CD4+ Th cell differentiation is crucial for protecting against blood-stage Plasmodium parasites, the causative agents of malaria. It has been known for decades that more than one type of Th cell develops during this infection, with early models proposing a biphasic Th1/Th2 model of differentiation. Over the past decade, a large body of research, in particular, reports over the past 2–3 y, have revealed substantial complexity in the Th differentiation program during Plasmodium infection. In this article, we review how several studies employing mouse models of malaria, and recent human studies, have redefined the process of Th differentiation, with a particular focus on Th1 and T follicular helper (Tfh) cells. We review the molecular mechanisms that have been reported to modulate Th1/Tfh differentiation, and propose a model of Th1/Tfh differentiation that accommodates observations from all recent murine and human studies.

Malaria has afflicted humans for millions of years, and remains a major cause of global mortality and morbidity, with 216 million cases and 445,000 deaths in 2016 (1). Malaria is caused by infection with protozoan parasites of the genus Plasmodium, and is transmitted by the bite of female Anopheles mosquitoes (2). Current malaria interventions such as insecticide-treated bed nets, rapid diagnostic tests, chemopreventive therapy in pregnant women, and antiparasitic drugs such as artemisinin-based therapies have helped to reduce incidence rates and deaths (1). However, the emergence of insecticide-resistant mosquitoes and drug-resistant parasites threatens to jeopardize efforts to control or eradicate malaria (1). The mammalian component of the Plasmodium life cycle is roughly divided into a pre-erythrocytic stage in the liver, and the symptomatic and potentially lethal erythrocytic stage when parasites in the bloodstream cyclically infect RBCs (2).

Immunity to malaria is difficult to attain, either via natural exposure, or through vaccination. For example, in malaria-endemic regions, multiple P. falciparum infections are required for children to develop robust antiparasitic immunity. Moreover, despite decades of intensive research and development, there is no preventative vaccine that elicits strong and long-lasting immunity to malaria. Nevertheless, various lines of evidence indicate that cellular and humoral immunity to Plasmodium is achievable. First, CD8+ T cell–mediated immunity can protect against liver-stage Plasmodium-infection [reviewed elsewhere (3, 4)], with some contribution by CD4+ T cells (5, 6). Crucially, CD4+ T cells can control and resolve erythrocytic Plasmodium infections, both in humans and experimental animal models (714). Additionally, it appears that the partial protection afforded by the only current malaria vaccine, RTS,S (Mosquirix), correlates with CD4+ T cell responses, both in assisting Ab production, and in driving poly-cytokine production (15). Thus, CD4+ T cells can control Plasmodium infection in multiple ways, in providing help for CTL responses in the liver, in driving Ab production in the spleen and bone marrow, and as Th1 cells secreting IFN-γ during blood-stage infection. Early models of Th differentiation during malaria adhered to the Th1/Th2 framework of Mosmann et al. (16), proposing that protective CD4+ T cell responses were biphasic, with Th1 responses occurring first, followed by Ab-promoting Th2 responses (17).

Since then, studies have suggested greater complexity in Th differentiation across all disease fields, with malaria being no exception. The addition of new Th subsets, such as T follicular helper (Tfh) cells (18, 19), and IL-10–secreting, type 1 regulatory (Tr1) cells (20) to more established Th subsets such as Th1, Th2, and induced T regulatory (Treg) cells raises questions of whether functionally diverse Th subsets are indeed generated within an individual experiencing Plasmodium infection, and if so, how are they generated from a pool of naive CD4+ T cells. In this review, we will discuss recent advancements in understanding Th responses during blood-stage Plasmodium infection, using data from murine and human studies. We aim to provide an up-to-date picture of how Th differentiation occurs during blood-stage Plasmodium infection, with a specific focus on Th1 and Tfh cell differentiation.

Upon TCR ligation and receipt of costimulatory signals from APCs, naive CD4+ T cells undergo a burst of proliferation termed clonal expansion. This places substantial extra energy requirements upon the proliferating T cell, which are met via aerobic glycolysis. In addition, at some point during clonal expansion, primed CD4+ T cells differentiate into one of various effector subtypes, including but not limited to Th1, Th2, Th17, Tfh, induced Treg, and Tr1 cells (21). The focus of this review will be on Th1 and Tfh cells, because evidence supports antiparasitic roles for these Th cells. However, we will first review evidence for differentiation of other Th subsets during blood-stage Plasmodium infection.

Many studies of Th responses to Plasmodium employed murine models of blood-stage infection to allow for detailed dissection of molecular mechanisms governing their development. P. chabaudi infection is a commonly used model. It is a nonlethal species that causes a short spike in parasitemia for ∼1 wk, followed by low-level parasite persistence for ∼60 d (reviewed in Ref. 22). It has been shown that Ab-dependent mechanisms are important for complete resolution of infection during the chronic phase. Another model, nonlethal P. yoelii infection results in high parasitemias for a longer period of time (∼30 d), followed by elimination of parasites thereafter. Similar to P. chabaudi, prolonged Th responses are detected during infection with P. yoelii. In addition, B cell responses are essential for protection against P. yoelii, because the infection is not resolved and can be lethal in B cell–deficient mice (23). P. berghei ANKA has mainly been used to study severe malaria, cerebral malaria, and immunopathogenesis caused by CD8+ T cells (reviewed in Ref. 24). Although some studies have employed P. berghei to study B cells during malaria, it is important to note that in wild-type mice, Abs cannot control parasitemia or prevent lethal pathology (25). Thus, although in this review we have attempted to reconcile data from several recent studies into a single model of Th development, we remind the reader that the choice of model infection should be taken into account in the interpretation of any murine malaria study. Moreover, this cautionary note will similarly apply to emerging human studies that variously examine infections with P. falciparum, P. vivax, or both.

