T helper cell subsets: diversification of the field

Polarized T helper cell (Th cell) responses are important determinants of host protection. Th cell subsets tailor their functional repertoire of cytokines to their cognate antigens to efficiently contribute to their clearance. In contrast, in settings of immune abrogation, these polarized cytokine patterns of Th cells can mediate tissue damage and pathology resulting in allergy or autoimmunity. Recent technological developments in single‐cell genomics and proteomics as well as advances in the high‐dimensional bioinformatic analysis of complex datasets have challenged the prevailing Th cell subset classification into Th1, Th2, Th17, and other subsets. Additionally, systems immunology approaches have revealed that instructive input from the peripheral tissue microenvironment can have differential effects on the overall phenotype and molecular wiring of Th cells depending on their spatial distribution. Th cells from the blood or secondary lymphoid organs are therefore expected to follow distinct rules of regulation. In this review, the functional heterogeneity of Th cell subsets will be reviewed in the context of new technological developments and T‐cell compartmentalization in tissue niches. This work will especially focus on challenges to the traditional boundaries of Th cell subsets and will discuss the underlying regulatory checkpoints, which could reveal new therapeutic strategies for various immune‐mediated diseases.


Introduction
The identification of polarized T helper 1 (Th1) and Th2 helper cell identities more than 30 years ago started with the inception of a dichotomous categorization into two mutually exclusive subsets with distinct cytokine profiles [1].This categorization was elegantly supported by later work, which demonstrated the antagonistic regulation of Th1 and Th2 cells by the other subset's signature cytokines and their coregulation by their respective autocrine signature effector cytokines.This dichotomy persisted as the so-called Th1/Th2 paradigm despite emerging data revealing IL-4 and IFN-γ coexpression at the single-cell level in mice and humans [2,3].The concept of T helper cell plasticity emerged, which consolidated these hybrid Th cell properties and also proposed that the flexibility and malleability of antagonistic cytokine expression patterns held therapeutic potential.Allergies mediated by Th2 cell responses were shown to dissipate upon transdifferentiation of Th2 cells into IFN-γ-producing Th cells, while autoimmunity was shown to subside upon induction of Th2 cytokines [4].
With the discovery of Th17 cells almost two decades ago, this dichotomous paradigm was challenged [5,6].Not only did this unique Th cell population emerge and compete with Th1 cells for the star role in the pathogenesis of autoimmune diseases, its emergence also heralded the search for additional Th cell subsets with distinct functions and molecular architectures.This resulted in an ever-growing family of Th cell subsets, now comprising Th9, Th22, and Th-GMCSF cells and other more or less established subsets [7][8][9][10].Not surprisingly, the need for consensus criteria for Th cell subsets has been raised [11].Expression of signature cytokines, master transcription factors, and specific priming cytokines has been and is still used to identify unique Th cell subsets.Additionally, the expression of chemokine receptors, which dictates migration abilities, delineates distinct Th cell subsets.Different combinations of the three markers CXCR3, CCR4, and CCR6 were shown to correlate with human Th1 (CXCR3 + CCR4 -CCR6 -), Th2 (CXCR3 -CCR4 + CCR6 -), and Th17 (CXCR3 -CCR4 + CCR6 + ) cells [12].The trend of increasing numbers of Th cell subsets was expected to continue, particularly with the advent of high-dimensional single-cell technologies such as CyTOF, spectral flow cytometry, and single-cell RNA sequencing.Interestingly, however, in the immunological community the notion has emerged that an asymptote has been reached.This belief does not overlook the vast heterogeneity of Th cells, which can now be visualized, but rather considers heterogeneity a reflection of functional or regulatory "states" lacking fixed stability and long-term maintenance.Th cells respond to external cues on the basis of their individual history, which yields an unprecedented heterogeneity of phenotypes and functional states.While cytokines have previously been considered the primary signaling molecules for T-cell differentiation and modulation, new factors, primarily from the peripheral tissue microenvironment, have been shown to have a powerful impact on Th cell identities.How intrinsic and external factors drive Th cell functionalities and induce the differential engagement of molecular programs is currently under intense investigation.

