Toxoplasma gondii (T. gondii) is an intracellular protozoan parasite of medical significance. The Centers for Disease Control and Prevention has reported that this important foodborne illness affects more than 30 million individuals in the United States with a prevalence rate of over 50% in parts of Europe and Africa (1–3). This opportunist infection is mostly asymptomatic in immunocompetent individuals but flu-like symptoms can sometimes be observed. Reactivation of this latent infection occurs in immunocompromised patients and is very common in AIDS patients with CD4 T cell counts below 200/μL (4). With the introduction of antiretroviral therapy in 1996, the incidence of toxoplasmosis and other opportunistic infections has decreased (5); however, Toxoplasma encephalitis is still the most common central nervous system infections in patients with AIDS in many parts of the world (6, 7) and can have fatal outcomes if not treated properly. In a recent study, latent toxoplasmosis was reported to be associated with the worst neurocognitive disorder in HIV-infected individuals (8). Other serious complications of T. gondii infection, such as myocarditis, are also observed in this population (9).

While both CD4 and CD8 T cells have been reported to play a synergistic role in immunoprotection against chronic toxoplasmosis, the effector role is primarily attributed to the CD8 population (10, 11), as depletion of CD8 rather than CD4 T cells results in the mortality of infected animals (10). The importance of effector CD8 T cells in controlling chronic infection has been demonstrated in studies from our laboratory when the functional exhaustion of these cells led to the reactivation of latent disease (12). The synergistic role of CD4 T cells can be defined in terms of the critical help they provide for the maintenance of long-term CD8 T cell immunity against the pathogen (13). Although the requirement for CD4 T cells during chronic toxoplasmosis is well appreciated, the precise role of these cells is not well defined. Studies conducted with viral pathogens, such as HCV, HBV, and LCMV, have reported that CD4 T cell dysfunction observed during the chronic phase of the infection affects CD8 T cell functionality (14–16). Similarly, studies performed in our laboratory have reported that chronic T. gondii infection causes CD4 T cell exhaustion, which is attributed to overexpression of transcription factor BLIMP-1. In these studies, we observed that CD4 T cell dysfunction severely compromises CD8 T cell functionality during chronic parasitic infection (17).

CD4 T cells comprise multiple subsets and the individual role of each population in the maintenance of CD8 T cell immunity during T. gondii infection has not been defined. T follicular helper cells (Tfh), a specialized subset of CD4 T cells, are essential for germinal centers formation and provide cognate help to B cells (18). These cells are an important source of IL-21, a cytokine that has been described to play a central role in the maintenance of CD8 T cell functionality (19, 20). Furthermore, studies have reported that IL-21R knockout (KO) mice failed to control various intracellular infections (21–23). Previous report from our laboratory has demonstrated that reversal of CD8 T cell exhaustion by blockade of PD-1–PDL-1 interaction led to increased IL-21R expression in chronically infected animals (24). In the current study, we demonstrate a strong induction of Tfh response during T. gondii infection, but this subset displayed evidence of exhaustion during the later phase of chronic infection manifested by increased expression of inhibitory receptors like LAG-3 and 2B4. Their dysfunction reduces IL-21 production in the infected host, which compromises CD8 T cell immunity leading to increased reactivation of the infection.

CD4 T cells play an important synergistic role and provide essential help to CD8 T cells, which is the primary effector population responsible for maintaining the chronicity of infection (10, 13). The CD4 T cells are an essential source of cytokines, which are important mediators of protective immunity against acute Toxoplasmosis (36). Earlier studies from our laboratory showed that in susceptible animals, CD8 T cells get exhausted during the chronic phase of Toxoplasmosis and are unable to prevent the reactivation of latent infection (12). Subsequently, we demonstrated that the underlying causes of CD8 T cell dysfunctionality were linked to CD4 exhaustion (17). In the present study, we demonstrate that Tfh are a critical CD4 T cell subset, which expand in response to T. gondii challenge and are an important source of IL-21, the cytokine that regulates CD8 exhaustion during the chronic phase of infection. However, these cells exhibit increased levels of inhibitory receptors during the chronic infection at the same time point when CD8 T cell exhaustion has been reported.

