T Follicular Helper Cell Heterogeneity.

T follicular helper (Tfh) cells specialize in helping B cells and are therefore critical contributors to the generation of humoral immunity. Tfh cells aid immunoglobulin class-switch recombination and support the germinal center response, thereby promoting immunoglobulin affinity maturation and the generation of humoral immune memory. Although their primary function is to promote B cell responses, Tfh cells also display phenotypic and functional diversity determined by the immunological and spatial contexts from which they emerge. We review recent advances in understanding the heterogeneity within Tfh cell subsets along with their differentiation and migratory trajectory, the phenotype they adopt, their ontological relationships with one another, and their function in their respective environments. Expected final online publication date for the Annual Review of Immunology, Volume 42 is April 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Development of Tbet- and CD11c-expressing B cells in a viral infection requires T follicular helper cells outside of germinal centers.

Tbet+CD11c+ B cells arise during type 1 pathogen challenge, aging, and autoimmunity in mice and humans. Here, we examined the developmental requirements of this B cell subset. In acute infection, T follicular helper (Tfh) cells, but not Th1 cells, drove Tbet+CD11c+ B cell generation through proximal delivery of help. Tbet+CD11c+ B cells developed prior to germinal center (GC) formation, exhibiting phenotypic and transcriptional profiles distinct from GC B cells. Fate tracking revealed that most Tbet+CD11c+ B cells developed independently of GC entry and cell-intrinsic Bcl6 expression. Tbet+CD11c+ and GC B cells exhibited minimal repertoire overlap, indicating distinct developmental pathways. As the infection resolved, Tbet+CD11c+ B cells localized to the marginal zone where splenic retention depended on integrins LFA-1 and VLA-4, forming a competitive memory subset that contributed to antibody production and secondary GC seeding upon rechallenge. Therefore, Tbet+CD11c+ B cells comprise a GC-independent memory subset capable of rapid and robust recall responses.

TFH cells progressively differentiate to regulate the germinal center response.

Germinal center (GC) B cells undergo affinity selection, which depends on interactions with CD4(+) follicular helper T cells (TFH cells). We found that TFH cells progressed through transcriptionally and functionally distinct stages and provided differential signals for GC regulation. They initially localized proximally to mutating B cells, secreted interleukin 21 (IL-21), induced expression of the transcription factor Bcl-6 and selected high-affinity B cell clones. As the GC response evolved, TFH cells extinguished IL-21 production and switched to IL-4 production, showed robust expression of the co-stimulatory molecule CD40L, and promoted the development of antibody-secreting B cells via upregulation of the transcription factor Blimp-1. Thus, TFH cells in the B cell follicle progressively differentiate through stages of localization, cytokine production and surface ligand expression to 'fine tune' the GC reaction.

Production of IL-10 by CD4(+) regulatory T cells during the resolution of infection promotes the maturation of memory CD8(+) T cells.

Memory CD8(+) T cells are critical for host defense upon reexposure to intracellular pathogens. We found that interleukin 10 (IL-10) derived from CD4(+) regulatory T cells (Treg cells) was necessary for the maturation of memory CD8(+) T cells following acute infection with lymphocytic choriomeningitis virus (LCMV). Treg cell-derived IL-10 was most important during the resolution phase, calming inflammation and the activation state of dendritic cells. Adoptive transfer of IL-10-sufficient Treg cells during the resolution phase 'restored' the maturation of memory CD8(+) T cells in IL-10-deficient mice. Our data indicate that Treg cell-derived IL-10 is needed to insulate CD8(+) T cells from inflammatory signals, and reveal that the resolution phase of infection is a critical period that influences the quality and function of developing memory CD8(+) T cells.

Cutting Edge: IL-21 and Tissue-Specific Signals Instruct Tbet+CD11c+ B Cell Development following Viral Infection.