Th cell regulation revisited
Historically, Th cells have been assigned master transcription factors and polarized effector functions, which emerge in the context of distinct polarizing and stabilizing cytokines.Th1 cell differentiation is driven by IL-12, which upregulates the master transcription factor T-bet, followed by secretion of the signature cytokine IFN-γ in mice and humans [13].Th1 and Th2 cells have traditionally been perceived as counterparts that antagonize each other.It therefore came as a surprise when the Th1 cell hallmark cytokine IFN-γ was shown to promote Th2 cell differentiation directly or by activation of dendritic cells (DCs) with Th2-priming capacity [14][15][16].In mice, it was shown that two distinct populations of Th2 cells, characterized by differential IL-4 and IL-5 expression levels, can be generated depending on IFN-γ levels at the time of priming [14].In an infection mouse model with the parasite Nippostrongylus brasiliensis (Nb), it was shown that type-I IFN signaling in DCs was relevant to promote Th2 cell development since blocking the IFN-I receptor at the time of Nb treatment blunted Th2 development [15].This was also observed with the parasite Schistosoma mansoni, which highlighted that the influence of IFN-I is not limited to antiviral or bacterial Th1restricted contexts, but also has a critical role in licensing DCs for the initiation of Th2 cell responses [16].Cumulatively, these findings blur the strict antagonistic regulation of Th1 and Th2 cells.

Regulation of Th1 cell responses
Eomesodermin (Eomes), a transcription factor of the T-box family closely related to T-bet, has been assigned roles in the imprinting of a Th1-like or cytotoxic Th cell profile beyond its classic role in CD8 T-cell and natural killer cell differentiation [17].Eomes favors the acquisition of pathogenic potential by T cells, for example, promoting IFN-γ and GM-CSF upregulation [7].Interestingly, the recent identification of a patient with complete inherited T-bet deficiency, which resulted in Mendelian inheritance of susceptibility to mycobacterial disease (MSMD), revealed several additional T-bet target genes beyond IFNG, such as CCL1, CCL13, CCL4, CSF2, CXCR5, GZMM, CD40, CD86, IL7R, IL10, LTB, ITGA5, and ITGB5 [18].As expected, T-bet deficiency causes increased production of Th2-associated cytokines such as IL-5 and IL-13 by Th cells [19].Absence of T-bet also results in increased IL-17 and IL-22 levels, supporting a counter-regulatory relationship of Th1 with Th2 and Th17 cell responses.Many other monogenic etiologies of MSMD are associated with impaired IFN-γ production, such as variants of IL12B, TYK2, IL12RB1, IL12RB2, and others [20].Surprisingly, IFN-γ expression is reduced in classic CCR6 -Th1 cells but not in CCR6 + Th1 cells, which have previously been reported to coexpress IL-17 in humans [21].CCR6 + Th1 cells are enriched for antigen specificities against mycobacterial antigens and Bacillus Calmette-Guérin (BCG) [22].Strikingly, BCG-specific Th1 cells with T-bet deficiency do not have compromised effector function.This human "experiment of nature" indicates some redundancy in the regulation of this effector cytokine in specific Th cell types with implications for human host defence against Mycobacterium tuberculosis and potentially other pathogens [18].How IFN-γ expression is maintained in the absence of T-bet remains to be explored.It will be interesting to see whether the IFN-γ permissive function of T-bet can be alternatively performed by Eomes.Another addition to the list of IFN-γ regulators in Th cells with in vivo relevance in humans is ITK, encoding the IL-2-inducible T-cell kinase (ITK) protein; inherited deficiency of ITK abrogates IFN-γ production in Th cells, which leads to insufficient protection from tuberculosis [23].

Regulation of Th2 cell responses
Th2 cells are induced by IL-4 and regulated by GATA-3, and the resulting Th2 cell responses are commonly associated with allergies.However, the primary purpose of Th2 cells is host defence against large helminths by means of detoxification and expulsion [24].Platelet-derived Dickkopf-related protein 1 (DKK1), a Wnt antagonist, has recently been proposed to be relevant for Th2 cell differentiation.This suggests links to tissue damage, platelet activation, and consecutive Th2 cell responses [25].Several studies support a relevant contribution of Th2 cytokineactivated macrophages to tissue homeostasis and resolution of inflammatory assaults [26].Amphiregulin has been shown to be coexpressed with the classic Th2 cell effector cytokines and to contribute to tissue hypertrophy and concomitantly to helminth parasite removal in mouse models [27].Alarmins, such as IL-33 and thymic stromal lymphopoietin (TSLP), which accumulate following tissue damage due to mechanical, allergic, or autoimmune insults, have been shown to have direct effects on the production of IL-13, inducing innate-like Th2 cell responses after exposure to tissue damage.Following the induction of epidermal growth factor receptor (EGFR) expression and the amphiregulin-dependent formation of a functional signaling complex between EGFR and IL-33R, Th2 cells secrete IL-13 in response to IL-33.Interestingly, this innate-like response can occur in an antigen-independent manner in TCR preactivated Th2 cells [28,29].However, the specific mechanisms by which tissue repair is regulated with the assistance of type 2 cytokines and their target cell types, including eosinophils, mast cells, basophils, and group 2 innate lymphocytes, remain largely unknown [26].Additionally, most insights regarding these Th2-mediated repair mechanisms have been generated in mouse models, leaving their translation into the human system largely unexplored.
Over the last few years, Th2 cells have been appreciated as a heterogeneous Th cell population [30].Although GATA-3 is known as the overall orchestrator of Th2 cell responses, the precise mechanism governing the selective production of individual Th2 cell effector functions has not been entirely revealed.Th2 cells expressing only IL-5 in the absence of IL-4 have been described, and they were associated with a more terminally differentiated subset of Th2 cells [31].IL-5-producing memory Th2 cells are exclusively present in the CD62L lo CXCR3 lo T-cell subpopulation and regulated by Eomes in memory Th2 cells.Eomes interacts with GATA-3 and prevents its binding to the IL5 promotor region in mice [32].CXCR6 + ST2 + memory Th2 cells and ST2 -memory Th2 cells have also been identified [33].Only CXCR6 + ST2 + Th2 cells expressed high amounts of major basic protein (MBP) and contributed to the reduction in helminth fecundity, while both populations were capable of inducing the accumulation of eosinophils in the lung as part of the antihelminth effector response in mice [33].Furthermore, human Th2 subsets expressing CRTh2, CD49d, and CD161 exhibit functional profiles of increased allergen-specific pathogenicity, which are distinct from those of conventional Th2 cells.These cells have been coined Th2A cells [34].These allergen-reactive Th2-biased Th cells with increased pathogenicity and terminal differentiation as well as reduced clonality can also be identified in individuals with allergies, as recently shown for dog allergen-reactive T cells by single-cell RNA sequencing [35].

