The CD4-centered universe of human T cell subsets.

Seminars in Immunology 25 (2013) 252–262

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Review

The CD4-centered universe of human T cell subsets J. Geginat a,∗ , M. Paroni a , F. Facciotti a , P. Gruarin a , I. Kastirr a , F. Caprioli b,c , M. Pagani a , S. Abrignani. a a b c

Istituto Nazionale di Genetica Molecolare (INGM), Autoimmunity Program, Via Sforza 35, 20122 Milan, Italy Department of Pathophysiology and Transplantation – University of Milan, Italy Unit of Gastroenterology 2, Fondazione IRCCS Cà Granda, Ospedale Policlinico, Milan, Italy

a r t i c l e

i n f o

a b s t r a c t

Keywords: CD4+ memory T cells T cell differentiation Cytokines Tissue homing

Humans are continuously exposed to a high number of diverse pathogens that induce different types of immune responses. Primary pathogen-specific immune responses generate multiple subsets of memory T cells, which provide protection against secondary infections. In recent years, several novel T cell subsets have been identified and have significantly broadened our knowledge about T cell differentiation and the regulation of immune responses. At the same time the rapidly growing number of incompletely characterized T cell subsets has also generated some controversies. We therefore review here the current knowledge on features and functions of human ␣/␤ T cell subsets, focusing on CD4+ T cells classified according to cytokine production and tissue localization. The principal helper and regulatory T cell subsets can be identified by a limited number of relevant surface markers, which are an integral part of the T cell differentiation programs because they are directly induced by the relevant lineage-defining transcription factors. In vivo occurring human T cell subsets can thus be purified directly ex vivo from relevant tissues for molecular and functional studies, and represent not only an ideal model to study T cell differentiation, but they also offer important clinical opportunities. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction

relevant tissues and to secrete the appropriate effector cytokines. Once the pathogen is cleared the majority of the activated effector T cells die, but some survive and become long-lived memory T cells that can mediate protection against secondary infections [6,7]. Although the effector-memory transition is not completely understood, it was shown that insufficient TCR stimulation results neither in the expression of critical survival molecules nor in the ability to respond to critical survival factors like IL-7, thus leading to a state of unfitness and abortive proliferation [8,9]. On the other hand, also excessive or chronic stimulation can compromise the fitness of primed T cells, since over-stimulated effector cells shut down IL-7R expression and become susceptible to activation-induced cell death [10–12]. Some of the surviving T cells become resident memory cells in non-lymphoid tissues and can mount immediate responses upon secondary infections, while others continuously circulate through the blood and secondary lymphoid organs [13]. Memory T cells can fight the same pathogen more rapidly and efficiently upon re-infection, because they rapidly migrate to the infected tissue and produce the appropriate effector cytokines [14]. At the molecular level this topological and cytokine memory is maintained by epigenetic mechanisms such as DNA de-methylation and permissive histone acetylations [15–18]. Consequently, memory T cells that recognize their specific pathogen can perform effector functions in a few hours, and since they are clonally expanded they also more efficiently generate secondary waves of effector T cells.

Through their lifetimes human beings are continuously exposed to highly diverse pathogens like viruses, bacteria and fungi. Since these pathogens have developed sophisticated strategies to avoid rapid elimination by the innate immune system, an adaptive immune response is often required to control these infections [1]. CD4+ T cells coordinate adaptive immune responses through the secretion of cytokines and cell-to-cell contacts, while CD8+ T cells are largely pre-committed to become cytotoxic effector cells. Naive T cells that are exported from the thymus express a highly diverse T cell antigen receptor (TCR) repertoire and are thus potentially able to recognize all kinds of pathogens [2]. In peripheral tissues pathogens are sensed by dendritic cells (DC), which then instruct an appropriate primary T cell response in secondary lymphoid organs [3]. DC that recognize a pathogen undergo a complex maturation program, i.e. they up-regulate MHC and co-stimulatory molecules, migrate to lymph nodes, interact with pathogen-specific T cells and secrete appropriate polarizing cytokines [4,5]. The strength of TCR-transduced signals and the type of cytokines that DC deliver to naive T cells determines their differentiation to various types of effector cells that have acquired the abilities to home to the

∗ Corresponding author. Tel.: +39 02 00660206. E-mail address: [email protected] (J. Geginat). 1044-5323/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.smim.2013.10.012