In examining evidence of Th responses other than Th1 and Tfh, previous murine studies identified small numbers of splenic Th17 cells in Plasmodium-infected mice, but no major role for IL-17A (2628). A recent study from last year suggested that IL-6, a known driver of Th17 differentiation in other systems, also contributed to modest Th17 activity in murine models (29). Nevertheless, there remains little evidence that IL-17A secreted by Th17 cells plays any role in controlling the outcome of blood-stage Plasmodium infection. However, it is important to note that IL-17A is not the only cytokine produced by Th17 cells. For instance, IL-22 can also be secreted. Previous studies have suggested a role for IL-22 in protecting against lethal liver damage during blood-stage infection (26). More recently, IL-22 has been reported to delay lethal cerebral symptoms in mice infected with P. berghei, and has been detected at elevated levels in P. falciparum–infected patients (30). A separate study using the P. berghei model showed that a major source of IL-22 was derived from γδ T cells rather than conventional CD4+ T cells. Nevertheless, existing data raise the possibility that IL-22, possibly produced by Th17 cells, newly defined Th22 cells, or semi-invariant T cells such as γδ T cells, could protect against organ damage during blood-stage infection. Future studies may clarify specific immune-suppressive roles for Th22 cells in malaria. Finally, in a study from 2016, a new subset of IL-27producing regulatory Th cells was reported, termed Tr27, which appeared to suppress T cell proliferation and IL-2 production in vitro (31). This report was particularly interesting because a series of earlier papers showed that IL-27 protected against immune-pathology during Plasmodium infection (3237). In this recent study, adoptive transfer of IL-27deficient polyclonal CD4+ T cells into TCRα−/− mice was employed to illustrate in vivo roles for Tr27 cells (31). Given that IL-27 is primarily thought to derive from myeloid cells [reviewed elsewhere (38)], this is an intriguing observation that warrants further study to cement the idea that Tr27 cells are generated during blood-stage infection, and that they can protect against immune pathology.

Given that immune responses must be tightly controlled to allow for pathogen control while minimizing immune pathology, previous studies first attempted to understand if Foxp3-expressing Treg cells were important during blood-stage infection in humans and murine models (39, 40). Although Tregs were shown to be activated in human patients infected either with P. falciparum or P. vivax, there were no clear associations between Treg cell responses and parasitemia or risk of developing clinical malaria (41, 42). Although Treg cell responses were also evident in Plasmodium-infected mice, Treg cell depletion in murine models, including our own studies, also suggested no major role for Treg cells unless their responses were amplified (20, 4345). Together, these observations suggested that other immunoregulatory mechanisms might act during malaria. Then 20 y ago, it was revealed that IL-10 played a crucial role in restraining Th1 responses during P. chabaudi infection of mice (46); ∼10 y ago, Foxp3 CD4+ T cells were identified as a major source of IL-10 (20, 33, 47), with these cells fitting the description of in vitro generated, immune-suppressive IL-10+/TGF-β+/IFN-γ+ CD4+ T cells, first described in 1997 (48). Although the original description of IL-10producing CD4+ T cells suggested they did not produce other Th cytokines (20), subsequent reports in mice and in humans indicated that IL-10producing Th1 cells, sometimes termed Tr1 cells, are capable of making both IL-10 and IFN-γ, and even IL-21 when stimulated in vitro (4952). We note that IL-10producing Th1 cells are not necessarily exactly the same as Tr1 cells as originally defined [reviewed elsewhere (53)]. However, in an attempt to draw useful distinctions between conventional Th1 and IL-10+/IFN-γ+ coproducing Th1 cells, we refer to IL-10producing Th1 cells as Tr1 cells for the purpose of this review. Recently, Montes de Oca et al. showed that abrogation of a Tr1 response by genetic depletion of prdm1 in T cells, which encodes Blimp-1, amplified Th1 responses, improved control of parasites, but elicited more pathology in the spleen. In addition, it was shown in two reports (50, 54) employing Ab-mediated blockade of the type I IFN signaling receptor chains, IFNAR1 in mice, and IFNAR2 on PBMCs from humans infected with P. falciparum, that these signaling pathways promote Tr1 responses in humans and mice. Notably, a recent study in Ugandan children showed that a higher proportion of Tr1 cells was associated with increased risk of parasitemia (49), consistent with a study by Boyle et al. (12) showing a markedly higher frequency of IL-10–producing CD4+ T cells in children residing in high malaria-transmission settings compared with low-transmission settings. Interestingly, these responses were comparable among immune adults residing in these same regions. Collectively, recent reports emphasize that IL-10–producing Tr1 cells are likely crucial for protecting host tissues from excessive damage during blood-stage Plasmodium infection, although they may also impair parasite control. Moreover, type I IFN-signaling via IFNAR1/2, as well as IL-27–signaling, may drive the generation of Tr1 cells, with IL-27 also being generated by a novel CD4+ T cell population, Tr27 cells (33, 50, 54). Thus, although Foxp3+ T cells may not play important roles during blood-stage Plasmodium infection, recent reports highlight clear roles for IL-10–producing Th1 cells, with exciting new roles for Tr27 cells and possibly IL-22 producing Th17/Th22 cells. In summary, recent reports support increased complexity in Th cell differentiation during blood-stage Plasmodium infection, with Tr1, Th17, Tr27, and Tfh cells (reviewed below) adding to the original biphasic Th1/Th2 model.

