We have identified a CD4 T cell population in RM secondary lymphoid tissues that resides in the B cell follicle and is characterized by a CD28hi
phenotype. Increased levels of ICOS, CTLA-4, CXCR4, BTLA, CD69, CD154, and BCL-6, as compared with the bulk of CD4 T cells, were typical of this population, which is similar to human and mouse TFH cells (6
). We have further identified GC structures in LN and SP, which are surrounded by CD4 T cells possessing a PD-1hi
phenotype. The relative distribution of PD-1, CD20, and Ki67 within these structures is indicative of GC “dark” and “light” zones, in which somatic mutation and affinity maturation of the B cell response is facilitated by TFH cells (2
). Although TFH cells defined by the multiple markers that we used expressed high CXCR5
mRNA levels, the use of CXCR5 to define TFH cells proved problematic (Figure A and Supplemental Figure 1). Despite this difficulty, the panel of markers that we used clearly identifies a population of CD4 T cells with a phenotype, function, and location indicative of TFH cells.
CD150 is an important regulator of TFH development, through its interaction with the adaptor SAP (14
). Reduced TBX21
transcription in CD150lo
TFH cells was associated with high production of IL-4, while TFH cells with high expression of CD150 predominantly produced IL-10. In addition, TFH cells produced significantly fewer Th1 cytokines compared with non-TFH cells, irrespective of CD150 expression (Figure B). Our data are therefore consistent with CCR7lo
CD4 T cells being the “GC TFH” cells in RMs (45
). Because IL-4 is induced upon CD150/SAP crosstalk and is mediated by FYN and PKC-θ/NF-κB signaling (13
), investigating the ratio of CD150 to SAP and the relative function of FYN- and PKC-θ–mediated pathways could inform the mechanisms underlying these polarized cytokine profiles. We found that sorted CCR7lo
CD4 T cells from chronically infected RMs were capable of providing in vitro help to sorted total or memory autologous B cells, further supporting their definition as TFH cells. Further experiments, using a larger cohort of noninfected and SIV-infected RMs, are needed to understand the nature of the in vitro help (30
) and compare it to the help provided by TFH cells from noninfected RMs.
TFH cells from SIV-infected and uninfected RMs were transcriptionally different. The increased expression of the IFN-induced genes in TFH cells from SIV-infected RMs suggests that these cells respond to the increased IFN production associated with the SIV microenvironment. In addition, the increased expression of TGF-β–associated genes suggests a role of TGF-β as a regulator of TFH dysfunction in SIV-infected RMs (47
). Other indicators of TFH dysfunction in SIV-infected RMs include the decreased expression of TNFSF14
. Finally, we show significant reduction in the expression of the IL4
gene in TFH cells from SIV-infected RMs; this gene plays a critical role in memory B cell survival and isotype switching. Our data therefore provide critical information regarding molecular pathways that regulate TFH cells in vivo and demonstrate that many of them are adversely affected by SIV infection, possibly leading to downstream problems with the B cell response to SIV and other infections.
Analysis of multiple CD4 T cell populations revealed loss of CCR7hi
accompanied by expansion of CCR7lo
(TFH) cells in secondary lymphoid tissues during chronic SIV infection. This was more evident within the CD150lo
TFH compartment. Our assays only allow a determination of the relative frequency of particular T cell populations. Because the relative accumulation of TFH cells was observed in the presence of total CD4 T cell loss, we cannot determine whether there is an absolute increase, or just less loss, of TFH cells compared with other CD4 T cells in LNs during SIV infection. We expected to find that TFH cells were less infected in vivo than other CD4 T cell populations in the LN. In fact, we found the opposite. During acute SIV infection, LN TFH cells were frequently in cell cycle (expressed Ki67) and had greater copy numbers of SIV than other CD4 T cell populations (Figure E and data not shown). No differences were found, however, during the chronic SIV infection, indicating that infection per se is not a major regulator of the TFH cell expansion. We found that CD150lo
TFH cells are not self-renewable, while their capacity for egression into the blood stream, judged by the expression of S1PR1
, is compromised. Interestingly, the expression of S1PR1
was low in TFH cells regardless the infection status, indicating that this is an intrinsic characteristic of these cells. However, it was apparent that chronic SIV infection results in increased S1PR1
expression on non-TFH CD4 T cells, potentially leading to increased egression of these cells into periphery. We hypothesize that such a process could affect the dynamics of CCR7hi
CD4 T cells in LNs from RMs with high TFH (Figure A). Furthermore, the rate of spontaneous cell death of TFH cells, as measured by active caspase-3 expression, was unaffected by SIV. The high level of spontaneous active caspase-3 expression in CD150lo
TFH cells suggests that they normally have a very short half-life in vivo, possibly explaining why SIV infection does not impact further upon an already short survival. In fact, our data argue that TFH cells may be less exposed to death signals mediated by PD-L1 expressed on B cells (33
) during the chronic SIV infection (Figure C).