J. Geginat et al. / Seminars in Immunology 25 (2013) 252–262

2. Constant pathogen exposure results in complex T cell pools Since humans encounter a high number of diverse pathogens throughout their lives, it is not surprising that the number of differentiation stages or T cell subsets that are required to protect us from these pathogens is also very high. It is well established that different classes of pathogens induce different types of T cell responses, and consequently CD4+ T cells directed against viruses, parasites or fungi secrete characteristic effector cytokines and belong to different subsets [19–23]. Notably however, T cell populations specific for the same pathogen can also belong to different subsets, and this can occur for several reasons: Different T cell clones can respond to different antigens from the same pathogen, as is the case for proteins expressed during the lytic cycle of Epstein Barr virus (EBV) that induce more differentiated CD8+ T cell populations as compared to proteins expressed during the latent stage [24]. T cells can also receive different levels of stimulation during priming at different time points, since the early stage of infection is normally accompanied by pro-inflammatory cytokines, while anti-inflammatory cytokines might be dominant at later stages to limit excessive tissue damage. A dramatic example for functional difference in the same T cell clone is the proposed asymmetric cell division of primed T cells, where the first cell division results in two specialized daughter cells with one becoming an effector and the other one a memory T cell [25]. Moreover, since primed T cells detach from DCs during cellular division, the two daughter cells can have distinct fates if for example one leaves the lymph node while the other one encounters a second DC and thus receives further stimulation [26–28]. Furthermore, T cells can be primed by several DC subsets, which express different levels of MHC molecules, distinct patterns of co-stimulatory receptors and have specific cytokine profiles [29–31]. Activated T cells with the same specificity can either home to B cell follicles in lymphoid organs and interact with antigen-presenting B cells, or migrate to non-lymphoid tissues where they encounter tissueresident macrophages or DC and their cytokine products [32,33]. Finally, as T cell cross-reactivity has been documented between different viruses [34] and between foreign and self-antigens [35,36], pathogen-specific memory T cells that cross-react with a different antigen could acquire additional properties in secondary responses.

3. Classification of T cell subsets according to cytokine production or surface markers Given the enormous heterogeneity of antigen-experienced T cells it is necessary to classify the pool of human memory T cells. Since the main T cell subsets produce characteristic cytokines, the secretion of effector cytokines is often the criterion of choice. Intracellular cytokine staining has the advantage to allow multiparameter analysis at the single cell level, but the disadvantage is that T cells have to be activated and killed for analysis, and a molecular or functional analysis of cells is therefore impossible. Viable T cells isolated according to cytokine production can be obtained [37], but only for selected cytokines and the cells have still to be activated for cytokine production. Large numbers of T cells producing different type of key cytokines can be generated in vitro, but the signals that drive T cell differentiation in vivo can only partially be recapitulated in culture. Consequently, in vitro generated T cells are partially unstable [16,38] and differ in several relevant aspects from their in vivo-occurring counterparts. However, several surface receptors such as many chemokine or cytokine receptors and co-stimulatory or inhibitory receptors are expressed on resting T cells, and their detection does consequently

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neither require in vitro activation nor fixation. Thus, specific surface markers allow the isolation and analysis of non-activated, viable and in vivo differentiated human T cell subsets that express characteristic genes and microRNAs [39]. Human peripheral blood is readily available for research and offers access to a high number of different T cell subsets. Nevertheless, T cells perform their functions in lymphoid or non-lymphoid tissues and some populations are exclusively found in selected tissues. It is therefore particularly relevant to study human T cells in solid tissues, although the availability of these tissues and the cell number may represent a limitation. 4. Heterogeneity of naive T cells Naive T cells have by definition not yet encountered their specific antigen and are the precursors of most antigen-experienced T cell populations. They circulate in peripheral blood and enter lymph nodes via high endothelial venules. For this process they require L-selectin (CD62L) and the chemokine receptor CCR7 [40,41]. Conversely, they have no access to non-lymphoid tissues because they lack the relevant adhesion molecules and chemokine receptors. Human naive CD4+ T cells are identified by the expression of the longest isoform of the common lymphocyte antigen CD45 (CD45RABC, “CD45RA”) and/or the lack the shortest isoform (CD45R0) [42]. CD45 is a tyrosine phosphatase that regulates the activation threshold of T cells, and the long isoform might contribute to the high activation threshold of naive T cells [43]. However, although CD45RA is lost upon T cell activation, it can be re-expressed on fractions of memory/effector cells, in particular in the CD8 compartment of HCMV-infected individuals [44]. For this reason expression of additional markers are required in combination with CD45RA and/or CD45R0 to discriminate between naive and antigen-experienced T cells. Co-expression of CD45RA and CCR7, CD62L or the co-stimulatory receptor CD27 is often used to isolate naive T cells, because these receptors are lost on fractions of effector and memory cells [45–47]. Naive T cells are often assumed to be a homogeneous population of uncommitted precursor cells that differ only in TCR specificities and recognize consequently different antigens. However, some naive T cells are already committed to a specific differentiation lineage, while others have acquired different characteristics upon antigen-independent homeostatic proliferation. In particular, CD31 expression was shown to discriminate between recent thymic emigrants and peripherally expanded naive T cells that accumulate upon aging in the CD4+ T cell compartment to compensate for the reduced thymic output in adults [48]. Uncommitted naive T cells do not secrete effector cytokines, but a fraction of them produces IL-2, and these IL-2 producing naive T cells have enhanced memory/effector functions in mice [49]. Finally, pre-committed natural regulatory T cells (Treg) and IL-17 secreting Th17 cells have been identified in the thymus in humans [50] and mice [51,52]. In humans, these pre-committed naive T cells can be discriminated among CD45RA+ cells by CD25 and CD161 expression from uncommitted naive T cells [50,53]. These pre-committed naive T cells and CD45RA+ memory T cells are a caveat of many human T cell differentiation studies, in particular when T cells are purified from adults and not from neonates. 5. Cytokine production identifies three major helper T cell subsets: Th1, Th2 and Th17 cells There is broad consensus in the field that CD4+ T cells can belong to a limited number of well-defined differentiation “lineages”, that have specific functions in protective but also in aberrant immune responses. These T cell subsets secrete a specific pattern of effector cytokines and have a characteristic phenotype