Regulation of Th9 cell responses
IL-9-producing Th cells represent a distinct specialized cell type within Th2 cells.Unique expression of IL-9 in the absence of other Th2 subset-defining cytokines has led these cells to be called Th9 cells.These cells are derived from naïve T-cell precursors via TGF-β signaling in the presence of IL-4 or "switch" at the effector cell level to IL-9-only producing cells if exposed to TGF-β in mice [8].Staphylococcus aureus derived enterotoxin B has also been reported to drive Th9 cell differentiation in both mice and humans, stressing the importance of microbial factors for the regulation of these cells [36,37].
B-cell activating transcription factor-like (BATF) has been reported to be required for the expression of IL-9 in Th cells [38].Other transcription factors, such as PU.1, STAT6, GATA-3, IRF4, and peroxisome proliferator-activated receptor-γ (PPAR-γ), are considered relevant for the identity of Th9 cells [39][40][41].Caution is warranted in the classification of human Th cells as Th9 cells since IL-9 production is activation dependent, transient, and accompanied by downregulation of other Th2 cytokines (and thus the functional bona fide Th2 cell state) in certain stimulatory conditions [41].

Regulation of Th17 cell responses
Th17 cells are regulated by ROR-γt and produce the signature cytokine IL-17 [42].Th17 cells have gained interest as culprits in the pathogenesis of autoimmune diseases.ROR-γt is relevant for both the differentiation and effector function of Th17 cells [43].Whereas IL-6 and TGF-β have been delineated as Th17 cell priming cytokines in mice, no satisfying consensus has been reached regarding the cytokines needed for human Th17 cell priming [6,44].In particular, the requirement for TGF-β for the de novo generation of human Th17 cells has been questioned since IL-1β and IL-6 were shown to robustly upregulate IL-17 in CD45RA + CCR7 + Th cells [45].Until now, the frequencies of IL-17-producing cells that can be generated from naïve Th cell precursors of different phenotypes, such as CD161-positive or CD161-negative naïve T cells [46], have remained disappointingly low.Despite initial enthusiasm and efforts in deciphering the differential requirements for this Th cell subset, there has been limited progress in the field.However, novel factors beyond cytokine signals, which will be discussed later, have emerged as potent players in the differentiation and stability of Th17 cells.The identity of Th17 cells has diversified amidst the challenge of identifying the mysterious Th17 polarizing cytokine "cocktail".Proinflammatory and anti-inflammatory Th17 cell subsets have been revealed in humans, which are generated in IL-1β-dependent and IL-1β-independent priming conditions, respectively [47].To exert these divergent effector functions, Th17 cells express either IFN-γ or IL-10.This also translates into distinct surface marker expression profiles with IL-17 + /IFN-γ + Th17 cells coexpressing CCR6 and CXCR3 and IFN-γ -/IL-10 + Th17 cells being CCR6 + CXCR3 - [44,48].IL-10 can exert selfregulatory feedback on Th17 cell pathogenicity via autocrine signaling.This mechanism has been shown to be compromised in autoinflammatory syndromes, in which increased systemic IL-1β levels block Th17 cell-intrinsic IL-10 production [49] in patients.How this IL-10-suppressive effect is regulated by IL-1β in T cells remains to be investigated in more detail.In particular, the mechanism underlying the sustained absence of IL-10 expression at the effector Th17 cell level in IL-1β-primed human Th17 cells remains poorly understood.In mice, self-regulatory feedback mechanisms by which Th17 cell pathogenicity is limited have also been proposed, such as the autocrine production of IL-24 [50].
Th17 cell functionalities also correspond to distinct T-cell receptor antigen specificities [22,47].IL-1β-dependent human Th17 cells were shown to recognize Candida albicans, whereas IL-1β independently primed Th17 cells, which instead displayed antigen specificities that were enriched for S. aureus.Accordingly, these C. albicans versus S. aureus specific human Th17 cells differ in the expression of IFN-γ versus IL-10 and thus have different overall functionalities [47].Interestingly, the absence of Th17 cells due to inborn genetic disorders such as those caused by STAT3, STAT1, or RORγt mutations leads to infections with both pathogens but not others [51].This is consistent with the increased risk for candidiasis upon treatment with IL-17 inhibitors [52].This clinical proof-of-concept highlights a restricted repertoire of microbial targets for human Th17 cells with implications for human health and disease.The antimicrobial function of human Th17 cells may also be exerted by IL-26, which kills extracellular bacteria via membrane pore formation and thus confers a broader spectrum of pathogen control [51].

