To date, only a few studies have attempted to address the role of IL-22 in the context of helminth infection. IL-22 is upregulated in the intestinal mucosa after infection by *Trichuris trichuria* or *Necator americanus* in human beings (122–124) and Th22 frequency in PBMC is higher in filarial-infected patients than in healthy controls (109). While helminth infection clearly induces IL-22, so far no role for Th22 in either immune-mediated protection or pathology has been proven. In fact, IL-22−/− mice infected with *S. mansoni* did not present significantly modified immune responses compared to wild type controls, neither did the absence of IL-22 modify the establishment of the parasite or the development of pathology (125). In filarial infection, Th22 frequency in PBMC was higher in lymphedema-positive people than in asymptomatic people, as was their frequency after antigen restimulation with both adult and microfilarial stages of the parasite (109). As reported in the above section, IL22 produced by Th17 cells plays a role in gut expulsion of *N. brasiliensis* and *T. muris* (126).

Th22 has also been reported to be involved in skin repair mechanisms and as such may be relevant to the pathology following skin penetration by helminths. Furthermore, IL-22 is known to have a role in the control of dysbiosis in the gut (127). As helminths have co-evolved with both the host and its microbiome (128), in order to observe the role of Th22 in helminth infection, it may be required to study the tripartite interaction of microbiome–macrobiome–host rather that the classical bipartite helminth–host interaction.

#### **Tfh CELL SUBSET IN HELMINTH INFECTION**

In distinction to the other CD4+ T-helper cell subsets, Tfh cells were not initially described based on cytokine production and transcription factor expression patterns, but rather by the expression of the surface marker CXCR5+ denoting its localization to the germinal center of human tonsils. Tfh have since been shown to promote germinal center formation and class switching of Bcells in mice and are further characterized by their expression of the inducible co-stimulatory molecule (ICOS), the inhibitory receptor programed cell death-1 (PD-1), and B and T lymphocyte attenuator (BLTA) (129). Bcl6 has been identified as the master transcription regulator for Tfh cells (130–132).

In the context of *N. brasiliensis* infection, Tfh cells have been demonstrated to express IL-4 but not IL-13 (20). This study also reports that these IL-4+ Tfh cells localize to the B-cells follicle, and not to tissue sites such as the lungs. Interestingly, work by Glatman Zaretsky et al. demonstrated that IL-4-expressing CXCR5+ Tfh cells could develop from adoptively transferred Th2 cells (CXCR5 and PD1- ) in B-cell-sufficient hosts during *S. mansoni* infection (133). Furthermore, IL-4 production by Tfh cells is essential for proper B-cell expansion and activation, as demonstrated by a reduction in B-cell activation in IL-4R−/− mice (21).

The role of Tfh was studied in the context of *S. japonicum* induced pathology model (134). In ICOSL−/− mice that are deficient in Tfh cells, a diminution of the liver pathology is associated with a decrease in Th1 and Tregs, whereas Th2 and Th17 are unaffected. Furthermore, adoptive transfer of Tfh cells to ICOSL−/− mice proved sufficient to re-establish pathology characterized by the accumulation of cells in granulomas in the liver (134). Furthermore, plasma cells have been shown to be present in the granuloma induced by *S. japonicum* infection in both pig and mice, and depletion of B-cells reduced pathology in this model (135, 136).

In the context of non-helminth infections, Tfh cells have been shown to produce IFN-γ and several studies suggest that Tfh cell production of Th1-, Th2-, and Th17-associated cytokines provides further evidence that they are potentially derived from these lineages (129). Whether these different patterns of cytokine expression reflect different subsets of Tfh cells, akin to those observed in T-helper subsets and ILCs, is still unclear. The use of Bcl6 reporter mice may help to distinguish Tfh cells from other T-helper subsets and to provide an answer to this question, and may allow the identification of other cytokine patterns in Tfh cells that are induced in helminth infection in parallel with the other T-helper subsets.

#### **HYPOTHETICAL ThGM CELL SUBSET IN HELMINTH INFECTION?**

ThGM are the most recently proposed CD4+ T-helper subset having been described in *in vitro* studies developing from naïve CD4+ T-cells stimulated with anti-CD3 and anti-CD28, in the absence of IL-4, IFN-γ, and IL-12 (137). It is important to note that to our knowledge, this subset has not been described *in vivo* so far and that its existence will have to be further confirmed.

Putative ThGM cells produce high levels of GM-CSF, while not producing Th1- or Th2-associated cytokines. The authors further show that they do not express T-bet, GATA3, RorγT, or Foxp3, thus supporting the idea that they indeed constitute a new CD4+ T-helper subset (137). GM-CSF is a pluripotent cytokine, which has been shown to induce T-cells proliferation and activate macrophages and neutrophils, among other cells, and the absence of this cytokine has been shown to negatively impact the differentiation of both Th1 and Th2 responses (138).

Contrasting roles have been shown for GM-CSF in helminth infection settings. In *N. brasiliensis* infection, mice deficient in GM-CSF show no reduction in worm burden in the lungs or gut in both primary and secondary infection when compared to wild type mice (139).

In *Onchocerca volvulus* infection, presumably immune individuals (negative for the parasite, but living in endemic area) have a mixed Th1/Th2 response to L3 and microfilariae antigen, contrary to infected individuals that present only a strong Th2 response to those antigens. In particular, GM-CSF, at that time considered as a Th1 cytokine, was greatly enhanced in the putatively immune individuals (140). Co-cultivating human PBMCs *in vitro* with the *Schistosoma* antigen SmGST28 has been shown to be sufficient to induce some granulomatous formations, and GM-CSF is needed for this reaction (141), suggesting a role for this cytokine in development of pathology.

#### **FURTHER T-HELPER CELL DIVERSITY AND PLASTICITY IN HELMINTH-INDUCED TYPE 2 IMMUNE RESPONSES**

In the original Th1/Th2 paradigm, it was proposed that the T-helper cell subsets were distinct and negatively regulated each others' activities, all underpinned by regulatory epigenetic methylation signatures interacting at IFN-γ, *IL4*, Gata-3, and T-bet gene loci (142, 143). However, this concept has to be revised in light of the recent discoveries of additional functionally diverse T-helper subsets including those with mixed Th1/Th2 signature cytokine phenotypes and by the observation that certain T-helper subsets can reverse their degree of polarization (144, 145).

Although it has been known for some time that double-positive IFN-γ +IL-4+ T-cells can be detected in experimental models of Th2 differentiation (146), when viewed in the context of the original Th1/Th2 paradigm, they were considered to be Th0 cells that were not yet committed to a polarized phenotype. However, several recent publications clearly demonstrated that these Th1/Th2 hybrid cells are stable both *in vitro* and *in vivo* after infection with either the trematode *S. mansoni* and the nematode *H. polygyrus* helminth infections (146, 147). Furthermore, this Th1/Th2 hybrid cells arise in a IL-18-dependent manner in *S. mansoni* infected mice (148). Interestingly, adoptive transfer of Th1/Th2 hybrid into Th1 or Th2 inflammatory models (LCMV and allergic airway inflammation) showed in both cases a reduced pathology associated with the inflammation (147). At the molecular level, these Th1/Th2 hybrid cells present with an intermediate expression of Gata-3 and T-bet as compared to Th2 and Th1 cells, respectively, due to an intermediate signature of methylation, for example, gata3 methylation was 36% in those cells, versus 60% in Th1 and 8% of Th2 (149).

Both T-bet and GATA3 can regulate each others expression (150), and in a recent study, RORγt and Foxp3 were shown to directly interact in a way that determines Th17 versus induced Treg lineage (151, 152). Also, it has been shown that Tfh cells can express both Gata3 or T-bet and that while Bcl6 decreases their relative expression it does not block it completely (153). Taking these observations together, it would seem reasonable to speculate that it is the ratio of transcription factors induced that may determine the fate of any developing T-helper subset and that as a T-helper cell differentiates there is available a broad contiguous range of gene expression patterns for shaping its ultimate phenotype during an immune response against a helminth (**Figure 2A**) (154).

The possibility of potential inter-conversions of different T-helper subsets in the context of a helminth infection has recently been studied (155). Both *in vitro*- and *ex vivo*-generated Th1 and Th17 cells, adoptively transferred into mice later infected with *N. brasiliensis*, were shown to convert into IL-4 producers while losing their own signature cytokine expression. Also, both the iTregs and the nTregs were found to be stable *in vivo* in this study, with only low number of cells converting to express IL-4 (155). It is interesting to note that the *in vitro*-generated Th1 and Th17 cells showed a lower propensity to convert after transfer, suggesting that *in vitro* culture has a strong impact on the degree of T-helper cell plasticity that can be observed and perhaps explaining why the

initial *in vitro* investigations into T-helper subset differentiation found subsets were highly stable and thus terminally differentiated.

Based on observations of the diversity and apparent plasticity of T-helper subsets phenotypes that can be detected now and on dynamic systems theory, we propose a landscape representation of the possibility of inter-conversions and intermediary states for T-helper cell subsets developing in the context of a helminthinduced immune response (**Figures 2B,C**). We have represented the dynamic of T-cells' fates on a quasi-potential landscape in which the different subsets constitute stable states, also called attractors. In this view, the subsets are in "valley" or "flat" areas. To transit from one stable state to another, the system needs to be perturbed, for example by an infection that would push cells toward certain attractors or away from others. Unstable states are usually represented as "hills." The Th1/Th2 hybrid state is stable enough, but less that the more terminally differentiated Th1 and Th2, and is thus represented by a well with less depth than Th1 or Th2.

Using such a "continuum of T-helper cell phenotypes" paradigm, it would be predicted that in host tissues responding to helminths there would be a gradient of activated and differentiated T-helper cell subsets with the most fully differentiated being stable and having lost much of their plasticity. Such terminally differentiated helper T-cells, maybe such as Th2, probably represent a small proportion of the pool of memory effector T-cells that maintain the helminth antigen specificity and the appropriate cytokine profile.

**FIGURE 2 | Dynamic of T helper differentiation**. **(A)** The dynamic of T helper differentiation can be visualized as a "potential landscape" in which each T helper subset represent a stable position or "valley" and the transition from one subset to another, would be a "hill", difficult to pass. Initially, the transition state between T helper subsets was considered as instable, and thus not observable in vivo. However, Th1/Th2 hybrid population has recently been reported to be stable after helminth infection. As this hybrid state is less abundant than Th1 or Th2, one could presume that the hybrid population is less stable that the Th1 or Th2 subsets, thus represented as a less deep well. **(B)** This transition between subsets can be further defined by the ration of transcription factor

participating in the fate determination of each subset. For example, the Th1/Th2 population has been shown to present intermediary level of gata-3 and T-bet expression as compared respectively to Th2 and Th1. Through similar transcriptomic approach, generalised on all the T helper subsets, it would thus be possible to define a ration of transcription factors necessary to enable the switch from one subset to another. **(C)** The plasticity of the T helper subsets is represented in a conceptual 3D potential landscape and illustrate that the diverse repertoire of T helper cell subsets, and its important plasticity, enable the host to have an array of fine-tuned adaptive responses to both control the parasite development and avoid and repair pathology caused by the worm migration.

#### **TYPE 2 IMMUNE RESPONSES TO HELMINTH INFECTION ARE A COMPROMISE BETWEEN PROTECTION, SUSCEPTIBILITY, TISSUE REPAIR, AND PATHOLOGY**

The original paradigm explaining resistance or susceptibility to helminths was described as a simple balance between Th1 (the susceptible, pathologic response), and Th2 (the response conferring parasite killing and elimination). However, this paradigm did not satisfactorily explain why so many parasites are able to establish themselves in hosts for extended periods of time (sometimes for decades) without causing any major clinical symptoms, nor why, in endemic environments, hosts are continually being reinfected with no apparent sign of disease. The hypothesis that the immune system would ignore the parasite infection was unconvincing,begging the question of whether these parasites themselves actively downregulate the host immune response and control pathology. Consequently, much research on helminth infection has focused on understanding how the parasite could regulate immune responses and teasing apart what is the physiological purpose of the type 2 effector responses in terms of benefit to host survival.

With the emergence of data showing that helminths and their products could be used to prevent/cure both allergic and autoimmune (156), our understanding of the immune response against helminth has changed to take into account the regulatory mechanisms induced by the parasites. From this emerged a new concept, the "modified Th2," characterized by a decrease IL-5 and IL-13 expression and an increase of anti-inflammatory cytokines, such as TGF-b and IL-10. Other cells types, such as regulatory myeloid cells or regulatory B-cells have been shown to be involved in the downregulation induced by helminth.

The Th2 immune response has also been shown to be part of a wound repair response, with the ability to block a runaway pathology. This is clearly illustrated in IL-4−/− mice infected with *S. mansoni* that die from excess pathology, even if their worm burden was similar to the wild type control mice (157). Th2 immune response has been considered as having evolved to cope both with helminth infection and with damage repair mechanisms, necessary to the survival of the host against those multicellular metazoan parasite migrating through tissue (12, 158). New research has shown that IL-9 has roles in controlling fibrosis and wound repair (91, 92), and that IL-22 from Th22 and Th17 cells promotes healing by increasing proliferation and survival of keratinocytes and intestinal epithelial cells (118, 159, 160). These data indicate that Th2 may not be the only T-helper subset contributing to the repair of helminth-induced tissue damage.