Although various Th subsets have been reported during blood-stage Plasmodium infection, until recently the vast majority of studies focused on Th1 cells. This is most likely because Th1 cells are often robustly generated in Plasmodium-infected humans and mice, and can be detected via IFN-γ/T-bet intracellular costaining at high frequencies either with or without restimulation in vitro (8, 10, 5558). Perhaps most importantly, it has been known for some time that IFN-γ is often antiparasitic, although clear evidence that Th1 cells, rather than NK, NKT or CD8+ T cells for example, constitute the predominant source of antiparasitic IFN-γ is sparse (11). Over recent years, it has also been revealed by our laboratory and others that the type I IFN signaling pathway, and its canonical transcription factor IRF7, suppresses Th1 responses in murine malaria models (5961), as well as more recently in cultured PMBCs from humans undergoing experimental P. falciparum infection (62). Finally, recent data suggest that Th1 cells in humans may have contributed to the efficacy of the Mosquirix vaccine (6365). Thus, the literature as a whole continues to support a role for Th1 cells in controlling blood-stage infection, and suggests that subtle modulation of type I IFN signaling may represent a possible strategy for boosting this antiparasitic Th mechanism in vivo. Interestingly, we note that although it is often stated that Th1 cells may promote the activation of macrophages to clear parasites from the bloodstream, definitive proof of this remains elusive.

The most substantial conceptual shift regarding Th cells over the past few years has concerned the provision of help to B cells for driving parasite-specific Ab production. Previously, IL-4–producing Th2 cells were thought to mediate these protective humoral responses (17, 66). These studies measured Th2 activity indirectly via detection of malaria-specific Abs, indicative of T cell help, or IL-5, another cytokine known to be secreted by Th2 cells. In both studies, the authors noted these were not direct measurements of Th2 activity, and that future studies might cement a role of Th2 for B cell help during malaria. However, this evidence never really materialized. Thus, it became clear that the Th1/Th2 paradigm was an insufficient model, because IL-4 was later revealed to not be critical for driving Ab production during experimental malaria (67). This conundrum now has a solution with the description of Tfh cells in malaria, first in murine models (19), and more recently, in humans (18). These two seminal studies paved the way for an exciting burst of recent studies (14, 54, 6874), mostly in murine models, although studies in humans are emerging (70). Most recently, evidence now demonstrates an essential role for Tfh cells in resolving blood-stage Plasmodium infections (14, 51).

As with any emerging field, there has been considerable debate about defining Tfh cells properly using molecular markers (reviewed in Ref. 75), because a number of them including, but not limited to Bcl-6, IL-21, Cxcr5, PD1, and ICOS, are variously employed in combination to do so. The situation is made complex by the fact that some of these molecules are expressed by non-Tfh cells, such as PD1, a canonical marker of T cell exhaustion, and ICOS, which is often upregulated on T cells during activation. Furthermore, Tfh cell differentiation is likely to occur via a multistep or progressive developmental pathway, shaped by various spatiotemporally regulated extracellular cues within secondary lymphoid tissue (7680). Studies have shown that the early upregulation of Bcl-6 plays only a minimal role in initiating the Tfh program (77, 78, 81), consistent with an earlier report that Bcl-6 upregulation occurred in most activated CD4+ T cells shortly after TCR stimulation (82). This correlates with an observation of a shared Tfh-like transitional state for activated CD4+ T cells across different studies (76, 8386). Some T cells may continue to experience Tfh-promoting conditions and migrate to the T-B border, leading to further upregulation of molecules such as Cxcr5 and CD40L, while downregulating CCR7 and PSGL1. Further persistent interactions with germinal center (GC) B cells eventually stimulate high expression of molecules such as Bcl-6, PD1, ICOS, and SAP on Tfh cells, leading to formation of GC Tfh cells within the GC (8791). Thus, although numerous recent papers have universally employed the term Tfh cell, it is possible in some instances that they have examined different states along a common Tfh cell developmental pathway, from CD4+ T cells that have rapidly upregulated CXCR5 and Bcl-6 but are still located in T cell zones, through to those that have upregulated IL-21 and are localized within GC in B cell follicles. Therefore, a distinction exists between emerging Tfh cells located in or close to T cell zones, and those that have localized to GC in B cell follicles, referred to as GC Tfh cells (90, 91). In the context of recent malaria studies, early Tfh-like cells and GC Tfh cells may have been partly conflated, perhaps due to a reliance on flow cytometric analysis of a small number of markers rather than definitive assessments of microanatomical localization. It will be important in future studies of Th1/Tfh differentiation to make clear distinctions between Tfh and GC Tfh cells. Studying Tfh cells in humans has been even more challenging because secondary lymphoid tissue is relatively inaccessible. Tfh cells have been recently studied in Plasmodium-infected humans by drawing on observations that some peripheral blood CXCR5+ CD4+ T cells can display Tfh-like characteristics, including the capacity to produce IL-21, and drive differentiation of naive B cells in vitro using superantigens such as Staphylococcal enterotoxin B (18, 70). These exciting clinical data together with reports using mouse models strongly support the view that a Tfh cell differentiation program does occur during blood-stage Plasmodium infection.

