Asterisks represent P values at the following significance level: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.001, **** ≤ 0.0001. (C) Composite representative bright-field and pseudocolor fluorescent images of individual cells stained for CD3 and/or CD20 using Amnis imaging flow cytometry. Staining was performed on live lymphocytes isolated from spleens of healthy RMs. Top panel, punctate staining pattern for CD20 molecules on CD3⁺ T cells; bottom panel, CD3 molecules on CD20⁺ B cells. Merged CD3/CD20 images indicate that the patterns of the two molecules are distinct. (D) Representative micrograph of a paraffin-embedded LN section taken from a healthy RM showing CD3 and CD20 staining. LN sections were stained with anti-CD3 (red) and anti-CD20 (green) antibodies. White arrows indicate cell-to-cell interaction events and the presence of DP cells. Top right panel, zoomed-in area showing CD3⁺ T-cell and CD20⁺ B-cell interaction; bottom right panel, presence of a CD20 molecule on the surface of a CD3⁺ T cell (DP cell).

FIG 2

Phenotypic characterization of DP cells in secondary lymphoid organs of RMs. (A) Correlation between the frequency of T_FH cells within the CD4⁺ T-cell population and the frequency of T_FH cells within the DP population in RMs (n = 18); Spearman’s nonparametric correlation coefficient and associated P value are shown. (B) DP cells are enriched in markers associated with T_FH cells. Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, and CD3⁺ CD20⁺ cells from RMs (n = 8) expressing the T_FH markers CXCR5, PD1, and ICOS. (C) Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, CD3⁺ CD20⁺ cells, and CD20⁺CD3⁻ cells from RMs (n = 5) expressing CD40L after PMA and ionomycin in vitro stimulation. (D) Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, CD3⁺ CD20⁺ cells, and CD20⁺ CD3⁻ cells from RMs (n = 9) producing IL-21 (but not IL-17) following 4 h of in vitro stimulation with PMA and ionomycin. For all panels, each dot represents a single animal. One-way ANOVA with the Kruskal-Wallis test was performed for multiple comparisons. Asterisks represent P values at the following significance level: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.001, **** ≤ 0.0001.

FIG 3

CD3⁺ CD20⁻ cells express several B-cell surface markers associated with the TCR-MHC synaptic complex. Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, CD3⁺ CD20⁺ cells, and CD20⁺ CD3⁻ cells from RMs expressing HLA-DR (n = 7) (A), CD86 (n = 8) (B), CD79b (n = 9) (C), IgD⁺ (n = 8) (D), CD40 (n = 8) (E), and CD21 (n = 7) (F). Each dot represents a single animal. One-way ANOVA with the Kruskal-Wallis test was performed for multiple comparisons. Asterisks represent P values at the following significance level: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.001, **** ≤ 0.0001.

FIG 4

Surface expression of CD40L identifies DP cells that are of T_FH-cell or B-cell origin by specific gene expression signatures. Transcript signatures were determined in sorted CD20⁺ (brown), CD40L⁻ DP (orange), CD40L⁺ DP (blue), and conventional T_FH (green) cells; 2,197 differentially expressed genes were identified between T_FH cells and CD20⁺ B cells. Based on statistical significance, clustering analyses are presented for the top 210 genes (A) and, among those, for the top 50 genes (B). Z scores (blue to red) from −2 to 2 indicate the medium log₂ fold change in gene expression.

FIG 5

Kinetics and susceptibility to infection of CD3⁺ CD20⁺ cells in SIV infection. (A) Cross-sectional comparison of the levels of DP cells, expressed as the frequency of live lymphocytes, in RMs at different phase of SIV infection, including uninfected (SIV-, n = 9), SIV-infected (SIV⁺, n = 40), on ART (SIV⁺ + ART, n = 47), and after ATI (SIV⁺+ ATI, n = 7). (B) Longitudinal comparison of the frequencies of DP cells between chronic phase of infection and on-ART time point in RMs that started ART (n = 40). (C) Longitudinal comparison of the frequency of DP cells on ART and after ATI in RMs in which ATI was performed (n = 7). (D and E) CD4⁺ T cells and DP cells were isolated from the spleens of SIV-infected RMs (n = 5) to quantify their frequencies of infection; the numbers of proviral DNA⁺ cells (by gag quantification) are shown per million CD4⁺ T cells or DP cells (D) and normalized for CD4⁺ T-cell levels (E). (F) Correlation between SIV DNA content in CD4⁺ T cells and DP cells. The Spearman correlation coefficient and associated P values are shown. Asterisks represent P values at the following significance level: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.001, **** ≤ 0.0001.

FIG 6

CD3⁺ CD20⁺ cells are found in human lymphoid tissues and show a phenotype similar to those in RMs. (A) Representative flow cytometry plots depicting a subset of CD3⁺ T cells expressing CD20 (left plots) and a subset of CD20⁺ cells expressing CD3 (right plots) in LNs of people living with HIV, with very low levels of DP cells in PBMCs. (B) Frequency of DP cells, expressed as a percentage of CD3⁺ T cells, in human blood and LN cells isolated from ART-naive people (black symbols, n = 16) or ART-treated people (orange symbols, n = 3). (C) Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, and CD3⁺ CD20⁺ cells expressing the T_FH markers CXCR5, PD1, and ICOS. (D and E) Frequency (% of parent) of CD4⁺ T cells, non-T_FH cells, T_FH cells, and CD3⁺ CD20⁺ and CD20⁺ CD3⁻ B cells expressing the B-cell markers CD21, HLA-DR, and CD40 (D) and CD86 or IL-21 but not IL-17 (E) following 4 h of in vitro stimulation with PMA and ionomycin. Asterisks represent P values at the following significance level: * ≤ 0.01, ** ≤ 0.001, *** ≤ 0.001, **** ≤ 0.0001.