Type I interferons induce an epigenetically distinct memory B cell subset in chronic viral infection.

Memory B cells (MBCs) are key providers of long-lived immunity against infectious disease, yet in chronic viral infection, they do not produce effective protection. How chronic viral infection disrupts MBC development and whether such changes are reversible remain unknown. Through single-cell (sc)ATAC-seq and scRNA-seq during acute versus chronic lymphocytic choriomeningitis viral infection, we identified a memory subset enriched for interferon (IFN)-stimulated genes (ISGs) during chronic infection that was distinct from the T-bet+ subset normally associated with chronic infection. Blockade of IFNAR-1 early in infection transformed the chromatin landscape of chronic MBCs, decreasing accessibility at ISG-inducing transcription factor binding motifs and inducing phenotypic changes in the dominating MBC subset, with a decrease in the ISG subset and an increase in CD11c+CD80+ cells. However, timing was critical, with MBCs resistant to intervention at 4 weeks post-infection. Together, our research identifies a key mechanism to instruct MBC identity during viral infection.

TGF-ß specifies TFH versus TH17 cell fates in murine CD4+ T cells through c-Maf.

T follicular helper (TFH) cells are essential for effective antibody responses, but deciphering the intrinsic wiring of mouse TFH cells has long been hampered by the lack of a reliable protocol for their generation in vitro. We report that transforming growth factor-ß (TGF-ß) induces robust expression of TFH hallmark molecules CXCR5 and Bcl6 in activated mouse CD4+ T cells in vitro. TGF-ß-induced mouse CXCR5+ TFH cells are phenotypically, transcriptionally, and functionally similar to in vivo-generated TFH cells and provide critical help to B cells. The study further reveals that TGF-ß-induced CXCR5 expression is independent of Bcl6 but requires the transcription factor c-Maf. Classical TGF-ß-containing T helper 17 (TH17)-inducing conditions also yield separate CXCR5+ and IL-17A-producing cells, highlighting shared and distinct cell fate trajectories of TFH and TH17 cells. We demonstrate that excess IL-2 in high-density T cell cultures interferes with the TGF-ß-induced TFH cell program, that TFH and TH17 cells share a common developmental stage, and that c-Maf acts as a switch factor for TFH versus TH17 cell fates in TGF-ß-rich environments in vitro and in vivo.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

IRF2 integrates inflammatory signals to balance T cell exhaustion.

The transcription factor interferon regulatory factor 2 (IRF2) translates interferon signaling to regulate T cells. In this issue of Immunity, Lukhele et al. identify IRF2 in tumor-infiltrating T cells as a sensor for extrinsic signals that drives an exhaustion program.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

Type 1 conventional dendritic cells maintain and guide the differentiation of precursors of exhausted T cells in distinct cellular niches.

Reinvigoration of exhausted CD8+ T (Tex) cells by checkpoint immunotherapy depends on the activation of precursors of exhausted T (Tpex) cells, but the local anatomical context of their maintenance, differentiation, and interplay with other cells is not well understood. Here, we identified transcriptionally distinct Tpex subpopulations, mapped their differentiation trajectories via transitory cellular states toward Tex cells, and localized these cell states to specific splenic niches. Conventional dendritic cells (cDCs) were critical for successful αPD-L1 therapy and were required to mediate viral control. cDC1s were dispensable for Tpex cell expansion but provided an essential niche to promote Tpex cell maintenance, preventing their overactivation and T-cell-mediated immunopathology. Mechanistically, cDC1s insulated Tpex cells via MHC-I-dependent interactions to prevent their activation within other inflammatory environments that further aggravated their exhaustion. Our findings reveal that cDC1s maintain and safeguard Tpex cells within distinct anatomical niches to balance viral control, exhaustion, and immunopathology.

Transforming growth factor-ß-regulated mTOR activity preserves cellular metabolism to maintain long-term T cell responses in chronic infection.

Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-ß repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.

Antigen-driven EGR2 expression is required for exhausted CD8+ T cell stability and maintenance.

Chronic stimulation of CD8+ T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.

Early precursor T cells establish and propagate T cell exhaustion in chronic infection.

CD8+ T cells responding to chronic infections or tumors acquire an 'exhausted' state associated with elevated expression of inhibitory receptors, including PD-1, and impaired cytokine production. Exhausted T cells are continuously replenished by T cells with precursor characteristics that self-renew and depend on the transcription factor TCF1; however, their developmental requirements are poorly understood. In the present study, we demonstrate that high antigen load promoted the differentiation of precursor T cells, which acquired hallmarks of exhaustion within days of infection, whereas early effector cells retained polyfunctional features. Early precursor T cells showed epigenetic imprinting characteristic of T cell receptor-dependent transcription factor binding and were restricted to the generation of cells displaying exhaustion characteristics. Transcription factors BACH2 and BATF were key regulators with opposing functions in the generation of early precursor T cells. Overall, we demonstrate that exhaustion manifests first in TCF1+ precursor T cells and is propagated subsequently to the pool of antigen-specific T cells.