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Fig. 1. Lineage-defining transcription factors induce cytokine production and surface markers. Naive T cells (center, gray) can differentiate to the various indicated types of helper T cells (colored) upon priming in response to different cytokines. These helper T cell subsets express “lineage-defining” transcription factors that induce specific surface markers in the steady-state, and characteristic effector cytokines following TCR stimulation.

in humans. Helper T cells belonging to these lineages express a “master” or “lineage-specifying” transcription factor that regulates their generation, effector cytokine production, phenotype and function and inhibits the differentiation to an alternative lineage (Fig. 1) [54]. Nevertheless, it has become increasingly clear that cells belonging to these differentiation lineages are not exclusively terminally differentiated cells, but that some maintain a certain degree of plasticity and can acquire characteristics of alternative lineages upon antigenic re-stimulation [55,56]. The three principal helper T cell lineages are Th1, Th2 and Th17 cells [57]. 5.1. Th1 cells target intracellular pathogens Th1 cells are required to fight intracellular pathogens like intracellular bacteria and viruses [57], but an overshooting Th1 response can cause lethal immunopathology [58]. They are generated upon naive T cell priming in the presence of IL-12 and express the transcription factor T-bet, which induces the prototypical Th1 effector cytokine IFN-␥ [59]. IFN-␥ can activate macropahages to destroy intracellular bacteria and promotes IL-12 production by dendritic cells, inducing thus a positive feed-forward loop of Th1 induction. T-bet also induces the chemokine receptors CXCR3 that allow Th1 effector cells to enter peripheral tissues [60]. CXCR3 is expressed on virtually all in vivo occurring human Th1 cells and allows the isolation of high numbers of resting Th1 memory T cell subsets [39,61]. Ex vivo isolated CXCR3+ Th1 cells express T-bet, produce high amounts of IFN-␥ and proliferate in response to several

relevant viral and bacterial antigens, including influenza-, vacciniaand cytomegalovirus as well as mycobacteria (Fig. 2) [61]. Moreover, they have a specific gene and microRNA signature [39]. 5.2. Th2 cells fight extracellular parasites Th2 cells are important to fight extracellular parasites like worms and are involved in allergic diseases like asthma [62]. Th2 cells are induced by naive T cell priming in the presence of IL-4, and express the transcription factor GATA-3, which in turn induces the type 2 cytokines IL-4, IL-5 and IL-13 [63,64]. Moreover, GATA3 induces the chemokine receptor CCR4 and the prostaglandin D2 receptor CRTh2 in humans [65]. IL-4 inhibits Th1 and Th17 cells and induces B cells to produce IgE antibodies, while IL-5 activates eosinophils. Prostaglandin-D2 is produced by mast cells and orchestrates the interaction between Th2 and their target cells in allergic asthma. Ex vivo isolated human CRTh2+ cells express high levels of GATA-3, secrete very high levels of type 2 cytokines [66] and respond to allergens (Fig. 2) [67]. Moreover, they have a specific gene and microRNA signature that is distinct from Th1 cells [39]. While CRTh2 in peripheral blood is expressed on a small subset of highly pure Th2 cells, CCR4 is also expressed on subsets of regulatory T cells and Th17 cells and is therefore not suited to track Th2 cells in the absence of additional markers [68]. Although Th1 and Th2 differentiation were for a long time considered to be mutual exclusive cell fates, many T cell clones co-produce IFN-␥ and IL-4 [69]. Moreover, a small fraction of human T cells co-produces IFN-␥ and IL-4 ex vivo, but the function of these cells is unclear.


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Fig. 2. Human CD4+ T cell subsets specific for different pathogens can be identified by surface marker expression. Helper cells among total CD4+ T cells can be distinguished from regulatory T cell subsets by IL-7R (CD127) expression and absence of CD25. Co-expression of CD45RA and CCR7, and absence of CD95 allows the identification of naive cells among helper T cells. CD4+ memory cells are predominantly CD45RA− , and can be sub-divided into CXCR3+ Th1 cells and CRTH2+ Th2 cells. The majority of memory cells lack CXCR3 and CCRTH2 expression and contain CCR6+ Th17 cells, CCR10+ Th22 cells and a major fraction of non-polarized central memory T cells that lack these markers.