Many studies across different model systems have previously reported developmental relationships between Th1 and Tfh cells (76, 83, 9294). Studies of experimental malaria in mice recently also revealed Th cells capable of coexpressing Th1-associated IFN-γ and Tfh-associated IL-21 upon restimulation (51, 52), raising the concept of phenotypic mixing and plasticity between Th1 cells and Tfh cells in human malaria (18). To explore possible developmental relationships between Th1 and early Tfh differentiation, we recently used a murine TCR transgenic line, called PbTII (95), in which this single TCR clone opted for both Th1 and early Tfh fates within the same infected mouse (69). By performing single-cell transcriptomics on PbTII cells, we modeled the developmental trajectories taken by these cells as they moved from their naive state to either a Th1 or early Tfh state in vivo during P. chabaudi infection (69). In this review, we summarize recent findings from numerous murine studies, including from our own group, as well as from human malaria studies examining Th1 and Tfh differentiation. We propose a generalized model to describe the complex developmental relationship between these two subsets.

Having established that Th1 and Tfh responses likely occur in Plasmodium-infected mice and humans, recent studies also explored factors controlling Th1 and Tfh differentiation, and provided interesting information on the likely relationship between Th1 and Tfh cells in vivo. One common model to have emerged is that Th1 responses may suppress functional Tfh responses. We review this work below, and debate whether suppression must be invoked to explain the perceived reciprocal relationship between Th1 and Tfh responses in experimental malaria.

Ag-specific naive CD4+ T cells require a number of signals to proceed through clonal expansion and Th differentiation, which were traditionally grouped into three types of receptor on the T cell surface: signal 1, the TCR itself; signal 2, costimulatory receptors for interaction with ligands expressed on the surface of other cell types; and signal 3, cytokine receptors to receive soluble signals either locally or from a distance. This view of T cell activation recognized that ligation of a specific combination of these receptors, as well as the associated strength of signaling through any one receptor, directly influenced both the magnitude of clonal expansion (96), as well as the ultimate choice of Th fate [reviewed elsewhere (97)]. Given the multitude of costimulatory and cytokine receptors that have been identified on activated T cells, and the role of TCR affinity in governing Th fate (98), it would appear that the three-signal model of T cell activation was sufficiently complex to explain the diversity of Th outcomes in vivo. We discuss below recent evidence in malaria for a variety of different signals from the signal 1–3 model that appear to influence Th fate, as well as recognizing newer concepts that other families of cell surface molecules might extend models of T cell activation, and could perhaps be considered as signals 4 and 5.

Costimulatory molecules.

Previously, Butler et al. (19) showed the therapeutic potential of targeting the T cell costimulatory receptor, PD1, by blocking (after Th differentiation had likely already occurred) the inhibitory ligand, PD-L1, either alone or combination with blockade of LAG-3. More recently, they also showed that CD4+ T cells from malaria patients and experimental mice upregulated the costimulatory molecule, OX40 (encoded by tnfrsf4) (99). Interestingly, in vivo administration of an agonistic anti-OX40 Ab around the time when Th differentiation had likely already occurred promoted general CD4+ T cell activation, as judged by CD11a/CD49d staining and IL-2 production, which was coupled with increased numbers of both splenic Th1 and Tfh cells and increased parasite-specific IgG/IgM responses (99). Unfortunately, the therapeutic efficacy of OX40 stimulation was lost when combined with PD-L1 blockade, highlighting that complex relationships exist between costimulatory pathways in T cells during malaria (99). Further complexity was also revealed with regards to PD1 signaling in CD4+ T cells, because PD-L2 signaling was recently shown to support Th1 responses and immunity to experimental malaria (100). Thus, PD-L1 and PD-L2 have now been shown to play opposing roles in controlling Th responses in Plasmodium-infected mice, which makes the interpretation of experiments deleting both signaling pathways at the same time via PD1 deficiency more difficult (100, 101), although perhaps suggests that inhibitory PD-L1 signals tend to dominate PD-L2 signals in malaria.

Another costimulatory molecule receiving recent attention in malaria, including from our laboratory, has been ICOS (68, 71, 72). Findings in other models previously indicated B cells express ICOSL at various stages of the humoral response, for example to guide or capture emerging Tfh cells as they move toward B cell follicles (102), and for GC B cells to maintain contact with Tfh cells (90). Consistent with the importance of ICOS for T cell–dependent humoral immunity, we found ICOS was substantially upregulated on CD4+ T cells in mice (72), and was also detected on circulating Tfh cells in P. vivax–infected humans (70). ICOS was needed for sustaining Tfh and GC B responses, and parasite-specific Ab levels, during the later stages of P. chabaudi and P. yoelii infection in mice, although it was dispensable for early Tfh differentiation (71, 72). It should be noted, however, that ICOS upregulation was not restricted to emerging Tfh cells, but was readily and evenly observed on all activated CD4+ T cells (68, 71, 72), raising the possibility that ICOS signaling might influence all Th fates. Notably, the absence of ICOS-signaling promoted Th1 responses during the first week of infection (71). Thus, emerging themes are that ICOS is expressed by Tfh cells in mice and humans, and that it may play an important role in T/B interactions around and within the B cell follicle, as well as restricting Th1 responses.

Cytokines.

Th cell differentiation is strongly influenced by signaling via a number of cytokine receptors expressed on the T cell itself. This is traditionally illustrated by in vitro T cell culturing experiments in which the addition of cytokines (or cytokine-blocking Abs) such as IFN-γ, IL-12, IL-4, TGF-β, IL-6, IL-27, and others are known to drive particular Th fates in activated CD4+ T cells (97).