T follicular helper cell population, a specialized subset of CD4 T cells, is critical for providing help to B cells in germinal centers (37, 38) and for their differentiation into plasma cells. Studies conducted with LCMV infection have described the role of IL-21 in the maintenance of CD8 T cell polyfunctionality and regulation of the exhaustion (19, 39). Importantly, the role of CD4 T cells in the maintenance of CD8 T cell immunity against viral infection was linked to their ability to produce IL-21 (39, 40). In recent years, IL-21 has been associated with the development of CD8 T cell immunity against other pathogens (mycobacteria, microsporidia), including HIV infection (22, 23, 41). These findings support our observations that, in the absence of IL-21 signaling, polyfunctional CD8 T cell response against T. gondii infection is compromised. Decreased frequency of polyfunctional CD8 T cells in the IL-21R KO animals fails to control chronicity and as a result, an increased number of tachyzoites is observed in the brain, spleen, and blood of these animals. In earlier studies, Stumhofer et al. have reported that IL-21 plays a critical role for optimal antibody production and CD8 T cell numbers during chronic toxoplasmosis (42). Also, similar to our findings, the same group reported an increase in Tfh population during T. gondii infection (25). However, in both of these reports, the role of IL-21 and Tfh population was evaluated in the context of antibody production. On the other hand, the data presented in our manuscript emphasize the importance of IL-21 in regulating exhaustion of CD8 T cells and maintaining their polyfunctional ability during chronic toxoplasmosis. Also, it is important to note that reactivation of latent toxoplasmosis in HIV-infected individuals usually occurs when CD4 T cell count falls below 200 cells/ml (4). Therefore, based on our data, the role of Tfh in the reactivation of chronic infection is in an important area of investigation that will need further attention.

As mentioned above, maintenance of polyfunctional CD8 T cells (IFNγ and cytotoxicity) is critical for controlling chronic toxoplasmosis and loss of any of these functions compromises the host immunity against the parasite (43). Studies from our laboratory have shown that during chronic toxoplasmosis, CD8 T cells exhibit a progressive increase in the expression of the inhibitory molecule PD-1, which leads to their exhaustion and loss of function (12). The loss of polyfunctional CD8 T cells causes the reactivation of latent infection manifested by an increase in tachyzoites and decrease of bradyzoites in the tissues. In subsequent studies, it was observed that neutralization of exhaustion markers on CD8 T cells with anti PDL-1 treatment was correlated with an increased expression of IL-21R, suggesting the importance of the cytokine in maintaining functionality (24). Furthermore, increased expression of IL-21R by both CD4 and CD8 T cells was linked to CD40–CD40L intrinsic signaling.

Also, we demonstrated that treatment of toxoplasma-infected animals with surrogate marker-specific non-exhausted CD4 population reversed CD8 exhaustion and controlled the reactivation of latent infection (17). The studies presented in the current manuscript suggest an important role for the Tfh population in keeping chronic Toxoplasmosis under control. This important CD4 T cell subset shows a rise starting 1-week p.i., peaks at 5 and exhibits a decline at week 7 p.i. At week 7 p.i., the Tfh population exhibits increased expression of inhibitory receptors (2B4 and LAG-3) which correlates with decreased IL-21 levels in these animals. It is important to note that this is a time point when CD8 T cell exhaustion becomes apparent (12). Interestingly, adoptive transfer of non-exhausted CD4 T cells to chronically infected mice, which has been reported to rescue CD8 T cell function (17), also increased the survival of the Tfh population (Figure 3G). Interestingly, in our earlier report, the frequency of Tfh cells was increased after anti-PDL-1 treatment (when CD8 T cells were rescued) (24). Thus, the restoration of CD8 functionality following adoptive transfer is likely dependent on increased Tfh survival and higher IL-21 levels. Therefore, in a T. gondii model of chronic infection, Tfh appear to be the primary source of IL-21 and exhaustion of these cells compromises CD8 T cell functionality leading to reactivation of the latent infection.