5.3. Th17 cells control fungi and extracellular bacteria Th17 cells are important for immune responses against fungi and extracellular bacteria, and are thought to give rise to the pathogenic cells that drive chronic inflammation in organ-specific autoimmune diseases [21]. Th17 cells express the transcription factor RORC that in turn drives IL-17 production and the expression of the chemokine receptor CCR6, as well as of the IL-23R [22,70,71]. IL-17 acts via epithelial cells to attract neutrophils, while CCR6 is important to recruit Th17 cells to the inflamed gut or skin [72]. Th17 cells can be induced in the mouse by the combination of TGF-␤ and IL-6 [73], but IL-1␤, IL-21 and IL-23 also promote Th17 differentiation [74–77]. The same cytokines were found by several different groups to induce or expand also human Th17 cells, although inconsistent data has been published on the contribution of single cytokines, in particular TGF-␤ [50,78,79]. CCR6 is induced by the same cytokines as IL-17 [22,36], and its expression is stably imprinted in CCR6+ human T cells [16]. Moreover, it is expressed on virtually all IL-17 producing memory cells and is therefore the best available marker for human Th17 cells [80]. Nevertheless, CCR6 is also expressed on CD25+ regulatory T cells [81], and the large majority of CCR6+ CD25− T cells lack IL-17 producing capacities [36]. However, the majority of human Th17 cells also express CD161 and CCR4 [22,82], and these markers are therefore useful to enrich IL-17 producing cells among CCR6+ T cells. Ex vivo isolated human Th17 cells express a characteristic set of genes and microRNAs [39], and

they proliferate with antigens derived from Candida albicans and Staphylococcus aureus [83], the two pathogens that cause infections in patients with genetic defects in the Th17 pathway (Fig. 2). 6. Novel T helper subsets: Th1/17, Th22 and Th9 cells Besides Th1, Th2 and Th17 cells several additional subsets that are related to, but distinct from these principal subsets have recently been identified, but are less defined (Fig. 1). In particular, Th17 cells can acquire characteristics of Th1 cells under conditions of chronic inflammation [84,85]. These Th1/17 cells are pathogenic in murine autoimmune models and some co-produce IFN-␥ and IL-17, while others lose IL-17 producing capacities [84,85]. Moreover, T cells that produce IL-22 or IL-9 but not IFN-␥, IL-4 or IL-17 have been identified and appear to represent independent subsets of Th22 and Th9 cells [86–89]. 6.1. Th1/Th17 cells drive chronic inflammation in autoimmune diseases Several autoimmune diseases are strongly associated with polymorphisms in the IL-23R [90–92] that can impair IL-23R signaling [93], suggesting a pivotal role of IL-23 in autoimmunity. IL-23 is not required for Th17 cell priming, but it is important to induce expansion and maturation of pre-committed Th17 cells [94]. Moreover, it promotes a switch from “classical” Th17 cells, which express

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IL-17, IL-10 and RORC, to “alternative” Th17 cells or Th1/Th17 cells, which express T-bet, IFN-␥ and GM-CSF in addition and drive experimental autoimmune diseases [95]. Importantly, in mice these cells are generated in chronic immune reactions, but not in response to acute infections [84]. In humans, IL-23 together with IL-1␤ promotes IL-17 and IFN-␥ co-production in pre-committed Th17 cells [50], and IL-17/IFN-␥ co-producing T cells are increased in several autoimmune patients [70,96]. Interestingly, also high salt concentrations promote pathogenic Th17 differentiation [97]. IL-17/IFN-␥ co-producing cells are present at low frequencies in circulating CD4+ T cells co-expressing the Th1 and Th17 markers CXCR3 and CCR6 [22], and these CXCR3+ CCR6+ memory cells respond to mycobacterial antigens and produce high levels of IFN-␥, but only low levels of IL-17. Interestingly, it was proposed that in inflamed tissues also IL-12 could convert Th17 cells to “nonclassic” Th1 cells that have down-regulated IL-17 production [85,98,99]. Finally, it was shown that Th17 cells primed by S. aureus or C. albicans have cytokine profiles of classical and alternative Th17 cells [83], but surface markers to distinguish between these two types of Th17 cells have not been identified so far. 6.2. Human Th22 cells are involved in skin immune defense Th17 cells also express IL-22 [100], a cytokine that induces proliferation of epithelial cells and that is involved in psoriasis [101,102]. However, IL-17 and IL-22 induction in human T cells have different requirements [86,87,103], and T cells that stably produce IL-22 but not IL-17 can be identified and cloned and were proposed to represent an independent subset of Th22 cells. Human Th22 cells home to the skin and can be identified among CCR6+ CCR4+ cells by CCR10 expression [86,87]. Moreover, they express the transcription factor AHR that drives IL-22 production, although AHR expression is not limited to Th22 cells [104,105]. IL22 is also a critical cytokine in the gut [106–109], but gut-homing Th22 cells are poorly characterized. Moreover, IL-17 and IL-22 have sometimes synergistic but also opposing functions [100,110,111], and how Th22 cells are related to IL-22 producing Th17 cells is currently unclear. 6.3. Th9 cells: a novel subset related to Th2 cells T cells producing IL-9 can be generated in response to TGF-␤ and IL-4 in mice and humans [88,89], although neonatal human T cells require IL-1␤ and IL-10 in addition [112]. IL-9 producing cells are involved in allergic asthma and autoimmunity and depend on the transcription factors PU.1 [112,113] and IRF4 [114], but the latter is also involved in Th2 and Th17 differentiation [115,116]. Nevertheless, it has been proposed that IL-9 producing T cells that do not produce IL-4, IFN-␥ or IL-17 represent an independent subset of Th9 cells. Interestingly, these cells have potent anti-tumor effects [117,118]. Th9 cells are present in human peripheral blood and in the skin, are decreased in melanomas [117] but increased in chronic helminth infections [119]. Moreover, they respond to peanut allergens selectively in allergic patients [120]. Surface markers to isolate human Th9 cells have not been identified so far, and human IL-9 secreting T cells are consequently poorly characterized.