Recently, the cytokines IL-21 and IL-6 were shown in two separate murine studies from the Langhorne group (51) and by us to be important, in distinct ways, for Tfh biology, GC B cell responses, and parasite-specific Ab production (68). Direct IL-21 signaling on T cells was crucial for controlling P. chabaudi infection in mice, not due to impaired Tfh development, but rather a likely failure in interacting with B cells within the developing GC (51). Our study demonstrated that IL-6 was important in supporting ICOS upregulation on CD4+ T cells and their localization close to B cells in the spleen during P. chabaudi infection, as well as showing that GC B cell responses and parasite-specific Ab production was impaired in two murine malaria models (68). Interestingly, neither paper noted a defect in Tfh cell generation. Therefore, the data support the concept that IL-6 and IL-21 do not influence the early differentiation of parasite-specific Tfh cells, but do influence their capacity to interact with splenic B cells.

In addition to cytokines that promote Th responses, it is clear that similar cytokine-mediated mechanisms exist to regulate Th responses and minimize immune pathology during Plasmodium infection. Several previous studies, particularly from the Couper laboratory, have demonstrated that IL-27 signaling plays an important role in regulating the magnitude of Th1 responses (3237, 103), with a recent paper suggesting that CD4+ T cells themselves may be an important source of IL-27 (31). Mechanistically, it was previously shown that IL-27 promoted the expression of IL-10 by Th1 cells during experimental malaria, and most importantly, given the many possible sources of IL-10, that Th cell–derived IL-10 was necessary and sufficient to minimize Th1 immune pathology (33). Given the recent report of Tr27 cells in murine malaria (31), which produce only IL-27, but not IL-10 or IFN-γ, it will be of interest to examine further this paracrine mechanism for regulating Th1 cells.

It was previously shown during P. chabaudi infection in mice that the most important source of IL-10 was in fact highly activated Th1 cells (33), a cellular phenotype similar to an earlier description of Tr1 cells (48). The current view is that prolonged exposure to IL-12 and chronic Ag stimulation induces IL-10 production by highly activated Th1 cells during infection (104, 105). Similarly, the presence of Tr1 cells in the circulating blood of children living in high malaria transmission areas (49, 74) and the emergence of Tr1 cells in mice after the first week of infection suggest that prolonged Ag exposure may be critical in their development (54, 69, 106). One possible hypothesis from these data is that Tr1 cells develop directly from Th1 cells during blood-stage Plasmodium infection. This remains to be definitively tested, although our recent transcriptomic study is consistent with this hypothesis (69). Another recent study provides further molecular detail, showing that the transcriptional repressor Blimp-1 (encoded by prdm1) was crucial for the appearance of Tr1 cells, though not for Th1 cells (106). Given that Blimp-1 is reported to inhibit the Th1 transcription factor, T-bet, an interdependent relationship between Th1 and Tr1 cells is conceivable. The emergence of Tr1 cells during Plasmodium infection could result from transcriptional changes in Th1 cells. We anticipate that examination of transcription factor binding at the il10 locus in Th cells will provide valuable information on this topic.

Another cytokine signaling pathway recently examined for its effects on Th cells in malaria is type I IFN signaling. Several previous studies by our group showed that type I IFN signaling suppressed Th1 responses during the first week of P. berghei and P. chabaudi infections in mice (5961, 72), and that this proceeded via type I IFN signaling in conventional dendritic cells (cDCs). Although the exact mechanisms by which cDCs were impaired were not elucidated, it was suggested that IL-10 expression and higher PD-L1/PD-L2 ratios were possible mechanisms. Type I IFN–mediated suppression of myeloid cells was also seen recently in PBMC samples from P. falciparum–infected humans, which again resulted in suppression of IFN-γ (50). Furthermore, our recent study suggested that type I IFN signaling via cDCs also suppressed humoral responses such as Tfh cell and GC B cell development, parasite-specific Ab production and parasite control (72). The immune-suppressive nature of type I IFN signaling in malaria was independently observed in another recent study (54), in which Tfh cells were again suppressed by IFNAR1 signaling. Interestingly, in this study type I IFN signaling was found to proceed not via cDCs, but via the T cells themselves, and moreover, was associated with the development of Tr1 cells. Thus, although there may be more than one mechanism by which type I IFN signaling suppresses Th1 and Tfh cells, recent studies collectively provide confidence that type I IFN signaling regulates Th cell responses during Plasmodium infection in mice and humans.

Beyond signal 3?

Numerous studies from the past 2 y have uncovered a large number of signal 2 (costimulatory) and signal 3 (cytokine signaling) pathways that profoundly influence Th differentiation during malaria, including ICOS, PD-L1, PD-L2, OX40, IL-21, IL-6, type I IFN, IL-27, and IL-10. Although signal 3 is not strictly defined in the literature, in this review we have adopted a common definition that signal 3 comprises inflammatory cytokine signaling. With this in mind, we now ask whether there other, noncytokine receptors expressed on CD4+ T cells that also influence Th differentiation. For example, given that the energy requirements for T cells undergoing rapid clonal expansion require employment of additional metabolic pathways, such as aerobic glycolysis, increasing attention has been paid to whether T cell amino acid transport and other metabolic pathways influence Th differentiation (reviewed in Ref. 107). To our knowledge this has yet to be examined during malaria, although our recent study confirmed that Plasmodium-specific CD4+ T cells dramatically upregulated aerobic glycolysis-related genes as they expand in vivo, and that the emergence of Th1 cells coincides with further acceleration in cell-cycling speed (69). In another study this year, a receptor for extracellular ATP, P2X7, was expressed by CD4+ T cells in mice, and was associated with promoting Th1 differentiation over Tfh development (108). Finally, although chemokine receptors have long been studied on T cells in malaria, their expression on Th cells has tended to be associated with their capacity to migrate to nonlymphoid tissues, such as to the brain, in experimental cerebral malaria (109113). We recently found that activated CD4+ T cells, which had yet to fully differentiate into Th1 or Tfh cells, variously expressed the chemokine receptors, Cxcr3 and Cxcr5, which afforded opportunities for interaction with cells expressing cognate chemokines (69). We subsequently showed that inflammatory monocytes expressed Cxcl9 and Cxcl10, and that depletion of these cells reduced Th1 differentiation, whereas B cell deficiency, as expected, reduced Tfh cell responses (69). Taken together, these studies provide evidence that during blood-stage Plasmodium infection, receptors that do not strictly belong to the signal 1, 2 or 3 families can nevertheless directly influence Th differentiation, thus widening the spectrum of molecular signals that control Th cells in malaria.

