Early loss of splenic Tfh cells in SIV-infected rhesus macaques

Follicular T helper cells (Tfh), a subset of CD4 T lymphocytes, provide crucial help to B cells in the production of antigen-specific antibodies. Although several studies have analyzed the dynamics of Tfh cells in peripheral blood and lymph nodes (LNs) during Aids, none has yet addressed the impact of SIV infection on the dynamics of Tfh cells in the spleen, the primary organ of B cell activation. We show here a significant decrease in splenic Tfh cells in SIVmac251-infected rhesus macaques (RMs) during the acute phase of infection, which persists thereafter. This profound loss is associated with lack of sustained expression of the Tfh-defining transcription factors, Bcl-6 and c-Maf but with higher expression of the repressors KLF2 and Foxo1. In this context of Tfh abortive differentiation and loss, we found decreased percentages of memory B cell subsets and lower titers of SIV-specific IgG. We further demonstrate a drastic remodeling of the lymphoid architecture of the spleen and LNs, which disrupts the crucial cell-cell interactions necessary to maintain memory B cells and Tfh cells. Finally, our data demonstrated the early infection of Tfh cells. Paradoxically, the frequencies of SIV DNA were higher in splenic Tfh cells of RMs progressing more slowly suggesting sanctuaries for SIV in the spleen. Our findings provide important information regarding the impact of HIV/SIV infection on Tfh cells, and provide new clues for future vaccine strategies.This work was supported by CHIR (Canada) and ANRS grants (France). JE thanks the Canada Research Chair program for financial assistance. VR was supported by a doctoral fellowship from FCT (Fundacao para a Ciencia e Tecnologia); code SFRH/BD/64064/2009. We would like to thank the Nonhuman Primate Reagent Resource for kindly providing CXCR5 antibodies. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Correction: Early Loss of Splenic Tfh Cells in SIV-Infected Rhesus Macaques.

[This corrects the article DOI: 10.1371/journal.ppat.1005287.]

Mucosal T follicular helper cells in SIV-infected rhesus macaques: contributing role of IL-27

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Early infection of Tfh cells.

<p>(A) Frequencies of SIV DNA and (B) RNA in sorted Tfh and effector memory CD4 T cells isolated from LNs and spleen of RMs infected with SIV. Each dot represents an individual RM. Statistical analyses are performed using Mann Whitney test. *, p<0.05; **, p<0.01. At day>180, open circles represent fast progressor RMs, PB023 and PB028; and full diamonds represent slow progressor RMs, PB013 and PB044. (C) Correlation between the frequencies of SIV DNA⁺ Tfh cells and the percentage of B cell subsets (as defined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005287#ppat.1005287.g005" target="_blank">Fig 5</a>) in LNs and spleen of RMs. Each dot represents an individual macaque. Spearman analysis was used for correlations.</p

T and B cell dynamics in <i>SIV</i>-infected rhesus macaques.

<p>(A) Flow cytometry was used to determine the percentage of CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells, and CD3^-CD20⁺ B cells in the (Left panel) blood, (Middle panel) LNs and (Right panel) spleen. (B) Histograms show total number of T and B cells in LNs and the spleen, and cell number/mm³ in peripheral blood. Each dot represents an individual RM; Axillary and Inguinal LNs are represented separately by two distinct dots for each RMs at day>180. Statistical analyses were performed using Mann Whitney test. *, p<0.05; **, p<0.01. At day>180, open circles represent fast progressor RMs PB023 and PB028; and full diamonds represent slow progressor RMs, PB013 and PB044.</p

Distribution of Tfh cells in the spleen of rhesus macaque infected with SIV.

<p>Splenic tissue sections were stained with antibodies against CXCR5 (blue), CD4 (green) and PD-1 (red) and imaged by confocal microscopy. Representative pictures of the same animals as depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005287#ppat.1005287.g008" target="_blank">Fig 8</a> are shown. Higher magnification is shown on the right part of the picture. Scale bar is shown.</p

B cell dynamics in SIV-infected rhesus macaques.

<p>(A) Representative dot plots depicting gating strategy and the expression of CD21 and CD27 in B cells (CD3⁻CD20⁺) derived from blood, LNs and spleen is showed. B cell subsets were defined as naive (CD21⁺CD27⁻), resting memory (CD21⁺CD27⁺), activated memory (CD21⁻CD27⁺) and tissue memory (CD21⁻CD27⁻) B cells. (B) Percentages and (C) cell numbers for each B cell subset are shown. Each dot represents an individual RM. Statistical analyses were performed using Mann Whitney test. *, p<0.05; **, p<0.01. At day>180, open circles represent fast progressor RMs PB023 and PB028; and full diamonds represent slow progressor RMs PB013 and PB044. Axillary and Inguinal LNs are represented separately by two distinct dots for each RMs at day>180.</p

Genotypes of rhesus macaques included in our cohort.

<p>The table indicates the date of sacrifice, CD4 T cell loss (compared to the baseline), and viral load of uninfected (SIV-) and RMs infected with SIVmac251. Animals were genotyped for MHC class I <i>Mamu-A</i> and <i>Mamu-B</i> haplotypes.</p

Dynamics of germinal center in the spleen of rhesus macaque infected with SIV.

<p>Splenic tissue sections were stained with antibodies against Ki-67 (white), IgD (green), CD3 (blue) and CD20 (red) and imaged by confocal microscopy. Representative pictures of a naive RM and of two chronically SIV-infected RMs, slow and fast progressor RMs are shown. The picture is representative of two individuals animals performed independently. Higher magnification is shown on the right part of the picture. Scale bar is shown.</p