Furthermore, ES-62, a secretory product of the filarial nematode *Acanthocheilonema viteae*, may play with the balance of the different T-helper subsets in order to diminish the protective immune response (161). In a murine model of OVA-induced allergic airways inflammation, ES-62 decreased Th2 responses, as shown by a reduced level of IL-4 in the broncho-alveolar fluid, decrease eosinophils recruitment, and IgE. In parallel, the Th1 immune response is exacerbated, and neutralization of IFN-γ initiated the allergic inflammation blocked by ES-62 treatment. Th17 was also suppressed in this model, and that this suppression is responsible for the increase of the Th1 response. Importantly, ES-62 in this model did not induce an increase of Treg population,thus suggesting that the immunomodulatory effect of the molecule is

to modify the balance of CD4+ T-helper subsets. This is the key data to understanding that the regulation of immune responses is not solely controlled by Tregs, but the different CD4+ T-cells subsets themselves antagonize one another.

Considering the fact that all the currently recognized stable CD4+ T-helper phenotypes have been observed during helminth infections, and that these different subsets contribute variously to either protection, wound healing, susceptibility, or immunopathology, we have designed a new model that condenses what is known about the direct contributions of the subsets and their cross-regulation of each other. This model demonstrates our better understanding of the balanced immune responses during helminth infection **Figure 3**.

The early opposition of Th1 and Th2 is still clearly visible as a driving force for the trade-off between host and helminth survival. Th2 and Th9 segregate together to confer resistance, as they share a common activation pathway through IL-4. They are opposed to Th1 and Th17 in regard to pathology and to Th1 and Tregs in regard to susceptibility and chronicity. This model emphasizes that what may be best for the host is a compromise between elimination of the parasite versus containment and also the need for rapid repair of damaged tissue and avoidance of self destructive pathology. This all-encompassing

immune response protects against helminth by expanding ILCs, eosinophils and basophils all involved in parasite expulsion or by activating macrophages in AAM, playing a role in granuloma formation. Th9 rather protects by increasing goblet cells hyperplasia and muscle contractility in the gut. Th17 induced pathology is mainly mediated by neutrophils and inflammatory macrophages. In contrary, Tregs induced development of regulatory macrophages, which control pathology.

view of an immune response gives a better understanding of the host issues at stake and gives a context for further investigations to investigate roles for each of the T-helper subsets. Furthermore, the consideration of a multipartite balance, rather than a one in one balance, would be useful to the design of therapy against helminths (i.e*.*, by developing adjuvant to vaccine that could determine the right balance of Th susbsets to obtain sterilizing immunity, and define the timing to administer such treatment), and to understand how helminth infection or their product could use a therapy against inflammatory and autoimmune disease, caused by the deregulation of different T-helper subsets.

#### **DISCUSSION/REMARKS**

The new challenge to the investigation of type 2 immune responses is to determine how many subsets of T-helper cells exist and what mechanisms control the level and degree of plasticity that occurs between T-helper cell subtypes. The question arises as to what would be the benefit to the host to have such complexity and myriad of genetic events underpinning this plasticity of the T-helper cell response. We would argue that the benefit to the host in being able to generate so many T-helper subsets is to have the diversity of options for dealing with the myriad of parasitic forms, invasive routes, and environments that have an endless supply of parasites that invade by physical means. In effect, the diversity and plasticity of the repertoire of functional T-helper cell subsets enable the host to have an array of adaptive responses. While the response might not kill the worm, it will enable the host to repair the more serious damage caused by the migrating parasite, and avoid the fatal consequences of debilitating pathology.

We wish to point out that in order to study the type 2 immune response elicited by helminths, i.e., define the role of the various old and new discrete CD4+ T-cell subsets, both techniques and approaches will have to evolve. For example, as plasticity between different T-helper subsets become increasingly evident, it maybe of interest to define a subset by both the cytokine production/nonproduction patterns, as well as by the ratio of transcription factors they express. For this matter, engineered reporter mice for particular cytokines and even combinations of cytokines may help in *in vivo* studies but may not reflect the native mRNA or protein. New bioinformatics approaches, such as studies of the transittability (162) (that defines the lower number of molecule switch to go from one fate of differentiation to another one based on a network of molecules involved in shaping the cell fate), could provide in the near future a list of more appropriate marker necessary to define one particular T-helper subset. Moreover, due to the plasticity of the T-cells, we think it is important to remember that immune studies look at dynamic events, and as such looking at a precise time point in the model, may give a wrong picture of the actual mechanism, for example a subset "in transition," may be missed because of the lack of markers used for describing it, or the relative rarity compared with the currently defined stable subsets.

Hopefully, advances in single cell analysis (Fluidigm), sequencing, multiplex quantification of transcripts (such as nCounter, Nanostring that can detect up to 800 genes), advanced multicolor flow cytometry (such as panels up to 20 colors), the emergence of mass cytometry (such as CyTOF that allows multi-detection of

up to 34 parameters to date, but could potentially go up to 100), coupled with bioinformatic approaches may offer the new tools necessary for studying the dynamics of T-helper differentiation in the context of helminth infection.

#### **ACKNOWLEDGMENTS**

We are grateful to Dr. Alexander Smith for the preparation of the figures and comments on the manuscript and to and Dr. Kara Filbey for her comments on the manuscript.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 05 August 2014; paper pending published: 08 September 2014; accepted: 22 September 2014; published online: 15 October 2014.*

*Citation: Bouchery T, Kyle R, Ronchese F and Le Gros G (2014) The differentiation of CD4*+ *T-helper cell subsets in the context of helminth parasite infection. Front. Immunol. 5:487. doi: 10.3389/fimmu.2014.00487*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Bouchery, Kyle, Ronchese and Le Gros. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## AntifungalTh immunity: growing up in family

#### **Monica Borghi 1 , Giorgia Renga1 , Matteo Puccetti 2 , Vasileios Oikonomou1 , Melissa Palmieri 1 , Claudia Galosi 1 , Andrea Bartoli 1 and Luigina Romani 1\***

1 Pathology Section, Department of Experimental Medicine, University of Perugia, Perugia, Italy 2 Polo GGB, Perugia, Italy

#### **Edited by:**

Dragana Jankovic, National Institutes of Health, USA

#### **Reviewed by:**

Edward John Collins, The University of North Carolina at Chapel Hill, USA Cosima T. Baldari, University of Siena, Italy

#### **\*Correspondence:**

Luigina Romani, Pathology Section, Department of Experimental Medicine, University of Perugia, Polo Unico Sant'Andrea delle Fratte, Perugia 06132, Italy e-mail<EMAIL_ADDRESS>
Fungal diseases represent an important paradigm in immunology since they can result from either the lack of recognition or over-activation of the inflammatory response. Current understanding of the pathophysiology underlying fungal infections and diseases highlights the multiple cell populations and cell-signaling pathways involved in these conditions. A systems biology approach that integrates investigations of immunity at the systems-level is required to generate novel insights into this complexity and to decipher the dynamics of the host–fungus interaction. It is becoming clear that a three-way interaction between the host, microbiota, and fungi dictates the types of host–fungus relationship. Tryptophan metabolism helps support this interaction, being exploited by the mammalian host and commensals to increase fitness in response to fungi via resistance and tolerance mechanisms of antifungal immunity.The cellular and molecular mechanisms that provide immune homeostasis with the fungal biota and its possible rupture in fungal infections and diseases will be discussed within the expanding role of antifungal Th cell responses.

**Keywords:Th cell subsets, immunity, tolerance, fungi**

#### **FUNGAL INFECTIONS AND DISEASES IN THE METAGENOMICS ERA: A REAPPRAISAL**

Fungi can interact with their hosts (plants, animals, or human beings) in multiple ways, establishing symbiotic, commensal, or pathogenic relationships. Most fungi, such as *Aspergillus fumigatus* and *Cryptococcus neoformans*, and the thermally dimorphic fungi are ubiquitous in the environment, and human beings are exposed by inhaling spores or small yeast cells. In addition, more than 400 species of fungi associated with human beings have been identified (1). In this case, co-evolution of commensals, such as *Pneumocystis jirovecii, Malassezia* spp., and *Candida albicans*, with their mammalian hosts implicates the existence of sophisticated mechanisms to antagonize immunity in order to survive. Once considered pathogenic microbes, the commensal fungal microbiota is now an important component of the human intestinal ecosystem. Indeed, despite the intimate contact of fungi with the human host, fungal diseases in immunocompetent hosts are fairly uncommon, indicating that low-virulence fungi have evolved particular adaptation mechanisms that allow them to persist relatively unnoticed by the immune system (2). This "peaceful" coexistence may digress into overt disease under conditions of immune deregulation, such as in primary immunodeficiency human immunodeficiency virus infection and as a result of immunosuppressive therapies (2). In addition, invasive fungal diseases continue to be a serious problem in patients with hematologic disorders, solid, and hematopoietic organ transplantation as well as in non-highrisk, *sensu strictu*, patients, such as patients with *Mycobacterium tubercolosis* infection, hyper IgE syndrome, and anti-TNF-alpha therapy (3).

The increasing understanding of the importance of the microbiota in shaping the host immune and metabolic activity has rendered fungal interactions with the host and its microbiome more complex than previously appreciated (4) (**Box 1**). Indeed, the complex interactions between fungal and bacterial commensals, either directly or through the participation of the host immune system, all impact on the pathophysiology of a number of inflammatory disease that, in turn, may lead to secondary fungal infections (5, 6). Evidence is accumulating to support the exciting concept that the interaction between different biomes and between the host and the mycobiome are critical in the pathogenesis of fungal infections and other human diseases (1, 7, 8). Here, we will discuss recent findings on host- and microbial-dependent mechanisms of immune homeostasis with the fungal biota and its possible rupture in fungal infections and diseases.

#### **RESISTANCE AND TOLERANCE MECHANISMS OF ANTIFUNGAL IMMUNITY**

As the immune system has evolved to accommodate colonization by symbiotic microbes while retaining the capacity to oppose their infectivity, a fine balance between pro- and anti-inflammatory signals is a prerequisite for a stable host/fungal relationship, the disruption of which may lead to pathological consequences. Indeed, despite the occurrence of severe fungal infections in immunocompromised patients, clinical evidence indicates that fungal diseases also occur in the setting of a heightened inflammatory response, in which immunity occurs at the expense of host damage and

**Abbreviations:** AhR, aryl hydrocarbon receptor; APS-1, autoimmune polyendocrine syndrome type 1 patients; CF, cystic fibrosis; CLR, C-type lectin receptors; CMC, chronic mucocutaneous candidiasis; DCs, dendritic cells; DTH, delayed type hypersensitivity; IDO1, indoleamine 2,3-dioxygenase 1; ILC3, innate lymphoid cells 3; IRIS, immune reconstitution inflammatory syndrome; PRRs, pattern recognition receptors; RVVC, recurrent VVC; Th, T helper; Treg, regulatory T-cells; VVC, human vulvovaginal candidiasis.

#### **Box 1 The mycobiome at the host/microbiome interface**.

The development of culture-independent methods has expanded our knowledge of the mycobiomes found in different body sites, their interface with other biomes, and their association with human health and diseases (1). Alterations in the mycobiome are frequently reported to be associated with various diseases such as cystic fibrosis (9), inflammatory bowel diseases (6, 10, 11), atopic dermatitis (12), or mucocutaneous candidiasis (13). However, it remains to be elucidated whether this variation is primary or secondary to an imbalanced bacterial microbiome. Indeed, interactions of fungi with bacteria in vitro have been described [reviewed in Ref. (6)] as well as the clinical relevance of these interactions (14), such as the occurrence of intractable candidiasis in association with antibiotic-induced dysbiosis (15) and of mixed fungal–bacterial species in biofilms (14). Fungal–bacterial interactions can be antagonistic, synergistic, or symbiotic; regardless, they influence the physiological characteristics and survival of either one partner and, consequently, impact on host immune reactivity. Variations in the mycobiome can also be secondary to dysregulated host immune reactivity.The traditional view of a single direction by which bacteria stimulates the immune system, leading to inflammation or autoimmune disorders, has been challenged by a more complex view; the gut immune system does not simply protect from pathogens, but is actively involved in the maintenance of a rich and healthy community of gut bacteria (16). Faults in the immune regulation lead to changes in the bacterial community that in turn feed back into the immune system. Similar to the microbiome, the host/mycobiome interactions also lead to mutual influences. Not only is the host affecting the mycobiome composition and variations, by means of genotype, physiology, immune system, and lifestyle, but also the fungal microbiota may contribute to the balance of inflammation and tolerance at local mucosal surfaces and at distal sites (17).

pathogen eradication (18). A number of fungal diseases are critical examples of such bidirectional influences between infection and immune-related pathology, a condition that highlights the bipolar nature of the inflammatory process in infection. Early inflammation prevents or limits infection, but an uncontrolled response may eventually oppose disease eradication. This conceptual principle is best exemplified by the occurrence of severe fungal infections in patients with chronic granulomatous disease (19), cystic fibrosis (20), or with immune reconstitution inflammatory syndrome (IRIS) (21), an entity characterized by local and systemic inflammatory reactions that can result in quiescent or latent infections manifesting as opportunistic mycoses. Chronic mucocutaneous candidiasis (CMC) and chronic disseminated candidiasis also belongs to the spectrum of fungus-related IRIS (22). Thus, an immune response that limits both fungal infectivity and host collateral damage is required to maintain a homeostatic environment (23). This dual role has recently been accommodated within the conceptual framework of a two-component antifungal immune response, i.e., resistance – the ability to limit fungal burden – and tolerance – the ability to limit the host damage caused by either the immune response or other mechanisms (2). Resistance is meant to reduce pathogen burden through innate and adaptive immune mechanisms, whereas a plethora of tolerance mechanisms, despite less known relative to resistance mechanisms, protect the host from immune- or pathogen-induced damage (24).

