In this study, Yamamoto and colleagues (1) examine the association of T follicular helper (TFH) cell characteristics with the production of broadly neutralising antibodies (bNAbs) in rhesus macaques (RM) infected with simian immunodeficiency virus (SIV) AD8 as a model of human immunodeficiency virus (HIV) infection. TFH cells are specialised CD4+ T cells essential to the germinal centre (GC) reaction, promoting affinity maturation and class switching of their cognate GC B cell receptors (2). This process is essential to the generation of long-lived high-quality antibody responses which form the basis of vaccination. The HIV-1 envelope glycoprotein spike that mediates viral entry is the primary epitope of naturally occurring neutralising antibodies against the virus and therefore a central target for HIV vaccine studies (3). However, genes encoding the HIV-1 envelope glycoproteins are also highly mutagenic with subsequent amino acid substitutions, insertions/deletions, and glycan shifting all occurring in the most easily accessible regions (4). The consequence of this is that development of bNAbs in HIV infection typically occurs late in chronic infection.
Yamamoto et al. infected eight RM with SIV-AD8 and monitored them for up to 120 weeks with periodic peripheral blood lymphocyte, lymph node and plasma sampling. The total strength and diversity of anti-HIV antibody responses from each RM were summarily scored based on the 50% inhibitory dilution titres of their plasma against 19 simian-human immunodeficiency virus (SHIV) and HIV clade viral isolates. The authors reproduce the observation that higher SIV set point viral load correlate with eventual CD4+ T cell depletion (5), late accumulation of TFH and development of neutralising antibodies (6). TFH accumulation was observed from 20 weeks post-infection, it is not clear if this represents a relative or absolute increase as identified previously (7). However, it did not appear to have a temporal relationship with generation of neutralising antibody which peaked at around 40 weeks, suggesting elevated TFH numbers were not necessarily responsible for eventual antibody responses. At 44 weeks after infection Env-specific TFH, defined as TFH producing CD154 (CD40L), IL-4 or IFNγ in response to stimulation with HIV Env protein, were found in greater frequency in RM with higher scoring bNAbs. In particular the Env-responsive TFH expressing IL-4 and CD154 correlated with both the number of IgG+ Env-specific GC B cells and stronger antibody responses. These findings are perhaps unsurprising as IL-4 and CD154 are crucial to GC interactions and their absence profoundly impairs GC and antibody responses (8,9), whereas the contribution of IFNγ is less clear. Whilst excess IFNγ can enhance GC responses and contribute to autoimmunity, its deficiency does not substantially impair the GC responses (10) and it is also unclear whether IFNγ has a similar function in primates as reported in mice.
The authors examined the transcriptional profile of these Env-responsive TFH. Although absence of conventional normalisation makes interpretation difficult, several intriguing observations were made. Whilst conventional TFH genes such as BCL6, MYB and IL-21 were expressed as expected, surprisingly the transcriptional repressor PRDM1 was found at equivalent levels between Env-responsive CD4+CD44+CXCR5+PD-1+ TFH and non-TFH CD4+ cells. BCL6, as the master transcriptional regulator essential to TFH differentiation, is crucial to both the differentiation and promotion of many important TFH functions (11). BCL6 and PRDM1 are mutually repressive in CD4+ T cells for the purpose of directing Th1/Th2 versus TFH CD4+ effector lineage commitment (12). Thus it is perplexing that PRDM1 expression was equivalent between non-TFH and TFH effector CD4+ T cells; it would be expected that TFH have BCL6-mediated repression of PRDM1. Curiously, the transcription factors TBET (TH1), GATA3 (TH2), and FOXP3 (TREG) were also observed at equivalent levels between the non-TFH and TFH effector CD4+ T cells. Although TFH may potentially arise from other CD4+ effectors, non-BCL6 transcriptional regulators such as TBET are normally only found in subsets of TFH and at lower levels compared with non-TFH effector CD4+ populations (13). Given abnormally high levels of transcriptional regulators and cytokines typically associated with other CD4+ subsets within the Env-responsive TFH raises the possibility of either aberrant CXCR5/PD1 expression amongst effector CD4 sets, contamination of TFH sorted pools, or issues with gene expression data.
Finally, although sequencing of single-cell sorted Env-specific IgG+ B cells heavy chain variable genes (VH4) correlated the degree of mutation with the calibre of late antibody response, the overall rate of mutation was very low suggesting hypofunctional GC responses. Unfortunately, it was not clear whether infected RM developed the hypergammaglobulinemia associated with HIV and SIV infection and which accompanies TFH abnormalities. Thus it is uncertain whether the increase in bNAbs represented a smaller pool containing more higher-quality antibody, or simply a larger pool of antibody with bNAbs representing overwhelming hypergammaglobulinemia, a sign of the quality of the GC and TFH response.
This study reproduces several unusual aspects of the GC biology in HIV/SIV infection. Despite declines in CD4+ T cells, phenotypically bona fide TFH (ICOS+Bcl6+CD40L+PD1+) are known to accumulate in SIV infected RM (14). These TFH are correlated with increased GC B and plasma cells and a reduction in memory B cells as observed by Yamamoto et al. This data supports the observation that it is not total numbers of TFH, but rather the quality which may impair their function. It remains elusive as to why TFH do not function normally in HIV infection. The GC is an established reservoir for HIV, particularly follicular dendritic cells (15), and TFH are known to be infected and form sites of viral replication (14,16). Due to PD1-PDL1 interactions between TFH and GC B cells repressing IL-21, TFH in HIV-infected individuals may be unable to induce normal antigen-specific immune responses (17). This impairment in TFH function is also observed with impaired influenza vaccination responses in HIV infected patients (18). The expression of PRDM1 and other transcription regulators in TFH in this study by Yamamoto et al. suggests abnormalities in TFH function, highlighting the need to better understand the effect of HIV/SIV infection on TFH biology. bNAbs may represent high-quality antibody production arising from TFH cell with typical transcriptional responses and GC characteristics, and optimising TFH response may represent novel target for HIV vaccine development.
SH Jiang has received support from NHMRC, RACP and Jacquot Foundation grants.
Conflicts of Interest: The authors have no conflicts of interest to declare.
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