7. Regulatory T cell subsets Regulatory T cells (Tregs) have the task to prevent unwanted immune reactions and to maintain tolerance to self. Several subsets of T cells with regulatory activities have been described among CD4+ T cells and are currently tested in clinical trials [121,122]. Some regulatory T cell subsets secrete the anti-inflammatory

cytokines IL-10 or TGF-␤, but the identities of these cells are incompletely understood. 7.1. CD25+ Foxp3+ Tregs are critical to maintain self-tolerance The best-defined CD4+ Treg subset expresses constitutively the high affinity receptor for IL-2, CD25, and the transcription factor Foxp3 [123–125]. IPEX patients have mutations in the Foxp3 gene, consequently lack CD25+ Tregs and suffer from multiple organ autoimmune manifestations, suggesting that CD25+ Tregs are required to prevent autoimmunity [126,127]. CD25+ Tregs require IL-2 signaling for their survival and suppressive functions, and intriguingly Foxp3 directly induces CD25 expression but blocks IL-2 production [128]. Conversely, the IL-7R␣ chain CD127, which is highly expressed on memory/helper T cell subsets and required for their survival, is down-regulated by Foxp3 [129]. Thus Foxp3+ Tregs can be distinguished from helper T cells and purified by the combination of CD25 and CD127 (Fig. 2). More than 90% of ex vivo purified CD25hi CD127lo cells express Foxp3 and have very potent suppressive functions in vitro [130]. Foxp3+ Tregs are autoreactive [131], but they also recognize non-self antigens [132]. CD25+ Tregs are a heterogeneous population and it is debated if they are a stable population or if they can be re-programmed and become effector cells [133–137]. It is known that Tregs can be generated in the thymus [53] or in the periphery, and naive thymic-derived Tregs can be identified by CD45RA expression in humans [138,139]. In addition, the expression of the transcription factor Helios was proposed to identify thymic Tregs [140], but recent reports have questioned this notion [141,142]. Thus it is unclear how to distinguish thymic and peripherally induced Tregs among antigen-experienced CD45RA− CD25+ T cells [138]. Tregs are largely devoid of effector cytokine producing capacities, but produce suppressive cytokines including IL-10 [143,144] and TGF-␤ [145–148], while IL-35 production by human Tregs is discussed [149,150]. Nevertheless, chemokine receptors identify different antigen-experienced Treg subsets that express the characteristic transcription factors and also the cytokines of Th1, Th2 and Th17 cells [151]. In mice it was shown that Treg subsets suppress the effector subsets they are molecularly related to [152], but in humans the possible contamination with effector cells that have transiently up-regulated Foxp3 is a caveat [153]. However, human IL-17-producing Tregs that stably maintain high levels of Foxp3 as well as regulatory activities have been identified and cloned [154,155]. Moreover, demethylation of regulatory elements in the Foxp3 gene discriminates between regulatory T cells with stable Foxp3 expression and activated cells that only transiently express Foxp3 [38]. 7.2. Type 1 regulatory (Tr1) cells control intestinal homeostasis via IL-10 Tr1 cells are characterized by IL-10 production, a potent antiinflammatory cytokine, and IL-10-dependent suppression of T cell responses [156]. IL-10 produced by CD4+ T cells is critical to prevent colitis in response to commensals in humans and mice, and IL-10 producing Foxp3+ Tregs and Tr1 cells appear to be selectively important for the maintenance of immune homeostasis in the small and large intestine, respectively [143,144,157–160]. Based on their cytokine profile it was originally proposed that Tr1 cells may represent an independent CD4+ T cell lineage derived from naive T cells upon priming with IL-10 [161,162] or more recently with IL-27 [163,164]. However, the identification of IL-10-dependent regulatory T cells that co-express IFN-␥ or IL-17 [165–167] suggested that cells with Tr1-like properties could also be generated from different T helper subsets upon tolerogenic or chronic activation [168]. Notably, IL-10 is produced by several T cell subsets