#### **MECHANISMS OF ANTIFUNGAL RESISTANCE**

Innate immune mechanisms are used by the host to respond to a range of fungal pathogens in an acute and conserved fashion. The constitutive mechanisms of innate defense are present at sites of continuous interaction with fungi and include the barrier function of body surfaces and the mucosal epithelial surfaces of the respiratory, gastrointestinal, and genitourinary tracts. Microbial antagonism, defensins, collectins, and the complement system realize the strict fungus specificity of the constitutive mechanisms and provide opsonic recognition. Multiple cell populations and cell-signaling pathways are involved in the antigen-independent recognition of fungi by PRRs (2, 25). Both murine and human studies have confirmed the association of susceptibility to fungal infections and diseases with genetic deficiency of selected PRRs (2). Because PRRs not only mediate downstream intracellular events related to fungal clearance but also participate in activation of adaptive immunity, deficiencies on innate immune genes also reverberate on the type and quality of the adaptive immune response, including effector CD4+ T helper (Th), regulatory T (Treg), and CD8+ T-cells (2, 25–27).

#### **DENDRITIC CELLS**

It is well established that the adaptive immune response, in particular that of T-cells, plays a pivotal role in antifungal host defense (2, 25). Dendritic cells (DCs) play a key role in promoting T-cell differentiation and responses to ubiquitous or commensal fungi. Studies have shown that lung DCs can transport fungal antigens to the draining lymph nodes (28, 29), where they orchestrate T-cell activation and differentiation into effector cells. Through elaboration of distinct sets of cytokines and other mediators, DCs have the unique ability to elicit a robust T-cell response that can be either tolerogenic or pro-inflammatory in nature, based on anatomical location and local metabolic environment. The whole-genome transcriptional analysis of DCs stimulated with fungi evidenced the presence of peculiar transcriptional programs governing the recognition of fungi (30).

These include common signaling pathways involving Syk kinase, Card9 and NF-κB downstream CLRs and ERK kinase, PI3K/Akt downstream TLRs for Th1/Th2/Th17 priming by conventional, inflammatory DCs, as well as p38/TRIF/STAT3 for Treg priming by plasmacytoid DCs (2, 31). In a mutual interaction, the host and the fungus control each other to avoid potential harmful inflammatory response. The ability of a given DC subset to respond with flexible activating programs and activation of distinct intracellular signaling pathways to the different PRR/fungal molecules' combinations confers unexpected plasticity to the DC system and pivotally contributes in shaping adaptive Th cells responses in infection and vaccination. The capacity of DCs to initiate different adaptive antifungal immune responses also depends upon specialization and cooperation between DC subsets (32). The multiple, functionally distinct, receptor/signaling pathways in DCs, ultimately affecting the local Th/Treg balance,

**Table 1 | CD4**+ **Th cell subsets in fungal infections**.

are likely successfully exploited by fungi from commensalism to infection (33).

#### **TH1 CELLS**

CD4+ Th cells exist in a variety of epigenetic states that determine their function, phenotype, and capacity for persistence, and form long-term immune memory (34). Well-balanced Th1 and Th17 cell responses are crucial in antifungal immunity and facilitate phagocytic clearance of fungal recognition, mainly through release of cytokines such as TNF-α, IFN-γ, and IL-17A and IL-17F (**Table 1**). These cytokines stimulate the disparate antifungal effector functions of phagocytes, as well as the generation of optimal T-cell-dependent immunity (2, 25). A dominant Th1 response correlates with the expression of protective immunity to fungi (2, 35) and vaccines (36, 37). Through the production of the signature cytokine IFN-γ and help for opsonizing antibodies, the activation of Th1 cells is instrumental in the optimal activation of phagocytes at sites of infection. Therefore, the failure to deliver activating signals to effector phagocytes may predispose patients to overwhelming infections, limit the therapeutic efficacy of antifungals and antibodies, and favor fungal persistency (2). Patients who are deficient in IL-12Rβ are susceptible to CMC, which is frequently recurrent or persistent (38), as well as to deep paracoccidioidomycosis (39).

#### **TH17 CELLS**

Th17 are present in the human T-cell memory repertoire to fungi (2) and inborn errors of human IL-17 immunity underlie susceptibility to CMC (40) in which both Th17 (41) and Th1 (38, 42, 43) responses are defective. Combined deficiency of the Th1 and Th17 pathway predisposes to fungal diseases (44, 45), thus emphasizing the important role played by both pathways in resistance against fungi. This could be explained with the notion that Th17 cells, although found early during the initiation of an immune response, are involved in a broad range of Th1-, Th2- and Treg-dominated immune responses (2, 46). In terms of effector functions, the ability of IL-17A to mobilize neutrophils and induce defensins may contribute to a prompt and efficient control of the infection at the different body sites. In respiratory fungal infections, Th17 cell are dispensable for resistance to the primary infection caused by *A. fumigatus* (47), but are required for vaccine-induced immunity against systemic mycoses endemic to North America (48). Thus, both Th17 and Th1 (27) cells are required for vaccine immunity to respiratory fungal pathogens.

It is intriguing that Th17 responses are down regulated by *C. albicans* (49). Regardless of the contribution of this phenomenon to infection or commensalism, this finding suggests that Th17 responses are finely tuned by fungi, as the failure to downregulate Th17 may eventually result in chronic inflammation and failure to resolve the infection (47, 50). The mechanisms that linked inflammation to chronic infection have been credited to the offending potential of IL-17A that, although promoting neutrophil recruitment, impeded the timely restriction of neutrophil inflammatory potential (51) while directly promoting fungal virulence (52). Thus, the Th17 pathway could be involved in the immunopathogenesis of chronic fungal diseases where persistent fungal antigens may maintain immunological dysreactivity. This may happen in autoimmune polyendocrine syndrome type 1 patients (APS-1) and Aire-deficient mice (53) where an excessive Th17 reactivity was observed. This finding apparently conflicts with the presence of autoantibodies against IL-22, IL-17A, and IL-17F observed in these patients (54, 55). Although correlated to susceptibility to CMC, these antibodies were also present in patients without CMC. In addition, despite the presence of antibodies to type I IFN, APS-I patients do not appear prone to recurrent viral infections. It has instead been shown that autoantibodies to pro-inflammatory cytokines may act as beneficial autoimmunity in their ability to dampen pro-inflammatory mediators and restrict self-destructive immunity (56).

#### **TH2 CELLS**

IL-4 and IL-13 act as the most potent proximal signal for commitment to Th2 reactivity that, by dampening protective Th1 responses and promoting the alternative pathway of macrophage activation, favors fungal persistence, allergy, and disease relapse. Limiting IL-4 production restores antifungal resistance (2) (**Table 1**). In atopic subjects and neonates, the suppressed DTH response to fungi is associated with elevated levels of antifungal IgE, IgA, and IgG. In CF patients, heightened Th2 reactivity associates with allergic bronchopulmonary aspergillosis and is sensitive to vitamin 3 (57). However, alternatively activated macrophages may have a protective role in defense against some respiratory fungi (58, 59) and Th2-dependent humoral immunity may afford some protection, in part by promoting Th1 immunity (60) and by altering fungal gene expression and intracellular trafficking (61–63). The efficacy of certain vaccines that elicit protective antibody strongly indicates that antibody responses can make a decisive contribution to host defense to fungi (61).

#### **TH9 CELLS**

The realization that Th effectors can produce various other cytokines alone or in combination in patterns not fitting the preconceived definitions of Th1/Th2 or Th17 subsets has led to the description of additional Th cell lineages, including Th9 and Th22. Initially thought to be a Th2-specific cytokine by virtue of its role in the pathogenesis of asthma, IgE class switch recombination, and resolution of parasitic infections, IL-9 is now considered to be the product of a distinct Th subset, the Th9 (64). Despite its relationship with other subsets, such as Th2, Th17, and Treg cells, Th9 cell subset can mediate tumor immunity and participate in autoimmune and allergic inflammation. Recently,human memory Th9 cells were found to be skin tropic or skin resident. Human Th9 cells co-expressed TNF-α and granzyme B, lacked coproduction of Th1/Th2/Th17 cytokines, and many were specific for *C. albicans*. IL-9 production preceded the upregulation of other inflammatory cytokines, such as IFN-γ, IL-13, and IL-17. IL-9-producing T-cells were increased in the skin lesions of psoriasis, suggesting that these cells may contribute to human inflammatory skin disease in the presence of *Candida* (65). Recent findings demonstrated that IL-9 is predominantly produced *in vivo* by a novel subset of innate lymphoid cells termed ILC2 (66). It has been proposed that IL-9 might have a regulatory and prosurvival function for many lymphoid and myeloid cells (67). Our recent evidence suggests that different types of ILCs are defective in IL-9-deficient mice infected with either *C. albicans* or *A. fumigatus*, and this profoundly affects the outcome of either infection and the associated pathology (unpublished observations) (**Table 1**).

#### **TH22 CELLS**

Th22 cells producing only IL-22 but neither IFN-γ nor IL-17A have been identified in human beings (68). They are induced in the presence of TNF-α and IL-6 and require ligation of aryl hydrocarbon receptor (AhR). Th22 cells via IL-22 influence the function of mesenchymal and epithelial cells and have been implicated in the dermatopathology of psoriasis and atopic dermatitis (69, 70). Memory *C*. *albicans*-specific IL-22+ CD4+ cells are present in human beings and defective in patients with CMC (71). Recent evidence indicates that IL-22 may play a crucial role in the innate immune resistance and local protection in mucocutaneous fungal diseases (72–74). Through the exploitation of primitive anti-fungal defense mechanisms, IL-22 was crucially involved in the control of *Candida* growth at mucosal sites in conditions of Th1 and Th17 deficiency (72, 74). Produced by ILC3 cells expressing AhR, IL-22 directly targeted gut epithelial cells to induce STAT3 phosphorylation and the release of S100A8 and S100A9 peptides known to have anti-candidal activity and anti-inflammatory effects (72, 74). Thus, due to dominant-negative mutations of STAT3, patients with autosomal dominant hyper-IgE syndrome have a defective Th17 (41) that is likely amplified on ECs where STAT3 mutation compromises the IL-22 effects. IL-22 also mediates antifungal resistance and epithelial protection in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). In RVVC, functional genetic variants in IL22 genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22 (74). Thus, IL-22+ cells, employing ancient effector mechanisms of immunity, may represent a primitive mechanism of resistance against fungi under a condition of limited inflammation (**Table 1**). The fact that IL-22 production in the gut is driven by commensals (see below) also provides novel mechanistic insights on how antibiotic-related dysbiosis may predispose to candidiasis (75).

### **MECHANISMS OF TOLERANCE**

#### **TREG CELLS**

The exposure to fungi requires the generation of a controlled immune response in the host that recognizes and controls them, limits collateral damage to self-tissues, and restores a homeostatic environment. A number of clinical observations suggest an inverse relationship between IFN-γ and IL-10 production in patients with fungal infections. High levels of IL-10, negatively affecting IFN-γ production, are detected in chronic candidal diseases, in the severe form of endemic mycoses, and in neutropenic patients with aspergillosis. Thus, high levels of IL-10 have been linked to susceptibility to fungal infections (76). However, given its prominent effect on resolution of inflammation, IL-10 production may be a consequence, rather than the cause, of the infection. This predicts that, in the case of chronic fungal infections dominated by non-resolving, persisting inflammation, IL-10 produced by Treg cells acts as homeostatic host-driven response to keep inflammation under control. Treg cells with anti-inflammatory activity have been described in fungal infections of both mice and human beings (2, 25). In experimental fungal infections, inflammatory immunity and immune tolerance in the respiratory or the gastrointestinal mucosa were all controlled by the coordinate activation of different Treg cell subsets, exerting a fine control over effector components of innate and adaptive immunity. Seen in this context, the Treg/IL-10 axis is a dangerous necessity, the failure of which may lead to detrimental inflammation. However, as the Treg responses may handicap the efficacy of protective immunity, the consequence of Treg activity is less damage to the host but also fungal persistence and immunosuppression, eventually (**Table 1**). Thus, by controlling the quality and magnitude and effector innate and adaptive responses, the spectrum of Treg cell activities may go from "protective tolerance," defined as a host's response that ensures survival of the host in a trade-off between sterilizing immunity and its negative regulation limiting pathogen elimination to overt immunosuppression. Taking a step further, this suggests that the interaction between fungi and the host immune status may determine their position from commensals to pathogens, and this position can change continuously. The salutary effects of Treg cells may go beyond their anti-inflammatory properties, to include the polarization of protective Th17 cells (46).

