Chronic simian immunodeficiency virus infection is associated with contrasting phenotypes of dysfunctional Bcl6+ germinal center B cells or Bcl6−Bcl2+ non‐germinal center B cells

Abstract Human immunodeficiency virus (HIV) infection is characterized by dysfunctional B cell responses. Here we show that chronic simian immunodeficiency virus (SIV) infection is characterized by an expansion of either lymph node germinal center (GC) B cells that co‐express Bcl6, Ki‐67 and IL‐21R and correlate with expanded T follicular helper (Tfh) cells or B cells that lack Bcl6, Ki‐67 and IL‐21R but express high levels of anti‐apoptotic Bcl2 that negatively correlate with Tfh cells. The lack of Tfh cells likely contributes to persistence of dysfunctional non‐proliferating B cells during chronic infection. These findings have implications for protective immunity in HIV‐infected individuals who harbour low frequencies of Tfh cells.


| BACKGROUND
B cell dysfunction has been well documented during human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infections and is characterized by hypergammaglobulinemia or B cell exhaustion. 1 Numerous studies have implicated a role for expanded T follicular helper (Tfh) cells in driving the expansion of B cells in the germinal centers. [2][3][4] On the other hand, lack of help from Tfh cells has been reported to impair B cell immune responses during HIV infection. 5 Studies have shown that Bcl6 expression in T cells drives the differentiation of Tfh cells, whereas its expression is necessary for the maintenance of germinal center (GC) B cells. 6,7 In contrast, Bcl6 expression suppresses the expression of Bcl2 8 , an anti-apoptotic protein that has been implicated in the development of follicular lymphomas. Bcl2 is generally not expressed in GC B cells but inducing their expression can prevent apoptosis in GC B cells. 9 The contrasting roles of these two transcription factors during HIV infection are not well defined and may be important in understanding the mechanisms associated with B cell dysfunction during HIV infection. Using the rhesus macaque model for chronic SIV infection, we examined the expression of Bcl6 and Bcl2 in lymph node B cells and correlated changes in these subsets with Tfh cells. We observed two distinct groups of SIV infected animals with lymph node (LN) B cells that expressed either high levels of Bcl6 (SIV + Bcl6 hi ) or lacked Bcl6 expression (SIV + Bcl6 lo ). The lack of Bcl6 in B cells was accompanied by significantly lower frequencies of Tfh cells and a higher expression of Bcl2. Given the anti-apoptotic nature of Bcl2, it likely contributes to the expansion and persistence of dysfunctional B cells that display a hypo-proliferative and IL-21R − phenotype. These finding provide additional insights into B cell dysfunction observed during chronic HIV infection.

| Animals and samples
Archival mesenteric lymph nodes (LN) and plasma samples that were collected at necropsy from uninfected (n = 6) and SIVmac251 infected (n = 10) rhesus macaques (Macaca mulatta) were used in this study. All animals were chronically infected for over 6 months at the time of sample collection and housed at Bioqual, Inc. in accordance with the recommendations of the Association for Assessment and Accreditation of Laboratory Animal Care International Standards.

| Data analysis
Flow cytometric data was analysed using FlowJo version 9.8 (Tree Star, Inc., Ashland, OR). Statistical differences between groups were determined using one-way ANOVA and differences within each group were determined by post hoc analysis using Fisher's LSD multiple comparisons test. Linear regression analysis was performed to determine line of fit, and correlations were derived using Spearman's correlation. A P < 0.05 was considered significant. Error bars represent standard error.

| Differential expression of Bcl6 and Bcl2 on lymph node B cells during chronic SIV infection
Studies have shown that B cells that participate in GC reaction coexpress high levels of Bcl6, 6,10 whereas others have reported increased expression of anti-apoptotic Bcl2 on developmentally blocked GC B cells. 11 To assess if expression of Bcl6 and Bcl2 was altered during SIV infection, we examined their expression on B cells in the LN from chronically infected animals and compared them to uninfected animals. Our results showed that Bcl6 was differentially expressed in chronically infected animals ( Figure 1A) with one group of animals expressing high levels of Bcl6 (SIV + Bcl6 hi ) on CD20 + IgG + B cells that did not significantly differ from uninfected animals, whereas the other group of animals lacked Bcl6 expression (SIV + Bcl6 lo ). Interestingly, SIV + Bcl6 lo group of animals expressed significantly higher levels of Bcl2 ( Figure 1B) as compared to the other groups; the Bcl2: Bcl6 ratio was significantly higher in SIV + Bcl6 lo group of SIV-infected animals ( Figure 1C).
The plasma viral loads ( Figure 1D) and the frequency of CD4 T cells ( Figure 1E) did not differ significantly between SIV + Bcl6 hi and SIV + Bcl6 lo groups of animals. There was, however, a significant difference in the frequency of total B cells between the three groups of animals. Total B cells were discriminated based on the expression of CD20 on CD3lymphocytes ( Figure 1F). SIV + Bcl6 lo group of animals were found to have a significantly higher prevalence of CD20 + B cells as compared to both uninfected and SIV + Bcl6 hi group of animals ( Figure 1G). There was, however, no significant difference in frequency of IgG + B cells between the groups ( Figure 1H) likely because of variation between animals though the SIV + Bcl6 lo group of animals had a higher prevalence of IgM + B cells as compared to uninfected animals ( Figure 1I).

| High levels of Bcl2 expression negatively correlate with IL-21R and Ki-67 expression on LN B cells
Studies have shown that Bcl6 is critical for the differentiation and proliferation of GC B cells. 6 To determine if the proliferative capac-

| DISCUSSION
B cell dysfunction is a hallmark of chronic HIV infection that has been shown to persist even during suppressive highly active anti-retroviral therapy. Dysfunctional responses is thought to be driven by an expansion of Tfh cells that likely contribute to hyper-reactive GC reactions characterized by an expansion of GC B cells that express Bcl6 and hypergammaglobulinemia. 1,3,4,[14][15][16] In contrast to hyperreactive B cell responses, B cell exhaustion associated with chronic immune activation has been reported during chronic HIV infection. 22 We observed similar contrasting phenotypes in chronically infected rhesus macaques with significantly higher frequencies of Bcl6 + IgG +  Early treatment with reverse transcriptase inhibitors significantly suppresses peak plasma IFNalpha in vivo during acute simian immunodeficiency virus infection. Cell Immunol. 2016;310:156-164.

S U P P O R T I N G I N F O R M A T I O N
Additional supporting information may be found online in the Supporting Information section at the end of the article.