and also promotes B cell responses [169,170], suggesting that IL10 production per se is insufficient to identify Tr1 cells. Moreover, while IL-10 reporter mice allow the identification of Tr1 cells in vivo [171], in humans they are nearly exclusively generated in vitro and there are several different protocols to induce them [162,172–174]. However, it was recently shown that Tr1-like cells are present in humans among the rare circulating CD4+ T cells that express neither the memory marker IL-7R nor the Treg marker CD25 [166]. These in vivo-occurring Tr1-like cells co-produce IFN␥ and IL-10, are chronically activated by persistent antigens and suppress T cell responses in an IL-10-dependent manner [166]. IL10 producing regulatory T cells also respond to allergens [175], and both human and murine Tr1 cells were shown to express LAG-3 [176] together with CD49b [177]. Human Tr1 cells identified according to Lag-3 and CD49b produced less IFN-␥ than Th1 cells, but expressed CD226, a marker involved in cytotoxic functions of in vitro generated Tr1 cells [178]. How Tr1 cells are related to conventional cytotoxic T cells [179] is however unclear. Moreover, although several transcription factors that regulate IL-10 production in developing Tr1 cells have been identified, including AHR, c-MAF [104,105] and BLIMP-1 [180,181], they are not exclusively expressed in Tr1 cells, and no Tr1-specific transcription factor has been identified so far. A distinct subset of IL-10 producing T cells with regulatory functions is present in human blood and spleens. Unlike Tr1 cells they express IL-7R and CCR6 and are thus a population of memory T cells [36]. CCR6+ T cells produce IL-10 in response to self-MHC displayed by myeloid DC in the steady-state, and can inhibit auto-reactive T cells via IL-10. However, they produce IL-2 and up-regulate CD40L in response to optimal stimulation by recall antigens, and could thus acquire helper functions in secondary immune responses [36]. These IL-10 producing memory cells are therefore distinct from Tr1 cells and appear to have a context-dependent regulatory/helper function.

7.3. Th3 cells: a critical source of TGF-ˇ in the gut Oral tolerance induction exploits the tolerogenic properties of the mucosal immune system and is associated with the appearance of regulatory T cells that produce TGF-␤, a potent antiinflammatory cytokine [182]. T cells produce TGF-␤ in a latent form (LAP), which is displayed on the cellular surface by the GARP membrane receptor [147] and converted to the biologically active form by intestinal DC expressing ␣v/␤8-integrins [183]. Some Foxp3+ Tregs are LAP+ and require TGF-␤ for some of their suppressive functions [145,184]. In addition, both in humans and mice there is evidence for a LAP+ T cell subset that is distinct from Foxp3+ Tregs [185]. These Th3 cells can be directly isolated according to LAP or GARP surface expression and discriminated from LAP+ Foxp3+ Tregs by CD25. LAP+ T cell subsets are likely to represent a relevant source of TGF-␤, in particular in the gut.

8. T cell subsets classified according to their localization or homing potential Memory and effector T cells secrete different combinations of cytokines that are important for their function, but they also express different chemokine and homing receptors that guide them to the tissues where they are needed, adding an additional layer of complexity to T cell differentiation. In particular, some antigen-experienced T cell subsets recirculate preferentially to lymphoid organs, where they scan DC for antigen or provide help for B cell antibody production [13]. Conversely, others can be recruited rapidly to non-lymphoid tissues upon inflammation or