Tbet+CD11c+ B cells, also known as age-associated B cells (ABCs), are pivotal contributors to humoral immunity following infection and in autoimmunity, yet their in vivo generation is incompletely understood. We used a mouse model of systemic acute lymphocytic choriomeningitis virus infection to examine the developmental requirements of ABCs that emerged in the spleen and liver. IL-21 signaling through STAT3 was indispensable for ABC development. In contrast, IFN-γ signaling through STAT1 was required for B cell activation and proliferation. Mice that underwent splenectomy or were deficient in lymphotoxin α generated hepatic ABCs despite the lack of secondary lymphoid organ contributions, suggesting that the liver supported de novo generation of these cells separately from their development in lymphoid organs. Thus, IFN-γ and IL-21 signaling have distinct, stage-specific roles in ABC differentiation, while the tissue microenvironment provides additional cues necessary for their development.

Author Correction: Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Distinct modes of mitochondrial metabolism uncouple T cell differentiation and function.

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation1. In CD4 T cells, T-cell-receptor ligation-along with co-stimulatory and cytokine signals-induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (TH1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of TH1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses TH1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state.

CD4+ T cells that help B cells - a proposal for uniform nomenclature.

T follicular helper (Tfh) cells cognately guide differentiation of antigen-primed B cells in secondary lymphoid tissues. 'Tfh-like' populations not expressing the canonical Tfh cell transcription factor BCL6 have also been described, which can aid particular aspects of B cell differentiation. Tfh and Tfh-like cells are essential for protective and pathological humoral immunity. These CD4+ T cells that help B cells are polarized to produce diverse combinations of cytokines and chemokine receptors and can be grouped into distinct subsets that promote antibodies of different isotype, affinity, and duration, according to the nature of immune challenge. However, unified nomenclature to describe the distinct functional Tfh and Tfh-like cells does not exist. While explicitly acknowledging cellular plasticity, we propose categorizing these cell states into three groups based on phenotype and function, paired with their anatomical site of action.

Local triggering of the ICOS coreceptor by CD11c(+) myeloid cells drives organ inflammation in lupus.

The inducible T cell costimulator (ICOS) is a potent promoter of organ inflammation in murine lupus. ICOS stimulates T follicular helper cell differentiation in lymphoid tissue, suggesting that it might drive autoimmunity by enhancing autoantibody production. Yet the pathogenic relevance of this mechanism remains unclear. It is also unknown whether other ICOS-induced processes might contribute to lupus pathology. Here we show that selective ablation of ICOS ligand (ICOSL) in CD11c(+) cells, but not in B cells, dramatically ameliorates kidney and lung inflammation in lupus-prone MRL.Fas(lpr) mice. Autoantibody formation was largely unaffected by ICOSL deficiency in CD11c(+) cells. However, ICOSL display by CD11c(+) cells in inflamed organs had a nonredundant role in protecting invading T cells from apoptosis by elevating activity of the PI3K-Akt signaling pathway, thereby facilitating T cell accrual. These findings reveal a mechanism that locally sustains organ inflammation in lupus.

The Interleukin-2-mTORc1 Kinase Axis Defines the Signaling, Differentiation, and Metabolism of T Helper 1 and Follicular B Helper T Cells.

The differentiation of CD4(+) helper T cell subsets with diverse effector functions is accompanied by changes in metabolism required to meet their bioenergetic demands. We find that follicular B helper T (Tfh) cells exhibited less proliferation, glycolysis, and mitochondrial respiration, accompanied by reduced mTOR kinase activity compared to T helper 1 (Th1) cells in response to acute viral infection. IL-2-mediated activation of the Akt kinase and mTORc1 signaling was both necessary and sufficient to shift differentiation away from Tfh cells, instead promoting that of Th1 cells. These findings were not the result of generalized signaling attenuation in Tfh cells, because they retained the ability to flux calcium and activate NFAT-transcription-factor-dependent cytokine production. These data identify the interleukin-2 (IL-2)-mTORc1 axis as a critical orchestrator of the reciprocal balance between Tfh and Th1 cell fates and their respective metabolic activities after acute viral infection.