#### **TR1 CELLS**

T regulatory Type 1 (Tr1) cells are adaptive Treg cells characterized by the ability to secrete high levels of IL-10. Since their discovery, Tr1 cells have been proven to be important in maintaining immunological homeostasis and preventing T-cell-mediated diseases. Tr1 cells suppress T- and DC-dependent responses primarily via the secretion of IL-10 and TGF-β, release of granzyme B and perforin, and by disrupting the metabolic state of T effector cells. Tr1 cells have been demonstrated to have a role in infectious diseases, autoimmunity, and transplant rejection in different pre-clinical disease models and in patients (77). It has recently been shown that Tr1 cells play a distinct, yet complementary role, in response to *A. fumigatus* in human beings and mice. Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of *A. fumigatus* (Crf-1/p41) were identified in healthy human beings and mice after vaccination with Crf-1/p41+ zymosan. These cells produced high amounts of interleukin IL-10 and suppressed the expansion of antigen-specific T-cells *in vitro* and *in vivo*, thus limiting immunopathology (**Table 1**). *In vivo* differentiation of Tr1 cells was dependent on the presence of AhR, c-Maf, and IL-27. In comparison to Tr1 cells, Foxp3+ induced Treg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. These data provide evidence that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3+ Treg cells (78).

#### **TRYPTOPHAN METABOLISM**

The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) and its downstream catabolites sustain the delicate balance between Th1/Th17 pathways and Treg cells, by providing the host with adequate protective immune mechanisms without necessarily eliminating the pathogen or causing undesirable tissue damage (79). As a result of their ability to induce differentiation of Treg cells and inhibit Th17 cells, IDO1 is critical to cell lineage commitment in experimental fungal infections and contributes to the overall outcome of inflammation, allergy, and Th17-driven inflammation in these infections. Under these circumstances, the Th17 pathway, by inhibiting tryptophan catabolism, may instead favor pathology and provides evidence accommodating the apparently paradoxical association of chronic inflammation with fungal disease (19). IDO1 is a"metabolic" enzyme conserved through the past 600 million years of evolution. Initially recognized in infection because of antimicrobial activity ("tryptophan starvation" of intracellular parasites), IDO1 is now widely recognized as suppressor of acute inflammatory responses and regulator of mammalian immune homeostasis (79). Not surprising, IDO1 may represent an evasion mechanism for microbes that establish commensalism or chronic infection (79). In their capacity to induce Tregs and inhibit Th17, IDO1 expressing DCs and epithelial cells and kynurenines revealed an unexpected potential in the control of inflammation, allergy, and Th17-driven inflammation in these infections (51, 80).

#### **MICROBIOTA REGULATION OF RESISTANCE AND TOLERANCE TO FUNGI**

Commensal-driven mucosal responses are upregulated in IDO1 deficiency (81) and IL-22 responses are upregulated in conditions of defective adaptive immunity (72) and IDO deficiency (75). AhR is a ligand-activated transcription factor that mediates IL-22 production (82). A variety of indole derivatives act as endogenous ligands for AhR (83) and are generated through conversion from dietary tryptophan by commensal intestinal microbes (84). Recent evidence has shown that AhR is involved in the (patho)physiology of skin including the regulation of skin pigmentation, photocarcinogenesis, and skin inflammation (85, 86). Of interest, the ability of *Malassezia*-derived indoles to activate AhR correlated with local immunoregulation (87) and pathogenicity in seborrheic dermatitis (88). Similarly, metabolomics has revealed that bioactive indoles with Ahr agonist activity are also present in mice with candidiasis (75). Thus, the trpyptophan metabolism pathway is exploited by commensals and the mammalian host to increase fitness in response to fungi via induction of resistance and tolerance at the skin and mucosal surface. The new findings support a model in which the IL-22 axis controls the initial fungal growth (i.e., resistance) and epithelial cells homeostasis likely exploiting primitive anti-fungal effector defense mechanisms. In contrast, the exploitation of the IFN-γ/IDO 1 axis for functional specialization of antifungal regulatory mechanisms (i.e., protective tolerance) may have allowed the fungal microbiota to co-evolutes with the mammalian immune system, survives in conditions of high-threat inflammation, and prevents dysregulated immunity (79). The two pathways, although non-redundant, are reciprocally regulated and compensate each other in the relative absence of either one (72), consistent with the theme that adaptive immunity depends on innate immunity but innate immunity requires adaptive regulation. This finding not only helps to explain the association of fungal infections with dysbiosis but also points to the essential help the microbiota may provide in fungal colonization and pathogenicity in immunodeficient patients.

#### **CONCLUSION**

Vertebrates have co-evolved with microorganisms resulting in a symbiotic relationship, which plays an important role in shaping host immunity. However, intestinal inflammation also dictates the composition of gut-associated microbial communities (89), a finding indicating the reciprocal influence of the microbiota and the mammalian immune status. The mycobiome is not an exception to the rule. The activation of different Th cells with distinct effector and immunoregulatory functions may impact differently on the local mycobiome composition. Indeed, the findings that fungi oppositely react to IFN-γ (90) or IL-17A (52), in terms of growth and virulence, suggest that the local Th environment may contribute to the diversity of the mycobiome at different body sites. Ultimately, fungi have evolved a contingencybased system during co-evolution to adapt to host immunity and persist in an inflammatory host environment. In turn, this feeds back into the host immune fitness. For instance, manipulation of the regulatory network of the host by the fungal microbiota, resulting in the activation of Treg-dependent immune tolerance, is a mechanism to ensure fungal survival and commensalism at different body sites, as well as local immune tolerance (76, 91, 92). Thus, challenging existing paradigms with new perspectives from the crosstalk between fungi, the immune system, and the microbiota will eventually lead toward the development of multipronged therapeutic approaches for mucosal and systemic fungal diseases.

#### **ACKNOWLEDGMENTS**

This work is supported by the Specific Targeted Research Project FUNMETA (ERC-2011-AdG-293714), project FFC#22/2014, and project HYPOXINFECT no. Z100CD11A1 to Luigina Romani. The authors thank Dr. Cristina Massi-Benedetti for editorial assistance.

#### **REFERENCES**

with chronic mucocutanous candidiasis with or without APECED. *Clin Exp Immunol* (2008) **154**:406–14. doi:10.1111/j.1365-2249.2008.03778.x

in vitro and in vivo: comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. *Proc Natl Acad Sci U S A* (1991) **88**:9543–7. doi:10.1073/pnas.88.21.9543

92. Bonifazi P, D'Angelo C, Zagarella S, Zelante T, Bozza S, De Luca A, et al. Intranasally delivered siRNA targeting PI3K/Akt/mTOR inflammatory pathways protects from aspergillosis. *Mucosal Immunol* (2010) **3**:193–205. doi:10. 1038/mi.2009.130

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 22 August 2014; paper pending published: 15 September 2014; accepted: 28 September 2014; published online: 15 October 2014.*

*Citation: Borghi M, Renga G, Puccetti M, Oikonomou V, Palmieri M, Galosi C, Bartoli A and Romani L (2014) Antifungal Th immunity: growing up in family. Front. Immunol. 5:506. doi: 10.3389/fimmu.2014.00506*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Borghi, Renga, Puccetti, Oikonomou, Palmieri, Galosi, Bartoli and Romani. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## CD4T-cell subsets in malaria:TH1/TH2 revisited

### **Damian Perez-Mazliah and Jean Langhorne\***

Division of Parasitology, MRC National Institute for Medical Research, London, UK

#### **Edited by:**

Dragana Jankovic, National Institutes of Health, USA

#### **Reviewed by:**

Urszula Krzych, Walter Reed Army Institute of Research, USA Mauricio Martins Rodrigues, Federal University of São Paulo, Brazil David K. Cole, Cardiff University, UK

#### **\*Correspondence:**

Jean Langhorne, Division of Parasitology, MRC National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK e-mail<EMAIL_ADDRESS>
CD4+ T-cells have been shown to play a central role in immune control of infection with Plasmodium parasites. At the erythrocytic stage of infection, IFN-γ production by CD4+ T-cells and CD4+ T-cell help for the B-cell response are required for control and elimination of infected red blood cells. CD4+ T-cells are also important for controlling Plasmodium pre-erythrocytic stages through the activation of parasite-specific CD8+ T-cells. However, excessive inflammatory responses triggered by the infection have been shown to drive pathology. Early classical experiments demonstrated a biphasic CD4+ T-cell response against erythrocytic stages in mice, in which T helper (Th)1 and antibody-helper CD4+ T-cells appear sequentially during a primary infection. While IFN-γ-producing Th1 cells do play a role in controlling acute infections, and they contribute to acute erythrocytic-stage pathology, it became apparent that a classical Th2 response producing IL-4 is not a critical feature of the CD4+ T-cell response during the chronic phase of infection. Rather, effective CD4+ T-cell help for B-cells, which can occur in the absence of IL-4, is required to control chronic parasitemia. IL-10, important to counterbalance inflammation and associated with protection from inflammatory-mediated severe malaria in both humans and experimental models, was originally considered be produced by CD4+ Th2 cells during infection. We review the interpretations of CD4+ T-cell responses during Plasmodium infection, proposed under the originalTh1/Th2 paradigm, in light of more recent advances, including the identification of multifunctional T-cells such as Th1 cells co-expressing IFN-γ and IL-10, the identification of follicular helper T-cells (Tfh) as the predominant CD4+ T helper subset for B-cells, and the recognition of inherent plasticity in the fates of different CD4+ T-cells.

**Keywords: malaria, Plasmodium, multifunctional CD4 T-cells, CD4 T-cell subsets,Tfh,Th1,Th2,Th22**

#### **INTRODUCTION**

Malaria, caused by infection with *Plasmodium* transmitted via mosquito bites, represents a major global cause of morbidity and mortality (1). *Plasmodium* spp. are eukaryotic apicomplexan intracellular parasites with different life-cycle stages within the vertebrate host: an early clinically silent liver stage that can last approximately 7–10 days in humans and 2 days in rodents, followed by an erythrocytic stage, responsible for the pathology of malaria (**Figure 1A**). Species of *Plasmodium* that infect humans include *P. falciparum*, *P. vivax*, *P. malariae*, *P. ovale*, and *P. knowlesi*. A number of *Plasmodium* species that infect rodents, but not humans, are available for laboratory research, including *P. berghei*, *P. vinckei*, *P. chabaudi*, and *P. yoelii* (2), which allow the dissection of immune mechanism of protection and pathology (3).

In most cases, the host's immune system can eventually control a *Plasmodium* infection; however, exacerbated host immune responses and inflammation induced by the parasite, contribute to the pathology accompanying infection (4, 5). CD4+ T-cell responses have been associated with control of erythrocytic stage parasites, but a small number of studies indicate a helper role also in pre-erythrocytic immunity (6–11). Parasite biology, host cell and tissue tropism, and kinetics of parasite growth differ between pre-erythrocytic and erythrocytic stages within the vertebrate host and, accordingly, the particular CD4+ T-cell responses elicited also differ. Herein, we discuss activation of different CD4+ T-cell

subsets during malaria, their role in the control of the infection and the interplay between different subsets, with a particular emphasis on the concept of CD4+ T-cell plasticity.

#### **CD4**+ **T-CELL SUBSETS ACTIVATED BY PRE-ERYTHROCYTIC STAGES**

Very little is known about the CD4+ T-cell response to *Plasmodium* pre-erythrocytic stages, or its regulation in natural infection either in humans or in experimental models. Clearly, since IgG antibodies and memory B-cells are generated to a wide range of pre-erythrocytic antigens, including those with expression restricted primarily to these stages, such as circumsporozoite protein (CSP), liver-stage antigen 1 (LSA1), and sporozoite threonine–asparagine-rich protein (STARP) (12–14), CD4+ Tcells must be induced by these stages of the infection. Indeed, CD4+ T-cells specific for pre-erythrocytic antigens have been documented, and in some cases, have been shown to correlate with protection in humans following natural infection (15) and immunization (11). However, we have few details of their functional heterogeneity.

CD4+ T-cells of undefined Th1/Th2 phenotype have been shown to confer protection against the pre-erythrocytic stages of *P. yoelii* even in the absence of CD8+ T-cells (9), and CD4+ T-cell clones recognizing peptides of CSP protected against a *P. yoelii* sporozoite challenge in mice, irrespective of their Th1 or

toward the erythrocytic stage of Plasmodium. **(D)** Current understanding of

(53). DC, myeloid dendritic cell; iRBC, infected red blood cell; CTL, cytotoxic CD8+ T-cell; Tr1, Foxp3− regulatory T-cell.

Th2 phenotype (6, 7). The location of priming of CD4+ T-cell specific for pre-erythrocytic stages is still a matter of debate, and there is little evidence as yet on priming of CD4+ T-cell in the liver. As protective CD8+ T-cells specific for a peptide of CSP from *P. yoelii* can be primed by dendritic cells (DCs) in lymph nodes after infection with sporozoites (16), it is likely that DCs in lymph nodes might also be critical for priming CD4+ T-cell responses to *Plasmodium* pre-erythrocytic stages.

As well as possible direct killing of infected hepatocytes, subsets of CD4+ T-cells provide crucial help for both B-cell and CD8+ Tcell responses (**Figure 1B**). In a very few studies, CD4+ T-cells were shown to be necessary to ensure survival of protective effector and memory CD8+ T-cells induced by irradiation-attenuated sporozoites (8, 10). This mechanism is dependent on STAT-6 and IL-4, suggesting that Th2 CD4+ T-cells may be in charge of providing help to CD8+ T-cells. More recently, CSP-specific CD4+ T-cells expressing CD107a (LAMP-1), a marker for cytotoxic degranulation, were shown to be induced and associated with protection against the pre-erythrocytic stages after immunizations of healthy volunteers by bites from *P. falciparum*-infected mosquitoes during chloroquine chemoprophylaxis (11). Nothing is yet known about induction of Tfh cells, the subset that provides help for T-cell dependent B-cell responses (17), which are presumably activated by, and would be important for, generation of high affinity IgG antibodies.