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even become permanently resident in peripheral tissues to provide a first-line defense against already encountered pathogens [186]. 8.1. Central memory and memory stem cells recycle through secondary lymphoid organs and drive secondary expansions The lymph node homing receptors CCR7 and CD62L, which are typical for naive T cells are also expressed on a major fraction of human CD4+ CD45RA− and on a minor fraction of CD8+ CD45RA− memory T cells [45]. These central memory T cells (TCM ) are characterized by low effector functions and high secondary expansion potential and appear thus arrested at an intermediate stage of T cell differentiation [13]. Notably, also a subset of Foxp3+ regulatory T cells expresses TCM markers, and these cells are very potent to inhibit graft versus host disease in mice, because they can interfere with pathogenic T cell priming in lymph nodes [187]. TCM cells can generate rapidly large numbers of effector cells in secondary responses and contain pre-dominantly non-polarized cells that secrete IL-2. Nevertheless, they are heterogeneous and some TCM are pre-committed to the Th1 or Th2 lineage and secrete some IFN-␥ and IL-4, respectively [61]. These pre-Th1 and pre-Th2 cells can be identified by respectively CXCR3 and CCR4 expression, and they preferentially generate Th1 or Th2 effector cells upon TCR-driven or homeostatic proliferation. Nevertheless, pre-Th1 and pre-Th2 cells maintain plasticity to differentiate to the opposite fate upon TCR stimulation in the presence of the relevant polarizing cytokines [61], while more differentiated cells appear to be less plastic. Thus, T cells might progressively lose plasticity upon differentiation to effector cells [66]. More recently, memory T cells with a CD45RA+ CCR7+ naive phenotype but that express low levels of CD95 were described [188]. These memory stem cells (TSCM ) are characterized by low effector functions and a very high expansion potential, and appear to be closely related to the previously described early-branched antigen-experienced CD4+ T cells that express CXCR3 or CCR4 [189]. In addition, some TCM express CCR6 and produce IL-17 [36,190,191], but the function of these immature Th17 subsets remains to be understood. 8.2. Effector memory and tissue-resident memory cells provide immediate protection in peripheral tissues Memory cells that have lost CCR7 or CD62L expression efficiently perform immediate effector functions and express chemokine receptors like CCR5 or CCR2 that mediates homing to non-lymphoid tissues [13]. There is convincing evidence that effector memory (TEM ) T cells can be generated from TCM cells [13,27,28,61,192], but also other models have been proposed [193]. There are two types of memory T cells that can be found in peripheral tissues: TEM cells that recirculate in the blood and are recruited into tissues by inflammation, and tissue-resident memory T cells (TRM ) that reside stably in peripheral tissues as a first-line defense [186]. In contrast to TRM , TEM up-regulate CCR7 upon TCR stimulation, can be recruited to the spleen or to inflamed lymph nodes and thus shape secondary T cell responses [194]. TRM have been characterized in different organs including the skin, the gut and the lung in mice and men. Interesting differences can be observed in the healthy human liver, where the few resident lymphocytes are mostly innate lymphocytes such as NK, unconventional T cell as ␥/␦ T cells and NKT cells. In the inflamed liver, TEM ␣/␤ T cells are recruited in addition, and during chronic viral hepatitis the amount of liver infiltrating lymphocytes can reach the number of the lymphocytes circulating in the blood, i.e. 5 × 109 cells. Moreover, most of these lymphocytes are activated, creating a highly inflamed environment [195,196]. Circulating memory T cells with homing potential for the skin can be identified by the expression of the skin homing receptors cutaneous lymphocyte antigen (CLA)

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[197], CCR4 [198] and CCR10 [199], and these skin-homing cells respond to contact allergens [200]. Conversely, gut-homing T cells express the ␣4␤7 integrin and CCR9, but while human ␣4␤7+ T cells recirculate in peripheral blood and respond to intestinal viruses, CCR9+ cells are nearly exclusively found as TRM in the small intestine [201]. Notably, intestinal T cells can be classified into helper and regulatory T cells according to cytokine and chemokine receptor expression similar to their counterparts in peripheral blood (Fig. 2). In intestinal and other non-lymphoid tissues CD103 that binds to E-cadherin expressed on epithelial cells is characteristic for intraepithelial TRM . CD69 is also expressed on TRM cells [202] and probably contributes to the retention of TRM in non-lymphoid tissues by inhibiting sphingosin1-phosphate receptor-mediated T cell egress from tissues [203]. Both TEM and TRM are heterogeneous and can be further subdivided into different subsets of helper and regulatory T cells [86,198,204]. 8.3. Follicular helper T cells provide help for antibody production in B cell follicles B cells require help from antigen-activated helper T cells via the surface receptors CD40 and ICOSL and the cytokines IL-21, IL-10 or IL-4 to proliferate and differentiate to antibody secreting cells [205]. These helper T cells were originally thought to be Th2 cells, but more recently a specialized B helper T cell subset of follicular helper T cells (TFH ) was identified [206,207] that are present in B cell follicles and in germinal centers and provide potent help to B cells [208–210]. Nevertheless, it was shown that in murine helminth infections TFH cells are derived from Th2 cells [211], and also a fraction of TFH cells in inflamed human tonsils produces IL-4 and expresses CRTH2 [212]. Human TFH cells express the transcriptional repressor BCL-6 [213], the co-stimulatory molecule ICOS [208,209] and produce high levels of IL-21 [207], but little IFN-␥ or IL-17. Moreover, they express the chemokine receptor CXCR5, which is transiently up-regulated upon TCR stimulation [194] and induces migration to the B cell zone in lymphoid organs [214,215]. Coexpression of high levels of CXCR5 and ICOS is characteristic for TFH cells that enter germinal centers, while TFH that express lower levels of CXCR5 and ICOS are more efficient to help memory B cells outside of germinal centers [216]. Notably, follicular regulatory T cells that co-express Foxp3 and CXCR5 and control the germinal center reaction have been identified in humans and mice [217,218]. TFH cells are activated effector cells that are exclusively found in inflamed lymphoid organs, but they can become resting TCM cells when the immune response ceases [33,219]. Notably, CXCR5 is expressed on a fraction of resting TCM with high B cell helper functions in peripheral blood [220,221]. These CXCR5+ TCM have low effector cytokine producing capacities and respond to vaccination antigens such as tetanus toxoid [61]. Interestingly, CXCR5+ TCM that express PD-1 have recently been shown to correlate with neutralizing antibody levels in HIV patients [222]. However, also Th17 cells possess B helper functions [70,223] and can express CXCR5 [220], but the relationship between TFH cells and Th17 cells with B helper functions is incompletely understood. 9. CD8+ T cell subsets: not only committed killers CD8+ T cells are pre-committed to become cytotoxic effector cells that produce IFN-␥ and kill transformed or virus-infected target cells. Nevertheless, many aspects of the complexity of CD4+ helper T cell subsets also apply for CD8+ T cells. Thus, they can be subdivided into naive cells that co-express CD45RA and CCR7 or CD27 and into various subsets of memory/effector cells [45,46,224]. Similar to CD4+ T cells, subsets of TSCM , TCM and TEM with progressively decreasing secondary expansion potential and increasing