Human effector T cells express TOX-Not so "TOX"ic after all.

TOX expression is not restricted to exhausted T cells but a characteristic of all human effector CD8+ T cells.

Precursor exhausted T cells: key to successful immunotherapy?

Cytotoxic T cell immunity in response to chronic infections and tumours is maintained by a specialized population of CD8+ T cells that exhibit hallmarks of both exhausted and memory cells and give rise to terminally differentiated exhausted effector cells that contribute to viral or tumour control. Importantly, recent work suggests these cells, which we refer to as 'precursor exhausted' T (TPEX) cells, are responsible for the proliferative burst that generates effector T cells in response to immune checkpoint blockade targeting programmed cell death 1 (PD1), and increased TPEX cell frequencies have recently been linked to increased patient survival. We believe the recent discovery of TPEX cells not only represents a paradigm shift in our understanding of the mechanisms that maintain CD8+ T cell responses in chronic infections and tumours but also opens up unexpected avenues for the development of new and innovative therapeutic approaches. In this Opinion article, we discuss the differentiation and function of TPEX cells and suggest that targeting these cells may be key for successful immunotherapy.

MiR-23~27~24-mediated control of humoral immunity reveals a TOX-driven regulatory circuit in follicular helper T cell differentiation.

Follicular helper T (TFH) cells are essential for generating protective humoral immunity. To date, microRNAs (miRNAs) have emerged as important players in regulating TFH cell biology. Here, we show that loss of miR-23~27~24 clusters in T cells resulted in elevated TFH cell frequencies upon different immune challenges, whereas overexpression of this miRNA family led to reduced TFH cell responses. Mechanistically, miR-23~27~24 clusters coordinately control TFH cells through targeting a network of genes that are crucial for TFH cell biology. Among them, thymocyte selection-associated HMG-box protein (TOX) was identified as a central transcription regulator in TFH cell development. TOX is highly up-regulated in both mouse and human TFH cells in a BCL6-dependent manner. In turn, TOX promotes the expression of multiple molecules that play critical roles in TFH cell differentiation and function. Collectively, our results establish a key miRNA regulon that maintains optimal TFH cell responses for resultant humoral immunity.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Expression of FOXO1.

Immunity following an acutely resolved infection or the long-term equipoise of chronic viral infections often depends on the maintenance of antigen-specific CD8+ T cells, yet the ongoing transcriptional requirements of these cells remain unclear. We show that active and continuous programming by FOXO1 is required for the functional maintenance of a memory population. Upon Foxo1 deletion following resolution of an infection, memory cells rapidly lost their characteristic gene expression, gradually declined in number, and were impaired in self-renewal. This was extended to chronic infections, as a loss of FOXO1 during a persistent viral infection led to a rapid decline of the TCF7 (a.k.a. TCF1)-expressing memory-like subset of CD8+ T cells. We further establish FOXO1 regulation as a characteristic of human memory CD8+ T cells. Overall, we show that the molecular and functional longevity of a memory T cell population is actively maintained by the transcription factor FOXO1.

Continuous activity of Foxo1 is required to prevent anergy and maintain the memory state of CD8+ T cells.

Upon infection with an intracellular pathogen, cytotoxic CD8+ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.

Type I interferons induced by endogenous or exogenous viral infections promote metastasis and relapse of leishmaniasis.

The presence of the endogenous Leishmania RNA virus 1 (LRV1) replicating stably within some parasite species has been associated with the development of more severe forms of leishmaniasis and relapses after drug treatment in humans. Here, we show that the disease-exacerbatory role of LRV1 relies on type I IFN (type I IFNs) production by macrophages and signaling in vivo. Moreover, infecting mice with the LRV1-cured Leishmania guyanensis (LgyLRV1- ) strain of parasites followed by type I IFN treatment increased lesion size and parasite burden, quantitatively reproducing the LRV1-bearing (LgyLRV1+ ) infection phenotype. This finding suggested the possibility that exogenous viral infections could likewise increase pathogenicity, which was tested by coinfecting mice with L. guyanensis and lymphocytic choriomeningitis virus (LCMV), or the sand fly-transmitted arbovirus Toscana virus (TOSV). The type I IFN antiviral response increased the pathology of L. guyanensis infection, accompanied by down-regulation of the IFN-γ receptor normally required for antileishmanial control. Further, LCMV coinfection of IFN-γ-deficient mice promoted parasite dissemination to secondary sites, reproducing the LgyLRV1+ metastatic phenotype. Remarkably, LCMV coinfection of mice that had healed from L. guyanensis infection induced reactivation of disease pathology, overriding the protective adaptive immune response. Our findings establish that type I IFN-dependent responses, arising from endogenous viral elements (dsRNA/LRV1), or exogenous coinfection with IFN-inducing viruses, are able to synergize with New World Leishmania parasites in both primary and relapse infections. Thus, viral infections likely represent a significant risk factor along with parasite and host factors, thereby contributing to the pathological spectrum of human leishmaniasis.