Bringing T cells to higher dimensions: single-cell genomics
The emergence of high-dimensional single-cell technologies has allowed researchers to simultaneously analyze the coexpression of a multitude of proteins (flow cytometry) and gene expression patterns at the single-cell level (scRNAseq) in large heterogeneous cell populations.This has enabled researchers to identify Th cell properties and regulatory networks that have thus far been overlooked.In particular, the detailed analysis of human cells has been accelerated by the advent of these high-dimensional and highthroughput technologies.Tissue biopsies provide a snapshot of the asynchronous transitions between cellular states.Although it is difficult to assign biological relevance to individual functional cell states in such heterogeneous cellular assemblies, when enhanced by hypothesis-driven bioinformatic analysis, this ex vivo situation can yield insights into the differentiation states and trajectories of cellular players at the tissue site of interest even in the absence of repeated tissue sampling over time.

Revisiting Th1 cell diversity through the lens of single-cell genomics
Overall, a systematic analysis of published reports that focus on disentangling the heterogeneity of Th cells on the single-cell level reveals a clear bias for Th17 and Th2 cells at the expense of Th1 cells in both mice and humans.Th1 cells have previously been reported to co-express self-regulatory IL-10 and coinhibitory receptors upon prolonged stimulation to mitigate their inflammatory response [53].CD4 + LAG3 + CD49b + T cells display high and stable coexpression of IFN-γ and IL- 10 [54].This supported the classification of a putative Th cell subset, which has been coined Tr1 subset.ScRNAseq has now revealed distinct roles for the transcriptional regulators cMaf and Blimp-1 in the upregulation of IL-10 and coinhibitory receptors in human Tr1 cells, respectively [55].This was relevant in settings of malaria, where targeting these molecules seem to provide clinical advantages through improved parasitic clearance [55].In mice, it has been shown that type I IFNs regulate the expression of coinhibitory receptors in human T cells.Computational approaches further identified the transcription factor SP140 as a key regulator in this network in mice [56].Its role for the Tr1 cell identity was, however, not supported in human T cells by scRNAseq analyses [55].Another comprehensive scRNAseq and high-dimensional single-cell mass cytometry study identified a hybrid Th1-like cell subpopulation coexpressing GM-CSF with IFN-γ within human memory T cells [7,57], which through IL-12 induced T-bet upregulation exerted profound pathogenicity in a mouse model of neuroinflammation.This highlighted that non-Th1 cells, such as the GM-CSF producing Th-GMCSF cells, can, upon secondary events, adopt features of the Th1 cell subset such as IFN-γ and T-bet expression.This finding is in line with another scRNAseq study that demonstrated that levels of IFN-γ increase proportionally to the effectorness of Th cells.Even Th17-polarized cells acquired IFN-γ expression along their differentiation process upon stimulation through their T cell receptor and costimuli, stressing the concept that the Th1associated features, particularly the signature cytokine IFN-γ, can be adopted by alternative Th cell subsets along their effectorness trajectory [58].