Transcription factor STAT3 and type I interferons are corepressive insulators for differentiation of follicular helper and T helper 1 cells.

Follicular helper T (Tfh) cells are required for the establishment of T-dependent B cell memory and high affinity antibody-secreting cells. We have revealed herein opposing roles for signal transducer and activator of transcription 3 (STAT3) and type I interferon (IFN) signaling in the differentiation of Tfh cells following viral infection. STAT3-deficient CD4(+) T cells had a profound defect in Tfh cell differentiation, accompanied by decreased germinal center (GC) B cells and antigen-specific antibody production during acute infection with lymphocytic choriomeningitis virus. STAT3-deficient Tfh cells had strikingly increased expression of a number of IFN-inducible genes, in addition to enhanced T-bet synthesis, thus adopting a T helper 1 (Th1) cell-like effector phenotype. Conversely, IFN-αß receptor blockade restored Tfh and GC B cell phenotypes in mice containing STAT3-deficient CD4(+) T cells. These data suggest mutually repressive roles for STAT3 and type I IFN signaling pathways in the differentiation of Tfh cells following viral infection.

CD4+ T cell help guides formation of CD103+ lung-resident memory CD8+ T cells during influenza viral infection.

Tissue-resident memory T (Trm) cells provide enhanced protection against infection at mucosal sites. Here we found that CD4(+) T cells are important for the formation of functional lung-resident CD8(+) T cells after influenza virus infection. In the absence of CD4(+) T cells, CD8(+) T cells displayed reduced expression of CD103 (Itgae), were mislocalized away from airway epithelia, and demonstrated an impaired ability to recruit CD8(+) T cells to the lung airways upon heterosubtypic challenge. CD4(+) T cell-derived interferon-γ was necessary for generating lung-resident CD103(+) CD8(+) Trm cells. Furthermore, expression of the transcription factor T-bet was increased in "unhelped" lung Trm cells, and a reduction in T-bet rescued CD103 expression in the absence of CD4(+) T cell help. Thus, CD4(+) T cell-dependent signals are important to limit expression of T-bet and allow for the development of CD103(+) CD8(+) Trm cells in the lung airways following respiratory infection.

T follicular helper cell heterogeneity: Time, space, and function.

T follicular helper (Tfh) cells play a crucial role in orchestrating the humoral arm of adaptive immune responses. Mature Tfh cells localize to follicles in secondary lymphoid organs (SLOs) where they provide help to B cells in germinal centers (GCs) to facilitate immunoglobulin affinity maturation, class-switch recombination, and generation of long-lived plasma cells and memory B cells. Beyond the canonical GC Tfh cells, it has been increasingly appreciated that the Tfh phenotype is highly diverse and dynamic. As naive CD4+ T cells progressively differentiate into Tfh cells, they migrate through a variety of microanatomical locations to obtain signals from other cell types, which in turn alters their phenotypic and functional profiles. We herein review the heterogeneity of Tfh cells marked by the dynamic phenotypic changes accompanying their developmental program. Focusing on the various locations where Tfh and Tfh-like cells are found, we highlight their diverse states of differentiation. Recognition of Tfh cell heterogeneity has important implications for understanding the nature of T helper cell identity specification, especially the plasticity of the Tfh cells and their ontogeny as related to conventional T helper subsets.

Roquin paralogs add a new dimension to ICOS regulation.

Two studies in the current issue of Immunity (Pratama et al., 2013; Vogel et al., 2013) demonstrate that the RNA-binding proteins Roquin-1 and Roquin-2 have redundant function in the posttranscriptional repression of Icos messenger RNA by CD4(+) T cells.

Thymic self-reactivity selects natural interleukin 17-producing T cells that can regulate peripheral inflammation.

