T follicular helper (Tfh) cells are essential for germinal center B cell responses. The molecular mechanism underlying the initial Tfh cell differentiation, however, is still incompletely understood. In this study, we show that in vivo, despite enhanced non–Tfh cell effector functions, the deletion of transcription factor Bach2 results in preferential Tfh cell differentiation. Mechanistically, the deletion of Bach2 leads to the induction of CXCR5 expression even before the upregulation of Ascl2. Subsequently, we have identified a novel regulatory element in the murine CXCR5 locus that negatively regulates CXCR5 promoter activities in a Bach2-dependent manner. Bach2 deficiency eventually results in a collapsed CD4+ T cell response with severely impaired CD4+ T cell memory, including Tfh cell memory. Our results demonstrate that Bach2 critically regulates Tfh cell differentiation and CD4+ T cell memory.
The biomolecular mechanisms controlling latent HIV-1 infection, despite their importance for the development of a cure for HIV-1 infection, are only partially understood. For example, ex vivo studies have recently shown that T cell activation only triggered HIV-1 reactivation in a fraction of the latently infected CD4+ T cell reservoir, but the molecular biology of this phenomenon is unclear. We demonstrate that HIV-1 infection of primary T cells and T cell lines indeed generates a substantial amount of T cell receptor (TCR)/CD3 activation-inert latently infected T cells. RNA-level analysis identified extensive transcriptomic differences between uninfected, TCR/CD3 activation-responsive and -inert T cells, but did not reveal a gene expression signature that could functionally explain TCR/CD3 signaling inertness. Network analysis suggested a largely stochastic nature of these gene expression changes (transcriptomic noise), raising the possibility that widespread gene dysregulation could provide a reactivation threshold by impairing overall signal transduction efficacy. Indeed, compounds that are known to induce genetic noise, such as HDAC inhibitors impeded the ability of TCR/CD3 activation to trigger HIV-1 reactivation. Unlike for transcriptomic data, pathway enrichment analysis based on phospho-proteomic data directly identified an altered TCR signaling motif. Network analysis of this data set identified drug targets that would promote TCR/CD3-mediated HIV-1 reactivation in the fraction of otherwise TCR/CD3-reactivation inert latently HIV-1 infected T cells, regardless of whether the latency models were based on T cell lines or primary T cells. The data emphasize that latent HIV-1 infection is largely the result of extensive, stable biomolecular changes to the signaling network of the host T cells harboring latent HIV-1 infection events. In extension, the data imply that therapeutic restoration of host cell responsiveness prior to the use of any activating stimulus will likely have to be an element of future HIV-1 cure therapies.
The development of therapies to eliminate the latent HIV-1 reservoir is hampered by our incomplete understanding of the biomolecular mechanism governing HIV-1 latency. To further complicate matters, recent single cell RNA-seq studies reported extensive heterogeneity between latently HIV-1-infected primary T cells, implying that latent HIV-1 infection can persist in greatly differing host cell environments. We here show that transcriptomic heterogeneity is also found between latently infected T cell lines, which allowed us to study the underlying mechanisms of intercell heterogeneity at high signal resolution. Latently infected T cells exhibited a de-differentiated phenotype, characterized by the loss of T cell-specific markers and gene regulation profiles reminiscent of hematopoietic stem cells (HSC). These changes had functional consequences. As reported for stem cells, latently HIV-1 infected T cells efficiently forced lentiviral superinfections into a latent state and favored glycolysis. As a result, metabolic reprogramming or cell re-differentiation destabilized latent infection. Guided by these findings, data-mining of single cell RNA-seq data of latently HIV-1 infected primary T cells from patients revealed the presence of similar dedifferentiation motifs. >20% of the highly detectable genes that were differentially regulated in latently infected cells were associated with hematopoietic lineage development (e.g. HUWE1, IRF4, PRDM1, BATF3, TOX, ID2, IKZF3, CDK6) or were hematopoietic markers (SRGN; hematopoietic proteoglycan core protein). The data add to evidence that the biomolecular phenotype of latently HIV-1 infected cells differs from normal T cells and strategies to address their differential phenotype need to be considered in the design of therapeutic cure interventions. IMPORTANCE HIV-1 persists in a latent reservoir in memory CD4 T cells for the lifetime of a patient. Understanding the biomolecular mechanisms used by the host cells to suppress viral expression will provide essential insights required to develop curative therapeutic interventions. Unfortunately, our current understanding of these control mechanisms is still limited. By studying gene expression profiles, we demonstrated that latently HIV-1-infected T cells have a de-differentiated T cell phenotype. Software-based data integration allowed for the identification of drug targets that would re-differentiate viral host cells and, in extension, destabilize latent HIV-1 infection events. The importance of the presented data lies within the clear demonstration that HIV-1 latency is a host cell phenomenon. As such, therapeutic strategies must first restore proper host cell functionality to accomplish efficient HIV-1 reactivation.
Immunosenescence is the increase in immune defects that occurs during aging. To identify any intrinsic defects in old B lymphocytes, we devised a transfer system to introduce follicular B cells from old mice (FOO, >22 months) or young mice (FOY, 8–12 weeks) into young mMT mice which have no B cells. Using this system, we observed that, compared to FOY cells, FOO cells do not produce antigen-specific antibodies to nitrophenyl (NP)-chicken gamma globulin after immunization. Examination of the germinal center (GC) response shows that FOO cells produced increased numbers of GC B cells which have divided faster than their FOY counterparts, but they have significantly decreased NP+ λ+ antigen-specificity. To understand the differences between FOO and FOY cells, single-cell RNAseq was performed on naïve and day 14 GC B cells. Comparing naïve populations revealed very few differences with only 387 differentially expressed genes between FOO and FOY cells. Interestingly, Ccr6 was upregulated in the FOO cells, suggesting that the cells were pre-activated before transfer into mMT mice. Antibody repertoire analysis also confirmed the lack of antigen-specificity in FOO cells, as the canonical NP-specific IgH V-gene, 1–72, was utilized in fewer cells and had zero instances of the CDR2 W33L mutation known to increase affinity. Taken together, old mice produce B cells which are pre-activated and hyper-responsive upon immunization, causing a lack of proper selection and affinity maturation in the GC. This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging.
Therapeutic attempts to eliminate the latent HIV-1 reservoir have failed, at least in part due to our incomplete biomolecular understanding of how latent HIV-1 infection is established and maintained. We here address the fundamental question of whether all lentiviruses actually possess a similar capacity to establish latent infections or whether there are differences between the lentiviral lineages driving differential latency establishment that could be exploited to develop improved latency reversal agents.
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