Decreased T Follicular Regulatory Cell/T Follicular Helper Cell (TFH) in Simian Immunodeficiency Virus-Infected Rhesus Macaques May Contribute to Accumulation of TFH in Chronic Infection

Abstract

T follicular helper cells (TFH) are critical for the development and maintenance of germinal center (GC) and humoral immune responses. During chronic HIV/SIV infection, TFH accumulate, possibly as a result of Ag persistence. The HIV/SIV-associated TFH expansion may also reflect lack of regulation by suppressive follicular regulatory CD4(+) T cells (TFR). TFR are natural regulatory T cells (TREG) that migrate into the follicle and, similar to TFH, upregulate CXCR5, Bcl-6, and PD1. In this study, we identified TFR as CD4(+)CD25(+)FOXP3(+)CXCR5(+)PD1(hi)Bcl-6(+) within lymph nodes of rhesus macaques (RM) and confirmed their localization within the GC by immunohistochemistry. RNA sequencing showed that TFR exhibit a distinct transcriptional profile with shared features of both TFH and TREG, including intermediate expression of FOXP3, Bcl-6, PRDM1, IL-10, and IL-21. In healthy, SIV-uninfected RM, we observed a negative correlation between frequencies of TFR and both TFH and GC B cells, as well as levels of CD4(+) T cell proliferation. Post SIV infection, the TFR/TFH ratio was reduced with no change in the frequency of TREG or TFR within the total CD4(+) T cell pool. Finally, we examined whether higher levels of direct virus infection of TFR were responsible for their relative depletion post SIV infection. We found that TFH, TFR, and TREG sorted from SIV-infected RM harbor comparable levels of cell-associated viral DNA. Our data suggest that TFR may contribute to the regulation and proliferation of TFH and GC B cells in vivo and that a decreased TFR/TFH ratio in chronic SIV infection may lead to unchecked expansion of both TFH and GC B cells.

Figure 1. T_FR can be defined by flow cytometry and identified by confocal microscopy within the germinal centers of RM

(A) Representative flow cytometry plot of lymphocytes from lymph nodes of untreated uninfected RM showing the gating strategy used to define T_FR, T_FH and T_REG cell populations. (B) Representative confocal microscope image showing a single T_FR within the lymph node of an uninfected RM. The first image shows staining for DAPI (green) and FoxP3 (red). The second image shows the same section with CD4 (green) and FoxP3 (red), the third PD1 (blue) and FoxP3 (red) and the last image CD4 (green), PD1 (blue) and FoxP3 (red). (C) Representative images of lymph node biopsies from SIV uninfected and acutely infected RM showing cells stained with CD4 (green), FoxP3 (red) and PD1 (blue) within the GC regions.

Figure 2. T_FR share immunophenotypical features of both T_FH and T_REG populations

Mean fluorescence intensity, percent positive, representative flow cytometry plots and histograms (Panels A, B, C, D, E, F) for expression of various immunophenotypical markers (i.e., FoxP3, CD25, CXCR5, PD-1, CD127, CTLA4, Bcl-6 and Helios) among T_REG, non- T_REG, T_FR and T_FH populations from LN of healthy, unvaccinated and uninfected RM. Non-T_REG here are defined as all CD4⁺CD25⁻FoxP3⁻ T-cells. Significance was determined by Wilcoxon signed rank tests.

Figure 3. RNA expression patterns confirm that T_FR share T_FH and T_REG like phenotype

(A) Principal components analysis of RNA transcripts from five healthy, SIV-uninfected RM. Each circle represents the transcriptome of a sorted population of T_FH (blue), T_REG (green), or T_FR (red) from a single animal. (B) Expression in FPKM of select T_FH and T_REG genes in sorted populations from uninfected RM. (C) Heat map of log 2 transformed gene expression of transcripts in FPKM. Transcripts represent T_FH and T_REG signature genes. The T_FH gene signature was defined as transcripts that were significantly differentially expressed in sorted T_FH compared to bulk CD4⁺ T-cells. T_REG gene signature was defined as genes that were significantly differentially expressed in sorted T_REG compared to bulk CD4⁺ T-cells. (D) Expression pattern of key T_FH and T_REG genes in sorted bulk CD4⁺ T-cell, T_FH, T_FR and T_REG populations.

Figure 4. Kinetics of T_FR, T_FH and T_REG after SIV infection

(A) Frequency of T_FH, T_FR and T_REG as percentage of the total CD4⁺ T-cell population within lymph nodes of uninfected, acutely (week 2) SIV-infected and chronically (week 24) SIV-infected RM. (B) Frequency of T_FR as a percent of T_FH within the lymph nodes of the same animals. (C) Ratio of frequencies of T_FR to the frequency of T_FH (both calculated as a percent of the total CD4⁺ T-cell population). (D) Percent of proliferating, Ki67⁺, T_FH, T_FR and T_REG within the lymph nodes of uninfected, acutely SIV-infected and chronically SIV-infected RM. Statistical analyses were performed using Mann-Whitney U tests.

Figure 5. Comparable levels of SIV infection in T_FH, T_FR and T_REG isolated from the spleen of chronically SIV-infected RM

(A) Viral DNA copies per million sorted T_FH, T_FR and T_REG from spleens of unvaccinated chronically SIV-infected RM. (B) Percent of CCR5-expressing cells among T_FH, T_FR and T_REG in unvaccinated SIV-infected RM. Statistical analyses were performed using Mann-Whitney U tests.

Figure 6. T_FR frequencies negatively correlate with T_FH and GC B-cell frequencies in the lymph nodes of RM

Correlations between the frequencies of T_FR (calculated as frequency of T_FH) and the frequencies of T_FH calculated as percent of total CD4⁺ T-cells (left) and GC B-cells calculated as percent of total B-cells (center) and proliferating (i.e., Ki67⁺) CD4⁺ T-cells (right) within the lymph nodes of SIV-uninfected (A) and chronically SIV-infected (B) RM. Statistical analyses were performed using Spearman rank correlation tests.

Figure 7. RNA expression within T_FR cells in uninfected and infected RM

(A) Enrichment of gene pathways in T_FR derived from the lymph nodes of unvaccinated SIV infected RM as compared to T_FR from SIV-uninfected animals as determined by Ingenuity Pathway Analysis. (B) Log 2 fold change of expression of T_FH and T_REG related gene transcripts in T_FR sorted from unvaccinated SIV-infected RM and SIV-uninfected RM. Significantly upregulated genes are in red and significantly down-regulated genes are in blue.