Revisiting Th2 cell heterogeneity through the lens of single-cell genomics
Single-cell genomics analyses have also refined the identity of the Th2 cell subset.Properties that were previously masked by bulk transcriptomic analyses or by biased flow cytometric approaches have been revealed, such as the ability of Th2 cells to produce the immunosuppressive steroid hormone pregnenolone [59].ScR-NAseq analysis of Th cells in the house dust mite model of allergic airway disease has also revealed a role of genes not previously associated with Th2 cell-associated roles, such as Cd200r1, Il6, Plac8, and Igfbp7 [60].Interestingly, this finding suggests an association of IL-6 expression with Th2 cells but not any other Th cell subset.CD200R1 might be assumed to have an autoregulatory function given its receptor-ligand relationship in early Th2 cell differentiation, but more extensive studies are needed to corroborate this notion.ScRNAseq analysis of this mouse model also identified several genes with critical functions in fatty acid metabolism, suggesting that it is a characteristic feature of Th2 cells in the airways [60].Comprehensive dissection of the circuitry governing Th2 cell activation and differentiation has been achieved by combined RNA-seq, ATAC-seq, and ChIP-seq analyses, yielding a rich resource for studies of Th2-associated molecules in mice.This approach not only validated known regulators of Th2 cell identity, such as GATA3, BATF, and IRF4, but also revealed new players of a core regulatory Th2 cell network, such as Pparg and Bhlhe40 [61].
Additional layers of complexity of the classic ROR-γt-driven hub of Th17 cell regulation have been revealed over the last few years.Single-cell genomics analyses have, for example, revealed precursors of differentiating Th17 cells with expression of key regulatory genes such as Med12, Etv6, and Zfx in settings of experimental autoimmunity [62].In effector and memory Th17 cells, on the other hand, the genes Hif1a, Fosl2, Stat4, and Rel have been found to play prominent roles.These genes also promote a Th1-like phenotype, the stability of which remains to be tested further [62].Furthermore, new candidate genes such as Cd5l, Gpr65, Gem, Tmem109, and cd226 have been found to correlate with pathogenicity at the site of inflammation in single-cell genomics analyses in mice [62,63].In contrast, the expression of Foxp1, was found to covary with that of regulatory and other anti-inflammatory genes, consistent with the previously assigned function of Foxp1 in counter-regulating IL-12, the driver of Th1 cell differentiation [62,64].Similarly, transcriptional profiling demonstrated that FAS promotes the stability of murine Th17 cells and prevents their differentiation into Th1 cells, thus protecting against autoimmunity.FAS bound and sequestered STAT1, and thus controlled the reciprocal effects of STAT-1 and STAT-3 on the Th1-Th17 balance [65].Single-cell RNA profiling of pathogenic versus nonpathogenic murine Th17 cells further identified protein C receptor (PROCR) as a cell surface molecule whose expression is related to the regulatory module of Th17 cells.PROCR suppressed pathogenic signature genes such as IL-1 and IL-23 while preserving overall Th17 cell responses [66].Several more genes have recently been revealed using scRNAseq analysis following Th17 cell priming in proinflammatory versus anti-inflammatory cytokine conditions and by comparing Th17 cells from draining lymph nodes with those form the CNS at the peak of experimental autoimmune encephalomyelitis (EAE).Gpr65, Plzp, and Toso, for example, were found to be associated with Th17 cell pathogenicity in mice [62].CD5L/AIM is a regulator expressed in nonpathogenic murine Th17 cells.It serves as a functional switch by modulating the intracellular lipidome, altering fatty acid composition, and restricting cholesterol biosynthesis and thus ligand availability for Ror-γt without perturbing Th17 cell differentiation [63].
Taken together, the findings of these high-dimensional singlecell analyses have yielded complex atlas-like maps of T cells with spatial and temporal resolution.The challenge will be to complement these findings with hypothesis-driven problem-oriented questions and mechanistic validation to make sense of the vast amount of information beyond its descriptive nature and to eventually overcome unmet clinical needs.It is critical for the trustworthy interpretation of the emerging data to rely on good quality starting cellular material.This represents a challenge considering the multitude of existing procedures for the isolation of tissue immune cells.The Human Cell Atlas consortium aims to charts all the cell types of the human body and with this international collaborative initiative also aims for standardized and validated procedures and comparability of data.

Th cells -innate answers to adaptive signals
Immunological memory is an exclusive characteristic of the adaptive immune system and is based on the clonal selection, expansion, differentiation, and persistence of antigen-specific T or B cells [22].Th cells with immunological memory elicit polarized immune responses upon rechallenge with their cognate antigen.Interestingly, innate cells have previously been shown to have adaptive features that allow them to respond to exogenous or endogenous insults with an altered response, which is educated by previous experience and reprogramming.This phenomenon has been coined trained immunity and has blurred the distinction between adaptive and innate immunity [67].Whether adaptive immune cells also have innate functions is far less explored.It has, however, been demonstrated that human Th1 cells engage in NLRP3 inflammasome signaling upon complement activation via C5a receptor 1 (C5aR1), which equips them with the ability to produce IL-1β.This mechanism provides a stimulus for IFN-γ production [68].This example demonstrates how innate danger signaling, which is executed by an inflammasome, can be exploited by Th cells to promote their effector functions.