Within the group of SIV-infected RMs who had a high frequency of TFH cells, we found significantly elevated levels of sCD14, supporting the potential role of general immune activation in this process. In particular, our data point to increased IL-6 signaling that favors the development of TFH. The increased expression of the IL-6R complex found on TFH cells, especially in chronic SIV infection, was associated with increased plasma levels of IL-6. Treatment with IL-6 significantly increased the proliferation of naive and non-TFH cells but had no effect on their survival. In line with their general lack of in vivo cycling, no proliferation was observed in stimulated TFH cells in the presence or absence of IL-6. Furthermore, IL-6 was capable of inducing molecular markers (BCL-6, ICOS, CD95, PD-1) of TFH cells, especially after TCR stimulation. Our gene analysis revealed that CEBPA
, a major negative regulator of G1
/S transition (48
) and necessary factor for the expression of IL-6Ra (51
), was noticeably increased in the CD150lo
TFH cells. Proliferation and differentiation can be mutually exclusive processes, and CEBPA has been proposed to serve as a linker between these two biological pathways (53
). Therefore, we hypothesize that CEBPA could serve as a central coordinator of the TFH dynamics in vivo by regulating their proliferation/differentiation process as well as the activity of IL-6 during the development of TFH responses. IL-6 signaling is mediated by phosphorylation of both STAT-1, a major inducer of Th1 cells (54
), and STAT-3, a critical regulator of TFH (21
). Although ex vivo IL-6 treatment induced phosphorylation of both STAT-1 and STAT-3 in all populations tested, a significantly higher STAT-3 phosphorylation was found in TFH cells compared with that in non-TFH cells only in samples collected from chronically infected RMs. Furthermore, mobilization of STAT-1 in TFH cells, under IL-6 treatment, was selectively compromised during chronic SIV infection. Whether increased expression of IL-6Ra or altered intracellular interactions in LNs with high TFH cells are responsible for this skewed IL-6 signaling needs to be investigated. Our in vitro data indicate that IL-6 could affect the development of TFH responses in SIV infection at multiple stages by affecting the molecular program (i.e., BCL-6 expression) and proliferation of less differentiated CD4 T cells as well as the intracellular signaling (through STAT phosphorylation) in TFH cells. Thus, our data reveal altered IL-6 signaling during chronic infection that favors the development of TFH cells and further support the significance of IL-6 as a critical regulator of TFH cell dynamics.
Accumulation of TFH cells in chronic SIV infection was associated with expansion of the GC B cell compartment and increased circulating SIV-specific immunoglobulins, in agreement with recently described data (24
). Although not significant, the avidity of SIV-specific antibodies was higher in RMs with high TFH compared with that in RMs with low TFH. Our data show that the gene expression alterations (i.e., reduction of IL4
) imparted by SIV upon the TFH cells did not affect the ability of B cells to respond to SIV. Therefore, delineating the molecular mechanisms mediating the TFH–B cell interactions is critical for our understanding of the development of neutralizing activity against SIV and presumably HIV. Moreover, further work is needed to understand whether the higher infection levels of TFH cells in the acute phase contributes to the limited specificities of the initial HIV-1–specific (and SIV-specific) antibody responses (55
In summary, we have characterized TFH cells in RMs at the phenotypic, molecular, and tissue localization level. Our findings support a model of TFH cell dynamics in which a constant flow of activated CD4 T cells enter the TFH compartment followed by further differentiation to a status (CD150lo TFH) characterized by a polarized cytokine profile and reduced survival ability. SIV impacts upon the molecular gene signature of this cell population yet does not lead to their specific depletion and often results in their accumulation. This is likely because of the diverse populations of CD4 T cells that can serve to continually replenish this population, thereby assuring their persistence late into SIV infection.