Altogether, these data suggest a role of CD4+ T-cells in protective immunity to pre-erythrocytic-stage infection, the mechanisms of which are not yet completely understood. With the current emphasis on pre-erythrocytic vaccines, it is important that we understand more about the potential contribution of diverse CD4+ T-cell populations on direct killing of infected cells, in the B-cell and CD8+ T-cell responses, as well as their regulatory roles in immunity to the pre-erythrocytic stages of *Plasmodium*.

#### **CD4**+ **T-CELL SUBSETS ACTIVATED BY ERYTHROCYTIC STAGES**

The Th1/Th2 paradigm proposed by Mosmann and Coffman postulated stable lineages of activated CD4+ T-cells with distinctive cytokine production patterns and functional capacity; IFN-γproducing Th1 cells being crucial mediators of host immunity against intracellular pathogens, while IL-4-producing Th2 cells mediating immunity toward extracellular pathogens and collaboration with B-cells for antibody production (18). As *Plasmodium* invades red blood cells (**Figure 1A**), which do not express MHC class I or II, it was difficult to envisage parasites at this stage as direct targets of Th1 or Th2 cells. Nonetheless, it was possible to draw parallels with the original Mosmann and Coffman model; malaria researchers observed that the erythrocytic stages triggered a strong IFN-γ response during acute infections in *P. berghei*, *P. yoelii*, and *P. chabaudi* infections in mice, as well as in *P. falciparum* infection in humans (19–24).

Since that time, the Th1/Th2 paradigm has gained in complexity with the identification of novel CD4+ T-cell subsets with distinctive characteristics and transcriptional programs in charge of driving the different cell fates (25). These differentiation programs are governed predominantly by signals derived from antigenpresenting cells (APC) and the microenvironment at the time of CD4+ T-cell activation. DCs are necessary for effective priming of the T-cell response in erythrocytic-stage malaria (26), and two subsets of splenic DCs, CD8− and CD4+ classical DCs, have been shown to present antigen for the activation of CD4+ T-cells during an erythrocytic-stage infection with *P. chabaudi* and *P. berghei*, respectively (26–29). Although it is known that IL-12 is an important cytokine in the induction of a protective response in experimental malaria infections (30), understanding of the regulation of this cytokine or other factors in DCs necessary for effective *Plasmodium*-antigen presentation to different subsets of CD4+ T-cells is still lacking.

IFN-γ, a defining cytokine of Th1 cells expressing the transcription factor T-bet, has proven to be important for controlling the acute erythrocytic stage of *Plasmodium* infection in rodent models (31–34). IFN-γ-producing CD4+ effector (E) and effector memory (EM) CD4+ T-cells both confer partial protection from *P. chabaudi* infection (35). In general agreement with this, IFN-γ from CD4+ T-cells has been shown to be important in maintaining strain-transcending blood-stage immunity (36). However, IFN-γ is not only produced by T-bet+ Th1 cells but also by NK cells, NKT cells, and γδ T-cells (37, 38) as well as CD8+ T-cells, and it is not always clear whether Th1 cells, IFN-γ, or IFN-γ from Th1 cells *per se* are the main players in early protection or pathology in experimental malaria. Studies so far to address these questions have given conflicting results. One study using *P. berghei* ANKA has shown that the enhanced IFNγ + T-bet+ CD4+ T-cell responses observed in mice lacking Type I IFN signaling are associated with better control of *P. berghei* ANKA infections, resulting in lower morbidity and mortality (39, 40). In contrast, others have shown that in the absence of T-bet, essential for Th1 commitment, cerebral pathology of *P. berghei* ANKA infections is ameliorated, and the number of IFNγ + CD4+ T-cells is reduced. However, control of parasite replication is lost and mice succumb to hyper-parasitemia and anemia (41). In a different rodent model of erythrocytic-stage malaria, *P. yoelii* 17X(NL), although activation of T-bet was detected on CD4+ T-cells early in infection (42), the infection can still be controlled in T-bet-deficient mice (43).

The erythrocytic stages of *Plasmodium* are also able to activate CD4+ T-cells that are very effective helpers for *Plasmodium*-specific antibody production, but produce little or no IFN-γ (21). This response was shown to coincide with the appearance of IL-4-producing CD4+ T-cells (21). The association between IL-4-producing CD4+ T-cells and antibody responses toward the parasite was also observed in *P. falciparum* immune subjects (44). An erythrocytic-stage *P. chabaudi* infection, the only mouse model that generates a chronic phase of infection (3), presents a biphasic CD4+ T-cell activation, with a large IFN-γ-producing CD4+ T-cell response during the acute phase, followed by an antibodyhelper/IL-4-producing CD4+ T-cell response during the chronic phase (**Figure 1C**) (21, 24, 45, 46). These data were interpreted as an early activation of Th1 cells able to control parasitemia through the activation of effector mechanisms such as macrophages, followed by a Th2 response in charge of activating B-cell responses to complete the clearance of the parasite (47, 48). However, the frequency of CD4+ T-cells able to help B-cells to produce *Plasmodium*-specific antibodies was much higher than the frequency of IL-4-producing CD4+ T-cells (21). Furthermore, control of a *P. chabaudi* infection and specific IgG responses, including IgG1 antibodies, was possible even in the complete absence of IL-4 (45). Therefore, it was clear, despite its attractiveness as a model, that the simple Th1/Th2 paradigm was not sufficient to explain the full complexity of CD4+ T-cell activation in the erythrocytic stages of *Plasmodium*. More recently, a subset of CD4+ T-cells, Tfh cells, has been described that produce IL-21, as well as other cytokines originally associated with other Th subsets, such as IFN-γ and IL-4 (17). We believe that the Tfh program, and not a Th2 response, is the critical one for B-cell help and activation of protective B-cell responses against the erythrocytic stages of *Plasmodium* infection (**Figure 1D**). However, there are very few data on Tfh or its crucial signature cytokine, IL-21, in malaria. Lymphocytes closely resembling Tfh have been observed in peripheral blood of humans (49), although not yet in people exposed to malaria. However, IL-21-producing CD4+ T-cells have been demonstrated in blood of immune adults living in endemic areas of *P. falciparum* transmission (50–52). Given the importance of the humoral response in protective immunity to the erythrocytic stages of *Plasmodium*, understanding the activation and maintenance of Tfh cells during malaria is of outstanding interest for vaccine design.

Recently, the Th17 subset of CD4+ T-cells, defined by the expression of the transcription factor RORγt, has gained attention among malaria researchers because of its role in autoimmune diseases and chronic inflammation and in responses to extracellular pathogens such as bacteria and fungi (53). CD4+ IL-17A+ RORγt + Th17 cells are activated during acute *P. berghei ANKA* and *P. yoelii* infection, but the function of these cells during infection was not explored (54). Ishida and colleagues demonstrated no association of Th17 cells and cerebral malaria in *P. berghei ANKA*infected IL-17-deficient mice (55). We have also observed the presence of IL-17A and IL-17F-producing CD4+ T-cells mainly in the liver during acute erythrocytic-stage *P. chabaudi* infection; however, IL-17A-deficient mice showed no significant alterations in the course of *P. chabaudi* infection (56). Therefore, despite activation, Th17 cells have so far not been shown to have a defined role during *Plasmodium* infections (**Figure 1D**).

Additional CD4+ T-cell subsets, such as that producing IL-22 (Th22), continue to be identified (57, 58). IL-22 has been implicated in both host defense against bacterial infections and tissue repair (59). Interestingly, IL-22 single-nucleotide polymorphisms associated with resistance and susceptibility to severe malaria have been identified (60). We have observed that IL-22-producing CD4+ T-cells are activated, albeit in low frequency, in both spleen and liver during acute erythrocytic-stage *P. chabaudi* infections, and that IL-22-deficient mice infected with *P. chabaudi* show exacerbated pathology (56) (**Figure 1D**). Research is ongoing to explore in greater detail the origins of these cells, their location, and the mechanisms underlying the observed pathology.

With the discovery of this wide array of possible CD4+ T-cell subsets and their different activation requirements and functional capacities, it is becoming clear that CD4+ T-cells may not be simply defined as individual subsets of Th cells producing a single cytokine, but rather they represent components of a dynamic and interactive response, in which these cells can be multifunctional, flexible, and plastic depending on the disease/infection and activation environment (61). The multifunctional capacity of T-cells, or the ability to perform more than one function (e.g., production of different cytokines) at the single-cell level, and its association with the capacity to control infections was first recognized in HIVinfected subjects (62) and in a mouse model of vaccination against *Leishmania major* (63). This association between multifunctional capacity of T-cells and control of infections is not limited to HIV

and *Leishmania*, and was soon observed in several other chronic infections, including viral, parasitic, and mycobacterial infections (64). Immunization of subjects with *P. falciparum* apical membrane antigen 1 (AMA1) (65), and immunizations of mice with full-length *P. falciparum* CSP protein (66) also activates multifunctional CD4+ T-cells responses. Moreover, multi-parameter flow cytometric analyses of human PBMC from children and adults exposed to malaria infection reveal the existence of CD4+ T-cells co-expressing several cytokines characteristic of many CD4+ Tcell subsets (52, 67), demonstrating the complexity of the CD4+ T-cell response activated by the erythrocytic stages of *Plasmodium*.

There is an important body of evidence suggesting that,far from being terminally differentiated stable lineages, the different Th subsets have an extensive capacity to interconvert further between different phenotypes, a concept known as plasticity (68). The most recent studies suggest that CD4+ T-cell activation with overlapping characteristics of different Th subsets is the norm rather than the exception (61), and this is likely to be reflected in complex diseases such as malaria. Although this has not been explored in any detail in the context of malaria, the concept of Th plasticity opens new possibilities for studying the function and regulation of CD4+ T-cells in the control of *Plasmodium* infection and related immunopathology. One subset known for its remarkable plasticity is the Tfh subset. It has been shown that Th1, Th2, and Th17 cells can migrate into the B-cell areas of secondary lymphoid organs and acquire the functional capacity and biomarkers of Tfh cells and, conversely, the Tfh subset can become Th1, Th2, and Th17 (69). In the context of *Plasmodium* infection, this would imply that not only could *Plasmodium* parasites activate Tfh responses directly but also the Tfh subset could potentially arise from any of the CD4+ T-cell subsets already activated by the erythrocytic stages.

In light of the multifunctional and plastic capacities of the CD4+ T-cells, a scenario can be envisaged in which Th subsets required for the control of parasite burden, such as Th1 cells, have the capacity to acquire a regulatory phenotype depending on the context, contributing to control of the inflammation and thus to protection of tissues and organs and preventing any potentially harmful effects of the response. This would allow a fine-tuning of the CD4+ T-cell response to guarantee the control of *Plasmodium* infection without causing deleterious side effects. One mechanism of self-regulation by CD4+ Th1 cells in malaria is the induction of IL-10. IFN-γ +IL-10+T-bet+ Th1 CD4+ T-cells can prevent pathology during *P. chabaudi* infection (70) (**Figure 1D**). In addition, IL-10 from CD4+ T-cells distinct from regulatory T (Treg) cells is able to control pathology in a *P. yoelii* infection in mice, but in this case, these cells do not co-express IFN-γ (71). IL-10 is produced in these cells in response to IL-27 (70), although the signals responsible for the induction of IL-27 remain unknown. The occurrence of IFN-γ +IL-10+T-bet+ CD4+ T-cells during *Plasmodium* infections is not restricted to mouse models; they have been reported to be present in PBMC of children living in highly malaria-endemic regions (72–74) and their proportion is higher in children with uncomplicated malaria compared to children with severe malaria (72). IL-10 can also be induced in IL-17-producing CD4+ T-cells, as yet by unknown pathways (75–77), and thus, IL-10 may be a more general mechanism for regulating any subset

of CD4+ T-cells in malaria. CD4+ T-cells, particularly Th1 cells, can also be controlled by Type I IFNs. In the *P. berghei* ANKA model, Type I IFN signaling suppresses Th1 responses by directly acting on classical DCs (40). Given that type I IFN signaling can also promote the expression of IL-10 on CD4+ T-cells (78–81), we hypothesize that these two regulatory mechanisms might share some common activation signals during *Plasmodium* infection.

CD4+ T-cell responses may also be controlled by the expression of surface molecules associated with exhaustion. Elevated frequencies of PD-1+ LAG-3+ CD4+ T-cells have been reported in *P. falciparum*-infected subjects (82, 83), and combined blockade of PD-1 and LAG-3 accelerated clearance of erythrocytic-stage *Plasmodium* infection in a mouse model (83). In agreement with these observations, PD-1-deficient mice show better control of an erythrocytic-stage *P. chabaudi* infection with higher frequencies of IFN-γ + and T-bet+ CD4+ T-cells during the chronic phase (84). The kinetics of PD-1+ CD4+ T-cells during the acute erythrocyticstage *P. yoelii* 17X(NL) infection are similar to those observed during *P. chabaudi* infection (42, 84). However, some caution should be exercised in assuming that expression of PD-1 automatically means exhaustion, as in some subsets of activated CD4+ T-cells, in particular Tfh cells, PD1 is expressed without affecting their functional capacity. It may be that the triggering of PD-1 by its ligand PDL-1 (85) is the key to whether the cell is programed for cell death.