Regulation of Th cell diversity by the NLRP3 inflammasome
An unbiased global single-cell transcriptomic analysis of human Th17 cells recently revealed the ability of a Th17 cell subpopulation to produce the unconventional cytokine IL-1α [69].This was surprising considering the innate characteristics of this senescence-associated alarmin [70].IL-1α production propagated the proinflammatory Th17 cell fate through continuous autocrine self-amplification but also suppressed IL-10 expression at the effector T-cell differentiation level.This autocrine feedback loop is consistent with the previously described, but until now mechanistically unresolved, continuous IL-10 blockade that can be established by the initial IL-1β stimulus provided by antigen-presenting cells during the early Th17 cell priming phase [47].The previously overlooked IL-1α-producing property of Th17 cells may highlight a new treatment rationale: anti-IL-1αrather than anti-IL-1β-neutralizing antibodies might disrupt this chronic pathogenic IL-10-suppressive feedback loop at the Th17 cell effector differentiation stage to treat chronic inflammatory diseases.Interestingly, this IL-1α-producing property of T cells was found to be restricted to human Th17 cells with T-cell receptor specificity for C. albicans.IL-1α production even significantly contributed to the clearance of C. albicans by enhancing monocyte-mediated phagocytosis.IL-1α therefore partners with IL-17 for efficient clearance of C. albicans infections.Surprisingly, IL-1α production was found to be regulated via the NLRP3 inflammasome, a multimolecular complex known to perform IL-1β cleavage and release through caspase-1, although the IL-1a pro-form does not display any caspase-1 cleavage sites.The NLRP3 inflammasome, instead, engages an alternative cascade including caspase-8, caspase-3, and gasdermin E cleavage in human Th17 cells to permit the extracellular exit of IL-1α via gasdermin E (GSDME) pores [69].This discovery revealed a new molecular pathway for the release of a previously overlooked unconventional cytokine in human Th17 cells via pores [69] (Fig. 1).It is tempting to assign a unique Th cell identity, such as Th-IL1α, to this IL-1α-producing Th17 cell subpopulation, which displays an NLRP3-GSDME axis of cytokine release.The NLRP3 inflammasome has also been detected in murine Th17 cells [71].However, its engagement resulted in IL-1β, but not IL-1α, production, which promoted Th17 cell pathogenicity in EAE.Another role for NLRP3 was observed in the Th2 cell population.In Th2 cells, NLRP3 supported the Th2 transcriptional program in the absence of inflammasome activation by transactivating the IL4 promotor in conjunction with the transcription factor IRF4 [72].Together, these examples demonstrate how innate danger signaling or molecules with specific functions in innate immunity can be hijacked by T cells to exert new functions that blur the boundaries of innate and adaptive immune functions.