cytotoxic effector functions can be identified by the combination of CD45RA, CD95 and CCR7 expression [188,225]. Furthermore, there is an additional subset of CCR7− CD45RA+ effector cells (TEMRA ) with low secondary expansion potential that contains pre-stored perforin [13,225]. In addition, some effector memory CD8+ T cells stably reside in non-lymphoid tissues as TRM and express CD69 [202] and CD103 [186]. An alternative way to classify CD8+ T cell subsets is the expression of the co-stimulatory receptors CD27 and CD28 [46] or the IL-7R [226–228] that are progressively lost upon cytotoxic T cell differentiation. Interestingly, different persistent viruses induce distinct subsets of CD8+ memory T cells [229]. Notably, CD8+ T cells also contain minor subsets of Tc2 cells that express CCR4 and produce IL-4 [225,230,231], and of Tc17 cells that express CCR6 [232] and/or CD161 [82] and secrete IL-17, but the functions of these subsets are incompletely understood [233,234]. In addition, CD8+ T cells that produce IL-10 [235] and that possess regulatory functions [236,237] have also been described. Interestingly, some CD8+ T cells lack cytotoxic functions, but up-regulate CD40L upon TCR stimulation similar to CD4+ helper T cells and provide help to DC and B cells [230,238]. Reciprocally, CD4+ T cells contain a minor fraction of CD28− cytotoxic effector cells that express perforin and contribute to the efficient elimination of virus-infected or transformed monocytes and B cells [179].

10. Conclusions In recent years the number of human T cell subsets has progressively increased, and future research will clarify how these new T cell subsets are related to the established differentiation lineages. Important milestones for the characterization of new subsets are the identification of specific surface markers and transcription factors, which are crucial to define the molecular identities of T cell subsets and their roles in different immune-mediated diseases. The study of novel human T cell subsets will not only lead to a better understanding of T cell differentiation, but also offers exiting translational opportunities such as the identification of novel diagnostic tools or possible therapeutic targets.

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Plasticity of human CD4 T cell subsets.

The who's who of T-cell differentiation: human memory T-cell subsets.

β T cell model.

Heterogeneity of locomotion in human T cell subsets.

T-cell subsets in autoimmunity.

Induction of T helper cell subsets.

The distribution and function of human memory T cell subsets in lung cancer.

Studies of immune functions of patients with systemic lupus erythematosus. T-cell subsets and antibodies to T-cell subsets.

The dermatoscopic universe of basal cell carcinoma.

The HLA system and T-cell subsets in Bell's palsy.

A Comparative Study of the T Cell Stimulatory and Polarizing Capacity of Human Primary Blood Dendritic Cell Subsets.

Shared and Distinct Phenotypes and Functions of Human CD161++ Vα7.2+ T Cell Subsets.

OMIP-021: Simultaneous quantification of human conventional and innate-like T-cell subsets.

OMIP-030: Characterization of human T cell subsets via surface markers.

delta T cells.

Functional subsets of allergen-reactive human CD4+ T cells.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

Identification of Melanoma-reactive CD4+ T-Cell Subsets From Human Melanoma Draining Lymph Nodes.

Immunoregulatory role of different T cell subsets in murine candidiasis.

Cytokine secretion patterns and cross-regulation of T cell subsets.

Glycolysis determines dichotomous regulation of T cell subsets in hypoxia.

Differential alterations of tissue T-cell subsets after sepsis.

Premature senescence of T-cell subsets in axial spondyloarthritis.

The collaboration of T-cell subsets in the mitogenic stimulation of purified B-cell subpopulations.