Interleukin 17 (IL-17)-producing CD4(+) helper T cells (T(H)-17 cells) share a developmental relationship with Foxp3(+) regulatory T cells (T(reg) cells). Here we show that a T(H)-17 population differentiates in the thymus in a manner influenced by recognition of self antigen and by the cytokines IL-6 and transforming growth factor-beta (TGF-beta). Like previously described T(H)-17 cells, the T(H)-17 cells that developed in the thymus expressed the transcription factor RORgamma t and the IL-23 receptor. These cells also expressed alpha(4)beta(1) integrins and the chemokine receptor CCR6 and were recruited to the lung, gut and liver. In the liver, these cells secreted IL-22 in response to self antigen and mediated host protection during inflammation. Thus, T(H)-17 cells, like T(reg) cells, can be selected by self antigens in the thymus.

scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3' mRNA profiling.

Cellular barcoding of 3' mRNAs enabled massively parallel profiling of single-cell gene expression and has been implemented in droplet and microwell based platforms. The latter further adds the value for compatibility with low input samples, optical imaging, scalability, and portability. However, cell lysis in microwells remains challenging despite the recently developed sophisticated solutions. Here, we present scFTD-seq, a microchip platform for performing single-cell freeze-thaw lysis directly toward 3' mRNA sequencing. It offers format flexibility with a simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and cost per sample matching that of the state-of-the-art scRNA-seq platforms. Freeze-thaw, known as an unfavorable lysis method resulting in possible RNA fragmentation, turns out to be fully compatible with 3' scRNA-seq. We applied it to the profiling of circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis. Our results delineate the heterogeneity in the transcriptional programs and effector functions of these rare pathogenic T cells. As scFTD-seq decouples on-chip cell isolation and library preparation, we envision it to allow sampling at the distributed sites including point-of-care settings and downstream processing at centralized facilities, which should enable wide-spread adoption beyond academic laboratories.

Differential expression of Ly6C and T-bet distinguish effector and memory Th1 CD4(+) cell properties during viral infection.

CD4(+) T cells differentiate into multiple effector types, but it is unclear how they form memory T cells during infection in vivo. Profiling virus-specific CD4(+) T cells revealed that effector cells with T helper 1 (Th1) or T follicular helper (Tfh) cell characteristics differentiated into memory cells, although expression of Tfh cell markers declined over time. In contrast to virus-specific effector CD8(+) T cells, increased IL-7R expression was not a reliable marker of CD4(+) memory precursor cells. However, decreased Ly6C and T-bet (Tbx21) expression distinguished a subset of Th1 cells that displayed greater longevity and proliferative responses to secondary infection. Moreover, the gene expression profile of Ly6C(lo)T-bet(int) Th1 effector cells was virtually identical to mature memory CD4(+) T cells, indicating early maturation of memory CD4(+) T cell features in this subset during acute viral infection. This study provides a framework for memory CD4(+) T cell development after acute viral infection.

Human Extrafollicular CD4+ Th Cells Help Memory B Cells Produce Igs.

Follicular helper T (Tfh) cells are necessary for germinal center B cell maturation during primary immune responses; however, the T cells that promote humoral recall responses via memory B cells are less well defined. In this article, we characterize a human tonsillar CD4+ T cell subset with this function. These cells are similar to Tfh cells in terms of expression of the chemokine receptor CXCR5 and the inhibitory receptor PD-1, IL-21 secretion, and expression of the transcription factor BCL6; however, unlike Tfh cells that are located within the B cell follicle and germinal center, they reside at the border of the T cell zone and the B cell follicle in proximity to memory B cells, a position dictated by their unique chemokine receptor expression. They promote memory B cells to produce Abs via CD40L, IL-10, and IL-21. Our results reveal a unique extrafollicular CD4+ T cell subset in human tonsils, which specialize in promoting T cell-dependent humoral recall responses.