In this final section, we focus on the relationship between Th1 and Tfh differentiation, because many recent studies have illustrated that modulation of a particular signaling pathway had consequences for both Th1 and Tfh cells, which were sometimes, but not always, reciprocal in nature. Moreover, some recent studies raise important questions about the phenotypic similarities between Th1 and Tfh cells, and whether Th1-/Tfh-like cells exist that possess characteristics of both subsets.

Several studies using the P. chabaudi murine model, from 1989 onwards, revealed that Th cell differentiation displayed a biphasic dynamic, with Th1 cells peaking first and waning relatively quickly, whereas Ab-driving Th responses, likely Tfh responses, peaked afterward (17, 66, 67, 71, 72). Importantly, Langhorne et al. (17) first revealed diversification and complexity in the Th response, because differentiation of Th cells producing IL-2 or providing help for Ab production, or both, were present by the end of the first week of infection. This suggested that Th differentiation and diversification is initiated during the first week of infection, which is consistent with a large number of subsequent studies in mice already discussed above. Thus, although Th1 and Tfh differentiation appeared to be initiated at the same time, this seminal study indicated temporal differences between the peak of Th1 and Tfh cell responses during P. chabaudi infection, which should be considered in exploring the relationship between the two Th subsets (17). An interesting question posed by Langhorne et al. was whether the different Th subsets observed in their study were representative of different developmental stages of a single process that CD4+ T cells went through, or whether different TCRs drove early diversity. The suggested approach for answering this question was adoptive transfer of CD4+ T cells.

Recently, we employed the adoptive transfer of Plasmodium-specific TCR transgenic CD4+ T cells (95) to map on a genomic scale the differentiation of Th1 and early Tfh cells during the first week of P. chabaudi infection (69). We employed CD4+ T cells with a common TCR to minimize the influence of TCR diversity on Th1/Tfh differentiation (69). Using single-cell RNA-sequencing techniques and computational modeling to reorder individual transcriptomes according to their progression through clonal expansion and Th differentiation, we observed that Th1 and early Tfh cells emerged from an intermediate developmental stage, which was characterized by expression of large numbers of genes, rapid cell cycling, increased energy requirements, and perhaps most interestingly, a Th-like signature that more closely resembled Tfh cells than Th1 cells. These data are consistent with previous in vitro studies, in which Th1 differentiation appeared to proceed through a Tfh-like intermediate stage (83, 86). Thus, our study provided one possible answer to the question posed ∼30 y ago, suggesting that Th diversity is not necessarily driven by TCR diversity among a polyclonal population, and can be explained by other effects such as internal stochasticity, and differential interactions with other immune cells. In particular, variability in expression of chemokine receptors Cxcr3 and Cxcr5 by activated, intermediate developmental stages was identified as a possible mechanism for controlling the Th1/Tfh balance. Cellular depletion of inflammatory monocytes, which expressed Cxcr3 ligands, reduced Th1 responses but did not significantly improve early Tfh differentiation, whereas depletion of B cell follicles boosted Th1 differentiation and reduced Tfh development (69). More recently, we found that T cell extrinsic deficiency in the innate transcription factor, IRF3, impaired CXCL9 and MHC class II expression by inflammatory monocytes, and also altered the Th1/Tfh balance in PbTII cells (114). These data place chemokine receptor expression by intermediate developmental stages of CD4+ T cells as a possible contributor to a Th1/Tfh balance that is characterized not by a reciprocal or see-saw model, but as a fan (Fig. 1), where Th1 and Tfh fates are related and possibly mixed as a result of sharing an intermediate developmental state.

FIGURE 1.

Recent insights into Th cell differentiation during malaria and a proposed fan model for Th1/Tfh differentiation. We propose a fan model for Th1/Tfh differentiation in which naive Plasmodium-specific CD4+ T cells undergo a period of clonal expansion and aerobic glycolysis, leading to an intermediate state characterized by expression of many Tfh-associated genes and some Th1-associated genes. Intermediate-state cells are then coached toward a Th1 fate by myeloid cell interactions, with further differentiation toward a Tr1 fate. Some intermediate-state cells are supported toward Tfh and then GC Tfh fates via interactions with B cells, whereas others continue to exhibit a mixed phenotype of Th1 and Tfh cells. Three recent research-based insights can be viewed within the context of this fan model: (1) the discovery and functional importance of Tfh cells in blood-stage malaria; (2) evidence of phenotypic mixing between Th1 and Tfh fates in humans and experimental mice infected with Plasmodium; and (3) recognition of the primacy of Tr1 cells as a potent source of IL-10 during experimental malaria, as well as better understanding of the molecular drivers of their development.

FIGURE 1.