Many of the CD4+ T-cells activated in a *Plasmodium* infection may undergo interconversion between defined cell subsets depending on antigen dose, APC, location, and cytokine/ chemokine environment, such as that described for Treg and Th17 subsets (86, 87). Thus, it is possible that the Th17 cells found in the spleen of malaria-infected mice gain a regulatory phenotype in other organs or tissues such as brain and liver. The capacity to identify and manipulate these possible mechanisms of CD4+ T-cell plasticity during *Plasmodium* infections would be of great value for the design of novel therapeutic strategies.

#### **CONCLUDING REMARKS**

The identification of two CD4+ T-cell subsets with different welldefined functions represented an attractive organizational system with which to rationalize CD4+ T-cell responses to *Plasmodium* infections. However, such a model has not been sufficient to reflect fully the complexity of CD4+ T-cell biology observed in human or experimental malaria. In particular, control of *Plasmodium* infection requires strictly regulated immune responses that are able to prevent parasite replication without causing detrimental side effects of uncontrolled inflammation. *Plasmodium* species have different stages with different tissue tropisms and this complex life cycle challenges the idea that a single static group of terminally differentiated CD4+ T-cells would be able to perform all the tasks required to control this infection. In order to cope with these tasks, the CD4+ T-cell response has to adapt to the changing scenarios presented as the infection evolves. The newer concept of CD4+ T-cell plasticity would add substantially to our understanding of induction and regulation of CD4+ T-cell responses in malaria, and it is highly probable that some of the CD4+ Th programs not yet explored in depth, such as the Tfh response, might play critical roles in the outcome of the infection. The combination of potent

tools such as multi-parameter flow cytometry, *in vivo* imaging, systems analyses of transcriptome, proteome, and metabolome, together with T-cell receptor transgenic mice and peptide-MHC II tetramers will give us the chance to explore the complexity of the CD4+ T-cell responses to malaria in *in vivo* models (74, 88–97). In addition, confocal microscopy and intravital imaging techniques make now possible to follow sporozoites injected via the mosquito bite into the skin, or by injection of attenuated sporozoites through to their arrival in the lymphoid organs and liver (98), and to study the consequent activation of CD4+ T-cells and their subsequent effector functions. Field studies of natural human *Plasmodium* infections and mouse models should complement each other to get a deeper understanding of the complex CD4+ T-cell response activated by these infections. A detailed delineation on how CD4+ T-cells modulate the activation of effector cells such as CD8+ T-cells, macrophages, and B-cells in response to *Plasmodium* infection is critical to achieve the goal of generating protective treatments to control malaria.

#### **ACKNOWLEDGMENTS**

We thank Deirdre Cunningham, Jan Sodenkamp, Wiebke Nahrendorf, and Jingwen Lin for their helpful comments and critical reading of the manuscript. The excellent support of Biological Services, NIMR Flow Facility, and PhotoGraphics is much appreciated. Recent work cited from the authors' laboratory was funded by the Medical Research Council, UK (U117584248), the Wellcome Trust (048684), and the European Union (FP7/2007–2013) under grant agreement 242095-EVIMalaR.

#### **REFERENCES**

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 03 September 2014; accepted: 15 December 2014; published online: 12 January 2015.*

*Citation: Perez-Mazliah D and Langhorne J (2015) CD4 T-cell subsets in malaria: TH1/TH2 revisited. Front. Immunol. 5:671. doi: 10.3389/fimmu.2014.00671*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2015 Perez-Mazliah and Langhorne. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### **Christian R. Engwerda1\*, Susanna S. Ng1,2 and Patrick T. Bunn1,3**

1 QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia

2 School of Natural Sciences, Griffith University, Nathan, QLD, Australia

3 Institute of Glycomics, Griffith University, Gold Coast, QLD, Australia

#### **Edited by:**

Dragana Jankovic, National Institutes of Health, USA

#### **Reviewed by:**

Sid P. Kerkar, National Institutes of Health, USA Kevin Couper, University of Manchester, UK

#### **\*Correspondence:**

Christian R. Engwerda, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, QLD 4005, Australia e-mail<EMAIL_ADDRESS>
CD4+ T cells are critical for defense against protozoan parasites. Intracellular protozoan parasite infections generally require the development of aTh1 cell response, characterized by the production of IFNγ andTNF that are critical for the generation of microbicidal molecules by phagocytes, as well as the expression of cytokines and cell surface molecules needed to generate cytolytic CD8+ T cells that can recognize and kill infected host cells. Over the past 25 years, much has been learnt about the molecular and cellular components necessary for the generation of Th1 cell responses, and it has become clear that these responses need to be tightly controlled to prevent disease. However, our understanding of the immunoregulatory mechanisms activated during infection is still not complete. Furthermore, it is apparent that although these mechanisms are critical to prevent inflammation, they can also promote parasite persistence and development of disease. Here, we review how CD4+ T cells are controlled during protozoan infections and how these regulatory mechanisms can influence parasite growth and disease outcome.

**Keywords: protozoan parasites,Th1 cells, IL-10, IL-27, CD4**+ **T cells, immune regulation**

#### **INTRODUCTION**

Mammalian immune systems have evolved to recognize and control pathogens. This is achieved by the coordinated actions of innate and adaptive immune mechanisms [reviewed in Ref. (1, 2)]. CD4+ T cells play key roles in coordinating immune responses by producing molecules critical for the production of high affinity antibodies by B cells and promoting the production of mucous and tissue repair mechanisms. They also help to fully activate CD8+ T cells so they can kill infected and transformed cells, and assist innate immune cells to recognize and control pathogens and tumors. CD4+ T cells play critical roles in both the generation of anti-parasitic immunity and immune surveillance during concomitant immunity, which is associated with many parasitic infections (3).

#### **REGULATION OF T CELL RESPONSES**

The help provided by CD4+ T cells for various immune activities includes the production of potent pro-inflammatory cytokines such as TNF, IFNγ, and IL-17, and as such, CD4+ T cell responses need to be tightly regulated so they themselves do not cause tissue damage. The pathogenesis of autoimmune diseases often involves aberrant CD4+ T cell responses in tissue sites such as the central nervous system, pancreas, and brain. Therefore, mammals have evolved multiple ways to control the pathogenic potential of CD4+ T cells [reviewed in Ref. (4)]. These include indoleamine 2,3-dioxygenase (IDO)-catalyzed tryptophan metabolism by phagocytic cells (5), leading to immune cell stress and activation of the general controlled non-repressed 2 (GCN2) kinase pathway (6) and/or cytotoxic and regulatory effects on T cells caused by the catabolites from the associated kynurenine

metabolism pathway (7). In addition, the production of regulatory cytokines, such as IL-10 and TGFβ, by innate immune cells in response to pathogen-derived molecules can suppress both developing and established T cell responses (8–10), as can IL-10 produced by certain B cell subsets (11). Dendritic cells (DCs) can be an important source of regulatory cytokines in experimental models of leishmaniasis and malaria. In addition, over the course of these infections, DCs reduce levels of CD11c, increase expression of CD45RB, and promote the generation of T cell IL-10 production (12–14). Thus, the development of regulatory DC subsets that have a major influence on T cell responses is a feature of established protozoan infections. More recently, specialized monocytes and macrophage subsets have been identified that can modulate localized T cell responses during protozoan infections [reviewed in Ref. (15)]. Classically activated (M1) macrophages produce pro-inflammatory molecules, such as TNF and l-arginine-dependent nitric oxide, while alternatively activated (M2) macrophages use arginase 1 to convert l-arginine to polyamines, which along with production of IL-10 and TGFβ, enable this cell subset to suppress inflammation [reviewed in Ref. (16)]. Inflammatory monocytes have been reported to promote Th1 cell activity in mice infected with *Leishmania major* (17), *L. donovani* (18), and *Trypanasoma brucei* (19, 20), but with pathological consequences in the latter model that were reversed by administration of IL-10 (20). In contrast, the products from M2 macrophages suppressed lesional CD4+ T cell proliferation and IFNγ production in mice infected with *L. major* (21), while *T. gondii* can actively promote the arginase 1 pathway in macrophages to enhance pathogen survival (22). Thus, macrophages play important roles in conditioning local tissue

environments and determining the direction and effectiveness of T cell responses during protozoan infections. However, regulatory mechanisms increasingly recognized as being paramount for preventing T cell-mediated disease, and therefore, the main subject of this review, involve specialized sub-populations of CD4+ T cells themselves capable of inhibiting immune responses and suppressing inflammation.

#### **REGULATORY T CELLS**

Regulatory T cells can be broadly divided into two types. First, natural regulatory T (Treg) cells are CD4+ T cells produced in the thymus and express the transcription factor FoxP3 that is critical for their suppressive functions (23, 24). Second, inducible regulatory T cells emerge from the thymus as conventional T cells, but develop regulatory functions in the periphery following exposure to appropriate inflammatory stimulation. These include IL-10-producing Th1 (Tr1) cells (25), TGFβ-producing CD4+ T (Th3) cells (26), and conventional CD4+ T cells that have converted to FoxP3-positive cells in peripheral tissues (27). Under homeostatic conditions, Treg cells limit potentially self-reactive T cell responses, thus preventing autoimmunity (23). However, they can also impair effective pathogen clearance, while trying to prevent immune-mediated tissue damage during infection. The molecular mechanisms by which Treg cells perform these functions are incompletely understood, but involve production of cytokines such as IL-10, TGFβ, and IL-35, the expression of the negative regulatory molecule cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and the generation of adenosine and cyclic AMP [reviewed in Ref. (28)]. In addition, their expression of high affinity IL-2 receptor allows them to deprive conventional T cells of this critical growth factor and thereby induce them to undergo apoptosis (29). Thus, Treg cells may act directly upon conventional T cells or via accessory cells such as antigen presenting cells (APCs) to limit T cell activity. An emerging paradigm is that Treg cells adapt to particular inflammatory conditions in order to regulate specific CD4+ T cell responses by the generation and use of shared transcription factors to mimic certain aspects of T cell behavior, such as tissue homing, survival, and cytokine production. For example, STAT3, T-bet, IRF-4, and Bcl-6 are required for Th17, Th1, Th2, and follicular helper T (Tfh) cell differentiation, respectively, as well as by the Treg cells that control the actions of these specific CD4+ T cell subsets (30– 34). For example, in mice orally infected with *T. gondii*, IL-27 promotes the expression of CXCR3 on Treg cells that enables these cells to regulate Th1 cell-mediated immunity, as well as prevent infection-induced pathology at mucosal sites (35). However, Treg cells can block the generation of effective parasite-specific T cell responses in specific tissues. For example, Treg cell depletion with anti-CD25 mAb in mice infected with *L. major* dramatically enhanced anti-parasitic immunity (36), while adoptive transfer of antigen-specific Treg cells in the same model promoted parasite growth (37). In addition, depletion of Treg cells with an anti-CD25 mAb protects mice from lethal *Plasmodium yoelii* infection by enabling the generation of a potent anti-parasitic T cell response (38). Similarly, the removal of Treg cells from peripheral blood mononuclear cells isolated from humans infected with *P. falciparum* enhanced T cell proliferation and CD4+ T cell IFNγ

production in response to stimulation with parasite antigens (39). However, the importance of Treg cells in several protozoan infections has been questioned because of the potential "off-target" effects of the anti-CD25 mAbs used in many studies (40). For example, several groups reported significant changes in immune responses and disease outcome in mice infected with *P. berghei* ANKA (41–43), but subsequent experiments in this model, where Treg cells could be specifically depleted with diphtheria toxin via cell-specific expression of a simian diphtheria toxin receptor (44), showed little impact of Treg cells on disease outcome and associated T cell responses (45, 46). Thus, the roles of Treg cells in protozoan infections will require further studies before their impact on anti-parasitic immune responses can be fully appreciated.

The secretion of IL-10 by conventional CD4+ T cells can potently suppress inflammation and tissue damage (47, 48). Initially, IL-10 production was identified in Th2 cells (49), but has since been described in Th1 (50–52) and Th17 (53) cell populations. Thus, CD4+ T cell-derived IL-10 production is emerging as an important mechanism of auto-regulation, whereby IL-10 can both directly suppress T cell activities, as well as upstream activation pathways initiated by APCs [reviewed in Ref. (48)]. These IL-10-producing Th1 cells were identified in mice infected with *T. gondii* (54) and *L. major* (55). In the *T. gondii* infection model, these cells did not impact upon control of parasite growth, but were critical for limiting pathology (54, 56), while in mice infected with *L. major*, IL-10-producing Th1 cells promoted the establishment and maintenance of chronic infection (55). Similar observations have also been made in mouse models of *Plasmodium* infection (57, 58), *T. cruzi* (59, 60), and *T. brucei* (61) infections. Importantly, these IL-10-producing Th1 cells have been identified in humans with visceral leishmaniasis caused by *L. donovani* (62) and African children with *P. falciparum* malaria (63, 64). Although IL-10 has been clearly shown to suppress CD4+ T cell activation in humans infected with *L. donovani* (65) and *P. falciparum* (63, 66), it is not yet clear how much of this activity can be attributed to the IL-10-producing Th1 cells. Significantly, the prevalence of IL-10-producing Th1 cells in Gambian children with asymptomatic malaria was greater than in children with severe disease, indicating that these cells may protect against damaging inflammation during acute malaria (67). However, antigen-specific IL-10-producing Th1 cells were found in cord blood of babies whose mothers had malaria during pregnancy (66), suggesting that these cells might be able to influence anti-parasitic immunity from very early in life. Hence, the kinetics of the emergence of IL-10-producing Th1 cells during malaria may be critical in determining the impact they have on the outcome of infection.