Th cells in tissues
It is estimated that the great majority of human T cells reside in peripheral tissues.Tissue-resident memory T cells are sequestered from the circulatory pool of T cells in the blood.It has recently been demonstrated that these T cells are stored in peripheral tissues such as the skin, lung, or gut for several years [73][74][75].Clinical situations such as allogeneic stem cell transplantation or solid organ transplantation can be taken advantage of to track individual immune cells of host versus donor origin in the blood and tissue, providing insights into the spatial compartmentalization of the human immune system over time [73][74][75]76].This approach has identified LGALS3, the gene encoding Galectin-3, as a marker for bone fide human skin resident Th cells.This finding extended the repertoire of available tissue resident marker molecules such as CD69, CD103, and absence of surface S1PR1 and CCR7 [73,77] (Fig. 2).Blood, which contains only 2% of human T cells, can therefore no longer serve as a representative surrogate for the overall human T-cell pool, and the understanding of Th cell diversification needs to be revisited taking tissue-specific factors into account (Fig. 2).
Most insights into the regulation of tissue resident memory T (T RM ) cells have been derived from CD8 + T cells, but more data have begun to emerge for CD4 + T cells.By using IL-17A fate-tracking mouse models, it was demonstrated that effector Th17 cells were capable of persisting as a long-lived exTh17 population, creating a resident cellular network in the lung airways [78].Tissue maintenance was supported by IL-7 derived from lymphatic endothelial cells.Interestingly, a significant fraction of these long-lived CD4 T RM cells lost their original effector cytokine expression and instead reverted to IFN-γ-producing cells upon restimulation, which is in line with the functional flexibility and plasticity previously described for circulating Th cells [78].Another elegant study identified T RM cells with a Th17 cell polarization pattern in the kidney following infection with S. aureus or C. albicans in a mouse model.These cells were shown to persist in loco after clearance of infection and to contribute to autoimmune tissue damage upon reactivation by local inflammatory cytokines [79].The same study also reported Th1-and Th2-polarized T RM cells in the human kidney, whose mode of generation remains to be further explored [79].Tissue-resident Th2 cells have also been described to have potent functions in peripheral tissues such as the lung.In adipose tissue, these cells have recently been found to be associated with tissue remodeling following intestinal infection [15].IL-2 was identified as a requirement for residency and for directing a tissue migratory program [80].Transcriptomic trajectory analyses of single cells showed that the genes IL13 and IL5 were restricted to effector/resident memory populations of Th2 cells in the lung, supporting previous data on their association with terminal differentiation [31,81].
The long-term storage of T cells in diverse tissue niches exposes them to tissue-specific extracellular microenvironments.This tissue imprinting is expected to functionally diversify Th cells with comparable differentiation based on their tissue cross-talk.One factor that has until recently been largely overlooked as an immunostimulatory agent for Th cells is sodium chloride (NaCl).Previous reports suggest that NaCl accumulates in peripheral tissues, such as the skin, in response to a high-salt diet or to infection [82].Negatively charged macromolecules such as hyaluronic acids, whose concentrations might be tissue specific, are thought to contribute to the spatial compartmentalization of NaCl by electrostatic attraction [83].NaCl promoted the differentiation of murine and human Th17 cells in the presence of polarizing cytokines in vitro, as seen by striking upregulation of IL-17 [84,85].In effector Th17 cells, NaCl induces upregulation of Foxp3 and several of its downstream anti-inflammatory target genes, such as LAG3, IL10, and TGFb [86].This result strongly suggests that NaCl induces anti-inflammatory conversion of fully differentiated Th17 cells, with implications for the amelioration of chronic inflammatory diseases.In fact, NaCl pretreatment of murine T cells resulted in a very strong reduction in EAE scores upon adoptive transfer in mice [86].It remains to be seen whether the compartmentalization of NaCl concentrations in various tissue niches affects Th cell polarization in vivo and to what degree these NaCl concentrations could be amenable to modulation by diet or other extrinsic processes.NaCl also had an impact on Th2 cell differentiation [30,87].It induced Th2 cell polarization even in the absence of exogenous IL-4 and promoted type 2 cytokine production in effector Th2 cells [30,87].In line with these in vitro observations in mice and humans, elevated NaCl concentrations were observed in the tissues of patients suffering from atopic dermatitis [87].Although NaCl signaling in T cells remains poorly explored, NFAT5 and SGK-1 respond to extracellular increases in NaCl.Both molecules determine the cytokine pattern of Th cells at high but not low NaCl concentrations [82], which reveals that the dependency of cytokine expression by Th cells on distinct transcription factors and other molecules can be determined by extracellular salinity.Considering that most of the in vitro studies that have provided fundamental insights into the regulation of Th cell functions were conducted in NaCl concentrations that mimicked the immunolog-ically inert low-salt blood compartment, revisiting basic concepts of Th cell immunoregulation in high-salt conditions, which better reflect physiological and pathological tissue conditions, is warranted.In addition to these tissue-associated stimuli, metabolites and microbial products, as well as physical stimuli such as temperature and pressure, have also been reported [88,89].This promotes the expansion and pathogenicity of Th17 cells [91].Genome-wide association studies have observed polymorphisms in IL23R in inflammatory bowel diseases and other chronic inflammatory diseases, such as psoriasis [92][93][94].Furthermore, IL-23 levels are increased in the gut mucosa of Crohn's disease patients [95].These observations are in line with the strong efficacy of IL-23-or IL-23R-blocking biologics in Th17 cell-mediated chronic inflammatory diseases.Similarly, direct blockade of the effector cytokine IL-17A or its receptor IL-17RA has proven highly efficacious in psoriasis and other Th17 cell-mediated diseases [96].Along with strategies targeting inducing, enhancing and effector cytokines of pathogenic Th cells, monoclonal antibodies targeting Th2 effector cytokines (IL-4Rα, IL-4, and IL-13) have also entered the clinic with great success in the treatment of atopic dermatitis and asthma [97,98].IL-13, in particular, increases mucus production and goblet cell hyperplasia, stimulates the activity of bronchial smooth muscle cells and promotes the extracellular deposition of collagen, thus promoting airway remodeling in asthma [99].Antibodies blocking IL-5 binding to IL-5R have also entered the clinic, thus inhibiting the maturation, activation, proliferation, and recruitment of eosinophils in the airways with beneficial effects in asthma [100].While these strategies were directly fueled by the systematic interrogation of the classic determinants of Th cell differentiation and maintenance, new potential biologics have entered the arena based on more specific alterative properties of the classic Th cell subsets.Surprisingly, IL-13 has recently been suggested to drive COVID-19, and anti-IL-13 therapy has been shown to protect against severe COVID-19 in both humans and mouse models despite the overwhelming Th1 cytokine-associated cytokine storm [101].This has been shown to be associated with a reduction in the gene expression of hyaluronan synthase 1 (Has1) and of the hyaluronan receptor CD44 [102].This finding encourages the continued application of dupilumab (IL4Rα antagonist) during the SARS-COV2 pandemic [103].IL-31, which is a product of Th2 cells, is being targeted by monoclonal antibodies (IL-31RA, nemolizumab) to treat itch in atopic dermatitis, prurigo nodularis and other pruritic diseases.Additionally, alarmins, such as TSLP and IL-33, are current targets in type 2 diseases.IL-1α, another alarmin, can also be depleted by direct neutralization or by blockade of IL1R1 with great clinical success in autoinflammatory syndromes and rheumatoid arthritis [104].The recent observation that the alarmin IL-1α confers pathogenicity to Th17 cells suggests a clinical benefit of targeting this proinflammatory Th17 cell mediator selectively while sparing overall IL-17 production [69].However, the differential contribution of IL-1α versus IL-1β, as well as the relevance of their cellular sources, remains to be dissected in detail [104].This endeavor is expected to be accelerated by current clinical studies with selective IL-1α-blocking therapies [104].