Recent insights into Th cell differentiation during malaria and a proposed fan model for Th1/Tfh differentiation. We propose a fan model for Th1/Tfh differentiation in which naive Plasmodium-specific CD4+ T cells undergo a period of clonal expansion and aerobic glycolysis, leading to an intermediate state characterized by expression of many Tfh-associated genes and some Th1-associated genes. Intermediate-state cells are then coached toward a Th1 fate by myeloid cell interactions, with further differentiation toward a Tr1 fate. Some intermediate-state cells are supported toward Tfh and then GC Tfh fates via interactions with B cells, whereas others continue to exhibit a mixed phenotype of Th1 and Tfh cells. Three recent research-based insights can be viewed within the context of this fan model: (1) the discovery and functional importance of Tfh cells in blood-stage malaria; (2) evidence of phenotypic mixing between Th1 and Tfh fates in humans and experimental mice infected with Plasmodium; and (3) recognition of the primacy of Tr1 cells as a potent source of IL-10 during experimental malaria, as well as better understanding of the molecular drivers of their development.

Close modal

Let us now examine whether recent experimental observations can be reconciled with our fan model. First, an emerging theme in several recent studies is that IFN-γ production, perhaps by Th1 cells, may suppress the Tfh differentiation program, perhaps specifically affecting GC Tfh cell development. However, reports show that increased Th1 responses triggered by OX40 or IFNAR1 targeting did not necessarily correlate with reduced Tfh differentiation (72, 99). Moreover, IFN-γ blockade alone had a modest effect on Tfh cells, GC B cell responses, or parasite-specific Ab production during either P. yoelii or P. berghei infections (54, 73). In contrast, IFN-γ blockade combined with either TNF or IL-10 blockade induced robust improvements in Tfh cell responses (54, 73). Given that TNF, IFN-γ, IL-10, and IL-21 can all be expressed by CD4+ T cells during P. chabaudi infection (62), these data remain consistent with the idea that Th1 can suppress Tfh differentiation, but emphasize that multiple inflammatory cytokines co-operate to obstruct Tfh responses. Of note, our transcriptomic study also examined lineage relationships between Th1 and Tr1 cells and provided genome-wide evidence that Tr1 cells developed directly from Th1 cells. Our data also demonstrated that the expression of il12rb2 was progressively increased along the Th1 pathway, consistent with previous findings that prolonged IL-12 stimulation in vitro stimulates Th1 cells to produce IL-10 (104). In addition to known roles for prdm1 in driving Tr1 development, several other genes, including trib2, which encodes a pseudokinase expressed mostly in human CD4+ T cells (115), were upregulated on the inferred path from Th1 to Tr1 cells (69). Functional studies on trib2 and many other predicted Tr1 genes may help decipher molecular mechanisms underlying possible Th1 to Tr1 conversion in malaria. These data can be incorporated into the fan model with Tr1 cells emerging not as a completely separate lineage, but as the next developmental step made predominantly by Th1 cells. However, the existence of Th cells with a capacity to coexpress IL-10 alongside multiple cytokines including Th1-associated IFN-γ and Tfh-associated IL-21 (52) suggests that IL-10 induction may occur in any Th cell regardless of their accompanying Th1 or Tfh phenotype.

Next, it was shown by Zander et al. (54) that combined blockade of IFN-γ and IL-10 boosted Tfh cell numbers and qualitatively improved ICOS expression, consistent with early effects of IFNAR1 signaling on ICOS reported in that same paper and elsewhere by our group (72). The authors demonstrate a role for type I IFN in driving Blimp-1 expression, a molecule shown to mediate Tr1 responses in P. chabaudi infection in a recent separate study. Interestingly, IFNAR1 blockade had no substantial effect on Th1 responses (no difference in PMA/ionomycin IFN-γ staining ex vivo, and T-bet expression reduced by ∼20% at day 8 postinfection). Thus this paper indicates that type I IFN signaling during P. yoelii infection had a modest effect in promoting T-bet expression by Th1 cells, and suppressing ICOS expression by emerging Tfh. At a later time point, emerging Tr1 cells likely suppressed Tfh cell numbers and ICOS expression via continued coexpression of IL-10 and IFN-γ. In our similar study, using mostly P. chabaudi infection to explore Th1/Tfh cells, we revealed that type I IFN signaling appeared to simultaneously suppress IFN-γ production by Th1 cells, and mediated a strong effect on the percentage and absolute numbers of CD4+ T cells with an emerging Tfh phenotype (Cxcr5+ Bcl-6+) as well as on ICOS expression. Moreover, use of CD11c-Cre ifnar1f/f mice indicated that type I IFN signaling might suppress Tfh cell development via actions on cDC. In attempting to reconcile apparent differences between these two studies, it is perhaps important to recognize that for the most part two different infections are employed, which certainly display very different kinetics and magnitudes for Th1 and Tfh responses. Perhaps a general observation that can be made is that a reciprocal balance between Th1 and Tfh differentiation is not strongly apparent in either study, with one study suggesting that IFNAR1-signaling suppresses both Th1 and Tfh differentiation, whereas the other tends to emphasize effects on Tr1 development via Blimp-1 over more modest effects on Th1/Tfh balance toward the end of the first week of infection. We propose that type I IFN signaling does not control the balance between Th1 and Tfh differentiation, but may suppress other processes either or both before the fan begins (for ICOS expression), or after Th1 cells have developed (for expression of Blimp-1 and IL-10).