#### **THE ROLES OF IL-10 IN PROTOZOAN INFECTIONS**

IL-10 is one of the most potent regulatory cytokines produced by leukocytes in response to inflammatory signals (68). The importance of IL-10 for regulating immunity is highlighted by the observation that IL-10 deficiency or blockade causes the early development of colitis in mice (69). However, as described above, many protozoan parasites, such as those that cause toxoplasmosis, malaria, trypanosomiasis, and leishmaniasis (54, 55, 59–61, 70, 71), have evolved to exploit the functions of IL-10 to inhibit anti-microbial mechanisms and allow the establishment of chronic infection. In fact, the generation of IL-10-producing T cells following vaccination with protozoan antigen can be a robust predictor of vaccine failure (28). One proposed mechanisms for IL-10-mediated immune suppression is the promotion of T cell exhaustion. The PD-1 pathway plays an important role in T cell exhaustion during all of the chronic infections mentioned above, and there is strong evidence that IL-10 plays a key role in regulating the expression of the PD-1 ligands (PD-L1 and PD-L2) on APCs [reviewed in Ref. (72)]. Several other molecules known to be involved in promoting T cell exhaustion, such as Tim-3 and Lag3, have also been linked with IL-10 expression (73, 74), but their precise relationships are not known. IL-10 produced by macrophages can also inhibit the differentiation of surrounding cells into classically activated macrophages that are required for the production of inflammatory cytokines and metabolites required to kill many intracellular pathogens (75). It can also suppress inflammatory cytokine production by T cells and inhibits antigen presentation by APC [reviewed in Ref. (76)]. Thus, IL-10 can suppress host immune responses during infection by multiple mechanisms.

#### **REGULATION OF IL-10 PRODUCTION BY CD4**+ **T CELLS**

An important approach to understanding how IL-10 production might be modulated for therapeutic advantage or to improve vaccination is to gain a better insight into the transcriptional regulation and the signaling pathways involved in IL-10 production and establishing whether they differ between cell types and in various tissue locations. IL-27 has emerged as an important growth and differentiation factor for IL-10-producing Th1 cells (53, 77, 78). It is thought to primarily be a product of macrophages and DCs (79), and drives the production of IL-21 by CD4+ T cells, which in turn, acts as an autocrine growth factor for IL-10 producing Th1 cells (80, 81). IL-27 is a heterodimeric cytokine composed of IL-27p28 and EBI3 that signals via a receptor complex comprising a unique IL-27 receptor alpha chain (IL-27Rα) and gp130 (82, 83), a common receptor used by several cytokines including IL-6 (84, 85). IL-27 promotes these activities via the transcription factors STAT1 and STAT3 (53), and by inducing the expression of the transcription factors c-Maf (80) and aryl hydrocarbon receptor (AhR) (86), which then physically associate and transactivate the IL-10 and IL-21 gene promoters (80, 86, 87). Interactions between glucocorticoid-induced TNFR-related (GITR) protein and GITR ligand can also stimulate IL-27 production (88), which can induce expression of inducible T cell costimulator (ICOS) on IL-10-producing Th1 cells to enhance IL-27-mediated expansion of these cells (80). Interestingly, IL-27p28 can also function as a natural antagonist of gp130-mediated cytokine signaling, and thereby inhibit IL-6-mediated inflammatory pathways (89). The importance of IL-27 for the generation of IL-10-producing Th1 cells has now been reported in mouse models of malaria (58, 90), leishamaniasis (91), and toxoplasma (53), although, surprisingly, the generation of these cells was independent of IL-21 in mice infected with *P. chabaudi* (58). It should also be noted that IL-27 has IL-10-independent regulatory functions in mice infected with *P. berghei* NK65 (90), thus emphasizing

the complexity of IL-27-mediated immune regulation during protozoan infections. IL-27 produced by CD14 positive monocytes was also reported to be associated increased numbers of IL-10 producing Th1 cells in blood from visceral leishmaniasis patients (92). Thus, there is substantial evidence for IL-27 being a critical factor in the generation of IL-10-producing Th1 cells during protozoan infections.

In other studies, the transcriptional repressor B lymphocyteinduced maturation protein 1 (Blimp1; encoded by the *Prdm1* gene) was found to be expressed by a subset of Treg cells and played an essential role in their production of IL-10 (93). Recently, Blimp1 was implicated in IL-10 production by Th1 cells (94), and shown to be important for the generation of these cells in mice infected with *T. gondii* (95). IL-27 and T cell receptor signaling were found to promote the expression of the transcription factor Erg2, which was required for Lag3 expression and production of IL-10 by conventional CD4+ T cells (96). Subsequently, IL-27-dependent Egr2 expression was reported to be critical for the induction of Blimp1 and generation of IL-10-producing Th1 cells (94). Interestingly, only STAT3-deficiency impacted upon IL-27 dependent Egr2 expression, while both STAT1 and STAT3 were required for IL-10 production by Th1 cells. Thus, a model for IL-10 production by Th1 cells is emerging (**Figure 1**). Furthermore, cellular pathways such as the Notch-Jagged axis in plasmacytoid DCs promote CD4+ T cell IL-10 production (97), but their roles in protozoan infections have yet to be investigated. Thus, there are still many gaps to be filled, and importantly, we need to clearly define the signaling and transcriptional pathways that are activated during protozoan infections. In particular, there is a clear gap in our knowledge regarding differences in the regulation and maintenance of IL-10 production by Th1 cells in secondary lymphoid organs and peripheral tissue sites. This information is important if we want to target these regulators to selectively modulate IL-10 activity during parasitic disease. In the broader context of immune regulation, we need to establish whether the IL-10-producing Th1 cells are a distinct T cell subset capable of dynamic and sustained regulatory function or whether they represent exhausted T cells, as suggested by their expression of molecules such as PD-1 and Lag3. In the former, we can develop ways to manipulate them for therapeutic advantage (for example in inflammatory diseases) or transiently block their function, as might be required for effective vaccination. However, if they represent a terminally differentiated state, then different approaches may have to be devised to either promote or inhibit their development.

#### **OTHER MECHANISMS OF Th1 CELL REGULATION DURING PROTOZOAN INFECTIONS**

Although IL-10 is a potent regulator of Th1 cell responses, there is likely to be multiple mechanisms to control such potentially damaging inflammatory responses. Type I IFNs have recently emerged as import immune regulators during parasitic infections. They are produced by most cell types and play critical roles in anti-viral immunity (98, 99), but several studies have identified this family of cytokines as important determinants of disease outcome in protozoan infection. However, these effects depend on the virulence of the parasite and the stage of infection. For example, in

mice lacking type I IFN receptor, early control of *T. brucei* was impaired, but it appeared that IFNγ production later in infection was suppressed by type I IFN signaling pathways (100). In contrast, early control of a high dose *T. cruzi* infection was enhanced in type I IFN receptor-deficient mice and this was associated with increased IFNγ production, but not when a lower parasite dose was used (101). In liver stage *P. berghei* ANKA infection, parasite RNA triggers a type I IFN transcriptional program in hepatocytes that enhances innate immune responses in hepatic myeloid cells to control liver parasite load (102). In contrast, in mice infected with *P. berghei* ANKA blood-stage parasites, type I IFNs promoted susceptibility to severe disease (103, 104) by suppressing Th1 cell development (103) indirectly through inhibition of CD8− DC function (105). Interestingly, this latter effect of type I IFN on DC function was also associated with reduced IL-10 mRNA accumulation in CD8− DCs that lacked type I IFN receptor, potentially linking infection-induced IL-10 production with Th1 regulation once again (**Figure 2**).

TGFβ has also emerged as an important regulatory cytokine controlling Th1 responses during protozoan infections [reviewed in Ref. (106)]. In mice, susceptibility to lethal *P. berghei* ANKA infection correlated with reduced TGFβ levels (107), while high levels of TGFβ in malaria patients was associated with increased parasite growth. In mice infected with*T. gondii*, TGFβ produced by gut intraepithelial CD8+ T cells was critical for controlling inflammation and gut pathology (108), thus supporting a key role for this cytokine in regulating inflammation during protozoan infections. In both malaria and toxoplasma, it is likely that TGFβ acts by suppressing T cell activation and promoting Treg cell functions (109). However, this is yet to be formally demonstrated and there still remains much to learn about the functions of TGFβ during infection.

Both lipoxin A4 and glucocorticoids have also been identified as important regulators of Th1 cell responses in mice infected with *T. gondii* (110–112). Lipoxin A4 is an eicosanoid mediator capable of suppressing DC IL-12 production in response to parasite antigen *in vitro* (110) or during *T. gondii* infection in mice (111). In this latter study, infection of mice lacking lipoxin A4 resulted in a fatal, parasite-induced inflammation (characterized by a potent Th1 cell response), but reduced parasite loads. Importantly, results from this and previous studies (113), suggest that IL-10 was critical for regulating inflammation during the acute stage of infection, while lipoxin A4 was important for immune regulation during chronic infection (111). A novel, IL-10-independent pathway of immune regulation was also recently identified in this infection model, whereby glucocorticoids produced by the hypothalamic– pituitary–adrenal axis during *T. gondii* infection act directly on CD4+ T cells to prevent Th1 cell hyperresponsiveness and resulting pathology (112). Given the critical roles for both IL-10-dependent and IL-10-independent pathways in preventing inflammatory diseases associated with protozoan infections, temporal and/or transient blockade of one or the other pathways may be a viable way to enable sufficient pro-inflammatory immunity to control parasite growth, but also leave enough regulatory machinery in place to prevent disease.

#### **CONCLUDING REMARKS**

There are currently no vaccines to protect against or treat diseases caused by protozoan parasites. It has proven extremely difficult to generate robust and long-lasting CD4+ T cell responses against the responsible pathogens (3). An important impediment for generating sufficient immunity against these pathogens could, in some cases, be the accompanying regulatory immune response that aims to limit inflammation. Treg cell depletion can dramatically improve candidate malaria vaccine efficacy (114, 115), although as mentioned above, these studies must be interpreted with caution because of the use of anti-CD25 mAb for Treg cell modulation. Nevertheless, the blockade of IL-10 produced by antigen-specific Th1 cells improved anti-parasitic immunity generated by a candidate vaccine directed against *L. major* (116), while studies on *T. gondii* indicated that induction of IL-10-producing Th1 cells following vaccination caused a lethal infection upon secondary exposure to the parasite (117). Although the depletion of Treg cells and/or IL-10-producing Th1 cells is not a viable option for improving vaccine efficacy given the critical roles of these cells in preventing immune-mediated disease, a much better understanding about how regulatory immune responses can be locally and temporarily modulated to enhance vaccine-induced immune responses may be of significant benefit.

The regulation of Th1 cell responses during protozoan infections is clearly important to ensure both sufficient generation of inflammatory mediators to control parasite growth, as well as to prevent excessive production of these molecules in sensitive tissue sites. IL-10 has emerged as an important regulator of these responses, both produced in a highly regulated manner by Th1 cells themselves, as well as parasite-activated innate immune cells. However, IL-10 is not alone in this activity, and alternative mechanisms of Th1 cell regulation have been identified. Our challenge remains to fully define these mechanisms of Th1 cell regulation and to use this knowledge to improve therapeutic options and vaccine efficacy. Research in protozoan infections of both mice and humans is ideally placed to identify broad mechanisms of immune regulation that are relevant not only to parasitic infections but also for autoimmune and physiological diseases, as well as cancer.

#### **AUTHOR CONTRIBUTIONS**

Christian R. Engwerda, Susanna S. Ng, and Patrick T. Bunn all contributed to the planning and research in this paper. Susanna S. Ng conceived and produced both figures, while Christian R. Engwerda and Patrick T. Bunn wrote the paper.

#### **ACKNOWLEDGMENTS**

We thank members of the Engwerda Laboratory at QIMR Berghofer for assistance with researching this paper and for helpful discussions.