Although exhaustion of Tfh population has not been widely observed, a few reports in tumor models are beginning to emerge (3, 44). In one of these models, increased expression of Tim-3 was implicated in the dysfunctionality of these cells. Our report demonstrates for the first time that Tfh are exhausted during a chronic infection as manifested by increased expression of multiple inhibitory receptors and their reduced ability to produce IL-21. These findings suggest that in mice carrying chronic T. gondii infection, Tfh are an important source of IL-21 and their exhaustion likely contributes to CD8 dysfunctionality. The total absence of cytokine signaling in IL-21R KO mice further exacerbates CD8 T cell exhaustion leading to increased reactivation of latent infection in these animals. Although previous studies from our laboratory have implicated PD-1 as the major inhibitory molecule responsible for CD8 T cell exhaustion during chronic toxoplasmosis (12), in the present studies, no significant difference in the expression of PD-1 levels between the CD8 T cells from infected WT and IL-21R KO animals was noted. However, as compared to WT animals a higher frequency of CD8 T cells from KO mice displayed reduced functionality which can be linked to the increased co-expression of multiple inhibitory receptors. These findings are supported by our previous report that, although anti PDL-1 treatment reversed CD8 T cell dysfunction (12), PD-1^hi expressing cells could not be rescued. Thus, multiple inhibitory receptor expression may be one of the causes for anti PDL-1 treatment failure to rescue PD-1^hi cells. Therefore, it is likely that the role of IL-21 in the regulation of CD8 T cell dysfunction during chronic toxoplasmosis is linked to the expression of multiple inhibitory molecules, which lead to severe exhaustion of these cells and is difficult to reverse. Recent studies related to the blockade of inhibitory receptors on CD8 T cells have demonstrated that cumulative expression of multiple inhibitory receptors limits the ability of these cells to be rejuvenated (45). Thus, the role of IL-21 in regulating multiple inhibitory receptors during chronic Toxoplasmosis is very important and needs to be further investigated.

Overall, based on our observations, we postulate that the increase in the Tfh population during toxoplasmosis occurs at the chronic stage of infection. These cells are an important source of IL-21, which is needed for the maintenance of a robust polyfunctional CD8 T cell response. However, Tfh get exhausted and their decline compromises the levels of IL-21 in the infected animal. The absence of functional IL-21 signaling most likely deprives CD8 T cells of essential help and leads to their increased co-expression of multiple inhibitory receptors. Our findings raise an interesting question about the molecular events that follow the increase in specific inhibitory receptors in the absence of IL-21 signaling. Also, these observations suggest that blockade of PD-1–PDL-1 interaction alone may not be sufficient to prevent reactivation of latent toxoplasmosis. In infectious diseases and cancer models, it has been reported that different inhibitory receptors in addition to PD-1 need to be blocked for optimal reversal of T cell dysfunctionality (46, 47). Based on our findings, the absence of optimal levels of IL-21 during the later phase of chronic T. gondii infection may lead to the increased expression of inhibitory molecules other than PD-1 (2B4 and LAG-3) on CD8 T cells. The expression of multiple inhibitory receptors causes severe dysfunctionality in these cells. Whether combinatorial antibody treatment is needed for total and irreversible restoration of CD8 T cell functionality is an important question. Ongoing studies in our laboratory should provide answers to these important questions.

This study was carried out in accordance with the recommendations of the George Washington University Institutional Animal Care and Use Committee under Animal Use Protocol A052.

MM, SH, and IK conceived and designed the experiments. MM and SH conducted and analyzed the experiments. MM and IK wrote the manuscript.

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.