Another recent study explored the effect of ICOS on the balance between Th1 and Tfh differentiation (71). They found that Th1 responses increased in the absence of ICOS, whereas Tfh differentiation at that same time point was unaffected. Crucially, however, at later time points ICOS served to maintain the Tfh phenotype. These data are consistent with ICOS playing two temporally segregated roles. First, ICOS suppressed Th1 cell development, without impacting on early Tfh development. Secondly, ICOS played a crucial role and managed Tfh cell interactions with B cells, consistent with emerging paradigms (102, 116). Thus in the context of a fan model, ICOS appears to act early to restrict passage toward Th1 as well as acting later to reinforce cells that have traveled toward Tfh. Thus, this research emphasizes the importance of timing with regards to molecular and cellular processes influencing Th1/Tfh differentiation.

Our recent single-cell transcriptomic study focused on T cells with a single TCR specificity, and was not designed to explore the subtle diversity in Th1/Tfh phenotypes that might occur, for example, due to TCR diversity (98, 117). Moreover, we examined early Th1/Tfh responses during the first week of P. chabaudi infection, with only emerging appearance of IL-10 production, and negligible appearance of GC Tfh cells (69). Thus, although our study captured the early and intermediate stages of Th1/Tfh differentiation, future experiments are required to fully examine IL-10 induction and GC Tfh differentiation.

An important concept from recent malaria studies is that phenotypic mixing of Th1 and Tfh cells results not only from transient, intermediate developmental stages as highlighted in our study (69), but may also be a final outcome of differentiation (18, 73, 118). For example, circulating Cxcr3+ Th1-like Tfh cells in Malian children were likely to be fully developed memory Tfh cells, not early Tfh cells stalled in their development (18, 119, 120). These Cxcr3+ Tfh cells were less functional compared with Cxcr3 counterparts, suggesting different qualities of Tfh cells during Plasmodium infection. Similar observations were also made during P. vivax infection in adult humans (70). Different cytokine polarization programs were reported in these studies, hinting at possible differences between P. falciparum and P. vivax infection and between adults and children. In mice, a recent study showed that potent B cell activity could be recalled from Cxcr3+ Tfh memory cells that could also secrete IFN-γ ex vivo (121). Together, these studies suggest substantial complexity in the relationship between Th1 and Tfh phenotypes in malaria, with the likelihood of substantial overlap between Th1 and Tfh lineages.

Therefore, we propose a fan model (Fig. 1) in which activated, noncommitted Th cells are continually shaped by complex spatiotemporally regulated signals, such as Ag presentation and local cytokine production toward a varied mix of Th1 and Tfh states. This fan model builds upon the bifurcation model suggested by our TCR transgenic transcriptomic study (69), but takes into account mounting evidence of phenotypic mixing between Th1 and Tfh states. The main characteristics of our model are that Th1 and Tfh fates are developmentally related, but not necessarily in a reciprocal manner. Although immune modulation may boost Th1 outcomes, this does not necessarily mean that Tfh development will be negatively impacted at the same time. It remains possible, nonetheless, that favoring Th1 differentiation can negatively impact Tfh biology, and vice versa. The presence of a common early path and intermediate developmental stage can also explain early immune modulations that affect both Th1 and Tfh differentiation similarly. Finally, the potential of Th cells to be further fine-tuned by immunomodulatory factors as they travel across the Th1/Tfh fan, allows for a wide spectrum of heterogeneity during Plasmodium infection.

Th differentiation has been studied in malaria for several decades. Then 30 y ago it became clear that individual effector CD4+ T cells had diverse T helper characteristics, and that different Th responses were temporally segregated, at least in terms of their peak response. Many studies over the past decade have increased our understanding of Th differentiation, illustrating that the diversity first proposed as Th1 and Th2 cells has now multiplied into a large number of Th types. Possibly the most important finding of the past few years has been the recognition through the work of several independent groups that Tfh cells are generated in mice and humans infected with Plasmodium, and that these cells are crucial for generating Ab-mediated immunity. A second set of major findings has been to understand in greater detail the biology of IL-10–producing Th cells, such as transcription factors and cytokine signaling pathways molecules, which control their production, as well as their developmental relationship with Th1 and Tfh cells. A final major finding has been the realization that Th1 and Tfh development is intertwined. Taken together, we propose that Th1 and Tfh differentiation occurs along a common pathway toward a Tfh-like intermediate state that fans out toward a spectrum of Th1, Tfh, and mixed Th1/Tfh phenotypes, depending on TCR diversity, and continued, fine-tuning, interactions with other immune cells such as DCs, monocytes, and B cells. Our fan model implies a dynamic network of molecular and cellular processes that gradually shape CD4+ T cells in a spatiotemporal manner, rather than there being a stringent binary model controlled by lineage-specific transcription factors. Our model also allows for differential kinetics for Th1 and Tfh differentiation. Thus, we offer a conceptual framework within which most, if not all, recent human and murine datasets can be accommodated.

Although we have advanced in our knowledge of Th differentiation in malaria, many key questions still remain. Does plasticity really exist between Th1 and Tfh subsets? Are there epigenetic programs underlying their differentiation processes that hold cells to their lineage specification? Importantly, how do memory cells relate with these effector responses? Whereas the field of Th cells has received much attention, our current understanding of how memory CD4+ T cells develop during malaria is much less advanced. A detailed dissection of how genetic and genome-wide factors are modulated in CD4+ T cells during blood-stage malaria could provide avenues for better manipulation of Th cell–mediated immunity to malaria.

We thank Susanna S. Ng for figure design.

This work was supported by an Australian National Health and Medical Research Council project grant (GRNT1126399) to A.H.

Abbreviations used in this article:

     
  • cDC

    conventional dendritic cell

  •  
  • GC

    germinal center

  •  
  • Tfh

    T follicular helper

  •  
  • Tr1

    type 1 regulatory

  •  
  • Treg

    T regulatory.

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The authors have no financial conflicts of interest.