Outlook
In summary, the therapeutic approaches based on cytokine neutralization and receptor blockade exemplify in a nonexhaustive manner how potent immunomodulation induced by targeting polarization factors of Th cells can be used for the treatment of a wide spectrum of chronic inflammatory diseases ranging from allergies to autoimmunity.New insights gained from regulatory networks governing the differentiation and effector function of tissue-resident T cells at sites of inflammation will most likely shape the future of targeted Th cell-based therapies for immunemediated diseases.This will be facilitated by recent advancements in single-cell technologies as well as new tools for highdimensional hypothesis-driven data analysis.Overall, a significant increase in insights about the functionalities, regulatory networks, and thus overall complexity of Th cells can be expected in the near future.This will blur the rigid classical categorization into bona fide Th cell subsets.Multifunctionality and contextual cellular adaptations will probably define Th cell states and will superimpose their original lineage identity.In addition to polarizing cytokines, which have prevailed as the main inducers of Th cell functions for the last 30 years, new factors such as tissue site, residency status, microbes, metabolites, ionic signals, and other microenvironmental factors, have emerged as highly relevant factors in shaping the short-or long-term identity of Th cells.This opens up exciting opportunities for systems immunologists in the years to come.
Excellence Strategy (Balance of the Microverse, C.E.Z.) and the Carl-Zeiss Stiftung (C.E.Z.).Open access funding enabled and organized by Projekt DEAL.

Figure 1 .
Figure 1.Human Th17 cell pathogenicity is shaped by autocrine IL-1β induced IL-1α secretion via gasdermin E pores.Left, TCR-activated human Th17 cells produce self-regulatory IL-10 and the effector cytokine IL-17.Right, exogeneous IL-1β (provided during Th17 cell priming by APC) induces IL-1α, which is secreted via gasdermin E membrane pores.By sharing the same receptor with IL-1β, IL-1α suppresses IL-10 production and promotes IL-17 production, thereby serving the role to enhance Th17 cell pathogenicity.

Figure 2 .
Figure 2. The tissue microenvironment blurs and diversifies T helper cell (Th cell) subsets entering from the circulation.Previous work on human Th cells identified various Th cell subsets.They are induced from naïve Th cell precursors upon encounter of antigen, which is presented by specialized antigen-presenting cells.This occurs in a polarizing cytokine microenvironment including additional signals, which are discussed.Whether their identity is maintained upon seeding peripheral tissues such as the skin (shown here) and to what extent they adopt new phenotypes and functions in vivo upon receipt of tissue-related signals such as microbial antigens and microenvironmental factors (i.e.NaCl) are currently intensely investigated.Tissue resident T cells (TRM) can be identified by various surface markers (as indicated) whose functional role remains to be elucidated in more detail.
Biologics and other molecule-targeting drugs have started to be used in clinical medicine with great advances over the last few years due to their high efficacy and specificity and good safety profile with short-term use.Most of the currently used biologics target T-cell effector cytokines or T-cell polarizing cytokines, thus directly interfering with the establishment or execution of Th cell responses.Insights into the regulation of Th17 cells were rapidly translated into the application of IL-23-and IL-17A-neutralizing drugs.While initially considered a Th17 cell priming cytokine [90], IL-23 has now been established to be of relevance for the expansion and maintenance of Th17 cells because it provides positive feedback for IL-17, ROR-γt, IL-1, and IL-6 expression.