#### **REFERENCES**

Egr-2. *Proc Natl Acad Sci U S A* (2009) **106**:13974–9. doi:10.1073/pnas.<PHONE_NUMBER>

117. Shaw MH, Freeman GJ, Scott MF, Fox BA, Bzik DJ, Belkaid Y, et al. Tyk2 negatively regulates adaptive Th1 immunity by mediating IL-10 signaling and promoting IFN-gamma-dependent IL-10 reactivation. *J Immunol* (2006) **176**:7263–71. doi:10.4049/jimmunol.176.12.7263

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 05 August 2014; paper pending published: 08 September 2014; accepted: 25 September 2014; published online: 13 October 2014.*

*Citation: Engwerda CR, Ng SS and Bunn PT (2014) The regulation of CD4*+ *T cell responses during protozoan infections. Front. Immunol. 5:498. doi: 10.3389/fimmu.2014.00498*

*This article was submitted to T Cell Biology, a section of the journal Frontiers in Immunology.*

*Copyright © 2014 Engwerda, Ng and Bunn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### **Jens Geginat \*, Moira Paroni, Stefano Maglie, Johanna Sophie Alfen, Ilko Kastirr, Paola Gruarin, Marco De Simone, Massimiliano Pagani and Sergio Abrignani \***

Fondazione Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" INGM, Milan, Italy

#### **Edited by:**

Dragana Jankovic, National Institutes of Health (NIH), USA

#### **Reviewed by:**

António Gil Castro, University of Minho, Portugal Toshinori Nakayama, Graduate School of Medicine, Japan

#### **\*Correspondence:**

Jens Geginat and Sergio Abrignani, Fondazione Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" INGM, Via Sforza 35, Milano 20122, Italy e-mail<EMAIL_ADDRESS><EMAIL_ADDRESS>
Human beings are exposed to a variety of different pathogens, which induce tailored immune responses and consequently generate highly diverse populations of pathogenspecific T cells. CD4+ T cells have a central role in adaptive immunity, since they provide essential help for both cytotoxic T cell- and antibody-mediated responses. In addition, CD4+ regulatory T cells are required to maintain self-tolerance and to inhibit immune responses that could damage the host. Initially, two subsets of CD4+ helper T cells were identified that secrete characteristic effector cytokines and mediate responses against different types of pathogens, i.e., IFN-γ secreting Th1 cells that fight intracellular pathogens, and IL-4 producing Th2 cells that target extracellular parasites. It is now well established that this dichotomy is insufficient to describe the complexity of CD4+ T cell differentiation, and in particular the human CD4 compartment contains a myriad of T cell subsets with characteristic capacities to produce cytokines and to home to involved tissues. Moreover, it has become increasingly clear that these T cell subsets are not all terminally differentiated cells, but that the majority is plastic and that in particular central memory T cells can acquire different properties and functions in secondary immune responses. In addition, there is compelling evidence that helper T cells can acquire regulatory functions upon chronic stimulation in inflamed tissues. The plasticity of antigen-experienced human T cell subsets is highly relevant for translational medicine, since it opens new perspectives for immune-modulatory therapies for chronic infections, autoimmune diseases, and cancer.

**Keywords: CD4 T cells, cytokines, differentiation, tissue homing, plasticity**

#### **INTRODUCTION**

Human CD4+ T cells are critical regulators of the immune system, as drastically demonstrated by HIV-infected individuals that develop susceptibility to opportunistic infections and cancer when virus-dependent depletion reduces CD4+ T cell counts below critical thresholds (1). CD4+ T cells are very heterogeneous in human adults, because they have been generated in response to a high number of different pathogens and belong to a progressively increasing number of different subsets with specialized functions (2). Helper T cell subsets are defined by the production of cytokines and/or the expression of characteristic lineage-defining transcription factors (**Table 1**). Five principal subsets or lineages of CD4+ T cells have been identified so far: T helper (Th)1, Th2, and Th17 cells that target specific classes of pathogens (3–5), regulatory T cells that are required to maintain self-tolerance (6) and follicular helper T cells (TFH) that provide help to B cells for antibody production (7). Heterogeneity is generated upon T cell priming, since naïve T cells have stemcell-like properties and can differentiate into virtually all different types of effector, memory, or regulatory cells (**Table 1**). Antigenexperienced T cells are less flexible, but many subsets retain some plasticity and can acquire additional cytokine producing capacities upon antigenic re-stimulation, while others appear to be terminally differentiated (8). In some cases, T cell functions can even completely change from helper to regulatory functions (9) or vice versa (10). A caveat of these findings in particular in humans is the enormous heterogeneity of T cells (2), making it difficult to exclude a selective outgrowth of rare pre-existing precursor cells. Several excellent reviews on the plasticity of mouse T cells have been published in recent years (11–13), while human T cell plasticity is less understood, but highly relevant for new therapeutic strategies in immune-mediated diseases (14).

#### **TERMINALLY DIFFERENTIATED TH1 AND TH2 EFFECTOR CELLS: THE TIP OF THE ICEBERG**

Seminal studies have established that CD4+ T cells can differentiate into two types of effector cells with different cytokine producing capacities and functions in humans and mice (3, 4). Uncommitted naïve T cells that are activated by specialized dendritic cells that produce IL-12 (15, 16) acquire IFN-γ producing capacities.  In contrast, naïve T cells primed in the presence of IL-4 undergo a different fate and start to produce IL-4, IL-5, IL-10, and IL-13, but not IFN-γ. These Th2 cells are required to fight extracellular parasites like helminths, but since they induce IgE from B cells they are also involved in allergies (17). Importantly, it was shown that Th1 versus Th2 differentiation was a crucial decision to resist infections, since BL/6 mice that mount a Th1 response to leishmania were protected, while BALB/c mice that instead induce a Th2 response were highly susceptible (18). The characteristic cytokines produced by Th1 and Th2 cells, IFN-γ, and IL-4, were further shown to inhibit the differentiation to the opposite differentiation lineage and thus

reinforced the originalfate decision. The capacity to produce either IFN-γ or IL-4 is stably imprinted by epigenetic modifications like DNA methylation and histone acetylations, ensuring that the cytokine profile of T helper cells is preserved upon cellular division independently of the inducing polarizing cues (19–21). Moreover, the generation of Th1 and Th2 cells was shown to depend on the "master" transcription factors T-bet and GATA-3, which induced not only the characteristic cytokines of Th1 and Th2 cells, but also inhibited the differentiation to the alternative lineage. Based on this evidence, it was initially assumed that the differentiation to Th1 and Th2 cells are mutually exclusive and irreversible fate decisions.

#### **TH1 AND TH2 CELLS CAN ACQUIRE NEW PROPERTIES AND FUNCTIONS IN SECONDARY OR CHRONIC IMMUNE RESPONSES**

Early studies with human T cell clones showed that IFN-γ and IL-4 production were not necessarily two exclusive features, since some T cells co-produced IFN-γ and IL-4 (22). Notably, human Th1 memory cells are responsive to IL-4 stimulation, and acquire IL-4 producing capacities upon TCR stimulation in the presence of IL-4 without losing IFN-γ production *in vitro* (23).

In addition, some T cells in human blood co-express the Th1 and Th2 markers CXCR3 and CCR4 (24) or CRTh2 as well as the lineage-defining transcription factors GATA-3 and T-bet (25). Consistently, it was shown in mice that histones of these transcription factor genes had both repressive and permissive marks in opposing T cell lineages (13, 26). In mice, *in vivo* primed Th2 cells can acquire IFN-γ producing capacities in addition to IL-4 in response to IFN and IL-12 (27), while human blood Th2 cells seem to be less plastic (23). Moreover, the pathogens and the physiological conditions that induce Th1/2 cells in humans and their role in immune responses remain to be fully defined (25).

Another early finding that did not fit well into the fixed Th1/Th2 paradigma was the fact that IL-12 could induce IL-10 in Th1 cell clones (28). IL-10 has potent anti-inflammatory functions and inhibits maturation and T cell stimulatory capacities of APC (29), thus the concomitant expression of both IFN-γ and IL-10 by T cells was unexpected (30). Later it was shown that IL-10 produced by T-bet+ Th1 cells was required to inhibit lethal immunopathology upon infections with intracellular parasites (31, 32), indicating that IL-10-producing Th1 cells prevent overshooting immune responses and the resulting tissue damage in a negative feedback loop (9). Interestingly, although these IL-10 producing Th1 cells inhibited IL-12 production by APC, they were also able to restrict parasite growth via IFN-γ (31). However, IFN-γ has also been shown to have some negative effects on T cell responses (33, 34), providing a possible alternative explanation for IFN-γ production by regulatory T cells. Importantly, IFN-γ/IL-10 co-producing T cells with regulatory functions are present at low frequencies in peripheral blood of healthy donors and respond selectively to persistent pathogens (35), suggesting that similar to their mouse counterparts they inhibit overshooting immune responses in chronic infections. Thus, Th1 cells can switch from pro-inflammatory effector cells to IL-10 producing type 1 regulatory (Tr1)-like T cells (36, 37), and this switch is necessary to maintain the integrity of infected tissues in some infections. Complement receptor stimulation (38), production of IL-27 (39) or IL-12 (28) by myeloid cells (40), or generation of AHR ligands (41) are possible inductive cues, but also chronic or repetitive antigenic stimulation seems to be required to induce IL-10 production in Th1 cells (35, 42, 43). Interestingly, a recent paper suggests that IL-10/IFN-γ co-producing T cells can also be generated from Th17 cells under the influence of IL-12 or IL-27 in mice (44). If IFN-γ/IL-10 co-producing regulatory T cells are stably maintained or are short-lived, if they progressively lose IFN-γ production upon chronic stimulation or revert to Th1 cells upon pathogen clearance is currently unclear (**Figure 1**).

More recently, additional plasticity of Th2 cells was documented. Thus it was shown that TFH cells were derived from Th2 precursor cells in mouse models of helminth infections (45). This finding is relevant for Th2 stability, because TFH cells are professional B helper T cells that secrete IL-21 in B cell follicles, express the transcriptional repressor BCL-6 and are thus distinct from conventional Th1 and Th2 cells (7, 46, 47). Also in human tonsils a fraction of TFH cells express the Th2 marker CRTH2 and

produce IL-4 (48). The relationship of Th1 cells with TFH cells is less clear in particular in humans (49, 50). Some murine TFH cells produce IFN-γ (51), which induces IgG2a production by B cells (52), but TFH cells from human tonsils lack IFN-γ production.

Mouse Th2 cells can also switch from IL-4 to IL-9 production upon stimulation with TGF-β (53). These Th9 cells express the PU.1 transcription factor (54) and can also be directly induced from naïve and memory T cells upon stimulation with TGF-β and IL-4 in humans and mice (55, 56). Th9 cells can have a pro-inflammatory role in allergic asthma (57) and respond to helminth antigens and allergens in humans (58, 59). However, IL-9 induction by TGF-β is not restricted to Th2 cells (60).

Collectively, these findings indicate that both Th1 and Th2 cells can acquire different cytokine producing capacities and functional properties upon antigenic re-stimulation under the influence of cytokines, and are thus much more flexible than originally thought (**Figure 1**).

#### **STABILITY OF FOXP3**+ **TREGS IS DEBATED**

CD25+ regulatory T cells are required to maintain self-tolerance. They were first identified in mice (61) and later in humans (62), and the Foxp3 transcription factor was shown to be required for their generation and function (63, 64). Consistently, IPEX patients, who suffer from a devastating autoimmune disease, were found to have mutations in the Foxp3 gene (65). Although so-called natural or thymic Foxp3+ Tregs acquire regulatory lineage commitment already upon maturation in the thymus (66), adaptive,

or peripheral Foxp3+ Tregs can be induced from mature CD4+ helper T cells in the periphery under the influence of TGF-β (67, 68). The transcription factor Helios was proposed to distinguish between these two subsets of natural and induced Foxp3+ Treg, but this concept was not confirmed by others (69–71). In humans, CD45RA+CD25+Foxp3+ cells represent a population of *bona fide* "naïve" and thus thymus-derived Tregs, while CD45RA− Tregs are a mixed population that contain antigen-experienced Tregs of both thymic and peripheral origin (72). The stability of Foxp3+ Tregs is debated (73). Lineage tracing of Foxp3+ T cells in mice has lead to conflicting interpretations, since in several studies only very small fractions of Foxp3+ Tregs were found to lose Foxp3 and regulatory functions *in vivo* (74). In humans, CD45RA+ but not CD45RA− Tregs could be stably expanded *in vitro* (72, 75), suggesting different stabilities of thymic and peripheric Tregs. However, since human Tregs have to be purified according to surface marker expression, it is difficult to exclude a selective outgrowth of Foxp3− cells or of activated effector T cells that have transiently up-regulated Foxp3 upon stimulation (73).

The functional specialization of Foxp3+ Treg is shaped by the tissue microenvironment (76), and the induction of transcription factors characteristic for helper T cell lineages in mice allows Tregs to suppress the corresponding T helper cell responses (74). Thus, STAT3 in Tregs is required to suppress Th17 cells (77), IRF4 to control Th2 responses (78) while Tregs that regulate TFH cells and antibody responses express BCL-6 (79, 80). Foxp3+ Tregs also acquire T-bet and IFN-γ producing capacities upon

stimulation with IL-12, and these Th1regs might be specialized to suppress Th1 responses (14, 74). Tregs also inhibit anti-tumor CTL responses (81), and interestingly they can acquire cytotoxic properties in tumor-draining lymph nodes in mice (82) and *in vitro* in humans (83), and tumor-infiltrating Tregs are consequently cytotoxic (84). Similar to helper T cells, Tregs that secrete different types of effector cytokines can be identified according to chemokine receptor expression (2), and these Treg subsets might specifically suppress different types of immune responses (85). Human Foxp3+ T cells that produce IL-17 or IFN-γ can be isolated (86, 87), but while IL-17 producing Treg cells were normally suppressive (88), IFN-γ producing Tregs had reduced suppressive functions (87). The conditions that induce human Foxp3+ Tregs to secrete different effector cytokines and the role of these cells in infections, cancer, and autoimmune diseases remain to be fully established.

#### **HETEROGENEITY AND UNSTABILITY OF TH17 CELLS AND ITS RELEVANCE FOR AUTOIMMUNE DISEASES**