Exacerbated Leishmaniasis Caused by a Viral Endosymbiont can be Prevented by Immunization with Its Viral Capsid.

Recent studies have shown that a cytoplasmic virus called Leishmaniavirus (LRV) is present in some Leishmania species and acts as a potent innate immunogen, aggravating lesional inflammation and development in mice. In humans, the presence of LRV in Leishmania guyanensis and in L. braziliensis was significantly correlated with poor treatment response and symptomatic relapse. So far, no clinical effort has used LRV for prophylactic purposes. In this context, we designed an original vaccine strategy that targeted LRV nested in Leishmania parasites to prevent virus-related complications. To this end, C57BL/6 mice were immunized with a recombinant LRV1 Leishmania guyanensis viral capsid polypeptide formulated with a T helper 1-polarizing adjuvant. LRV1-vaccinated mice had significant reduction in lesion size and parasite load when subsequently challenged with LRV1+ Leishmania guyanensis parasites. The protection conferred by this immunization could be reproduced in naïve mice via T-cell transfer from vaccinated mice but not by serum transfer. The induction of LRV1 specific T cells secreting IFN-γ was confirmed in vaccinated mice and provided strong evidence that LRV1-specific protection arose via a cell mediated immune response against the LRV1 capsid. Our studies suggest that immunization with LRV1 capsid could be of a preventive benefit in mitigating the elevated pathology associated with LRV1 bearing Leishmania infections and possibly avoiding symptomatic relapses after an initial treatment. This novel anti-endosymbiotic vaccine strategy could be exploited to control other infectious diseases, as similar viral infections are largely prevalent across pathogenic pathogens and could consequently open new vaccine opportunities.

T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections.

Chronic infections promote the terminal differentiation (or "exhaustion") of T cells and are thought to preclude the formation of memory T cells. In contrast, we discovered a small subpopulation of virus-specific CD8(+) T cells that sustained the T cell response during chronic infections. These cells were defined by, and depended on, the expression of the transcription factor Tcf1. Transcriptome analysis revealed that this population shared key characteristics of central memory cells but lacked an effector signature. Unlike conventional memory cells, Tcf1-expressing T cells displayed hallmarks of an "exhausted" phenotype, including the expression of inhibitory receptors such as PD-1 and Lag-3. This population was crucial for the T cell expansion that occurred in response to inhibitory receptor blockade during chronic infection. These findings identify a memory-like T cell population that sustains T cell responses and is a prime target for therapeutic interventions to improve the immune response in chronic infections.

High antigen levels induce an exhausted phenotype in a chronic infection without impairing T cell expansion and survival.

Chronic infections induce T cells showing impaired cytokine secretion and up-regulated expression of inhibitory receptors such as PD-1. What determines the acquisition of this chronic phenotype and how it impacts T cell function remain vaguely understood. Using newly generated recombinant antigen variant-expressing chronic lymphocytic choriomeningitis virus (LCMV) strains, we uncovered that T cell differentiation and acquisition of a chronic or exhausted phenotype depend critically on the frequency of T cell receptor (TCR) engagement and less significantly on the strength of TCR stimulation. In fact, we noted that low-level antigen exposure promotes the formation of T cells with an acute phenotype in chronic infections. Unexpectedly, we found that T cell populations with an acute or chronic phenotype are maintained equally well in chronic infections and undergo comparable primary and secondary expansion. Thus, our observations contrast with the view that T cells with a typical chronic infection phenotype are severely functionally impaired and rapidly transition into a terminal stage of differentiation. Instead, our data unravel that T cells primarily undergo a form of phenotypic and functional differentiation in the early phase of a chronic LCMV infection without inheriting a net survival or expansion deficit, and we demonstrate that the acquired chronic phenotype transitions into the memory T cell compartment.