Concurrent helminth infection potently inhibits T cell immunity; however, whether helminthes prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligmosomoides polygyrus and Toxoplasma gondii, to investigate the negative impact of helminthes on the CD8 T cell response, we demonstrate helminth-induced suppression of IL-12–dependent differentiation of killer-like receptor G1+ effector CD8 T cells and IFN-γ production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8α+ dendritic cells, which occurred in STAT6-deficient mice, was not sufficient to normalize CD8 T cell differentiation. Instead, a combined deficiency in IL-4 and IL-10 was required to reverse the negative effects of helminth coinfection on the CD8 T cell response. Monoclonal T. gondii–specific CD8 T cells adoptively transferred into coinfected mice recapitulated the spectrum of helminth-induced effects on the polyclonal CD8 T response, indicating the lack of requirement for clonal skewing.
Helminth infection occurs worldwide with ∼2 billion people infected with intestinal helminthes (1). There has been much interest in whether helminth coinfection alters the course of infection, immunity, and disease manifestations of other infectious diseases (2), whether it negatively impacts the efficacy of vaccination in non-industrialized countries (3), and whether helminth products can serve as immunotherapeutic agents for treatment of inflammatory and autoimmune diseases (4). There is now also a growing appreciation that helminth infection is a key evolutionary and ecological determinant of the homeostatic set point of immune system (5, 6). How helminthes exert immunomodulation of T cell responses remains unclear. Helminth products can alter the innate activation of dendritic cells (DCs) and promote regulatory DCs but it is unclear whether the DC-directed modulatory effects of helminth products are sufficient to explain the immunomodulatory effects on lymphocyte responses (7, 8). It is not known whether helminth coinfection dampens immunity by altering the differentiation and clonal recruitment of Ag-engaged T cells into the effector lineage. In this study we address this issue by focusing on the effects of coinfection with the helminth parasite Heligmosomoides polygyrus on the mouse CD8 T response to vaccination with the protozoan parasite, Toxoplasma gondii.
Materials and Methods
Wild-type, IL-12p35−/−, STAT6−/−, and IL-10−/− mice on a C57BL/6 background were purchased from The Jackson Laboratory. IL4−/−IL-10−/− mice were a gift from Dr. T. Wynn, National Institute of Allergy and Infectious Diseases, National Institutes of Health and provided by Taconic Farms. Mice produced by somatic cell nuclear transfer from a single T. gondii (Tgd057)–reactive CD8 T cell specifically expressing TCRαβ (TCRβ V13-1,D2,J2-7 and TCRα V6-4, J12) chains specific for the Kb-restricted SVLAFRRL peptide were bred from stocks provided by Dr. H. Ploegh, Whitehead Institute, Massachusetts Institute of Technology (9), and used to derive a mouse line expressing Thy 1.1. All mouse experiments were approved by the Institutional Animal Care and Use Committee at Rutgers University.
Helminth infection and T. gondii vaccination
Mice were gavaged with 200 L3 larvae of H. polygyrus on day 0, as previously described (10). On day 7, helminth preinfected mice and control uninfected mice were vaccinated with 2 million irradiated T. gondii tachyzoites suspended in 0.5 ml PBS, as previously described (11). CD8 T cell responses in the spleen and peritoneal cavity of T. gondii-vaccinated mice were analyzed 7 d after T. gondii injection.
Analysis of CD8 T cells responses and IL-12 production by DCs
Spleen and peritoneal cell suspensions were stained with the appropriate cell surface Ab and tetramer (PE-labeled SVLAFRRL-Kb) as previously detailed (12, 13). For intracellular staining, cells were restimulated ex vivo by plating 4–6 million cells with live T. gondii tachyzoites (multiplicity of infection of 0.1) for 10–12 h at 37°C. To assay for innate IL-12 responsiveness, 6 h following T. gondii vaccination, the spleens of mice were harvested and subjected to collagenase D enzymatic digestion and intracellular staining for IL-12 production by I-Ab+ CD11c+ CD8α+ DEC205+ DCs, using published methods (14).
Adoptive transfer experiments
CD8+ T cells were enriched by negative selection from naive Thy 1.1+ tgd057-specific SCNT mice, as described (12). Then 500 donor monoclonal CD8 T cells were injected intravenously into mice on the same day as injection of T. gondii. The clonal descendants of the donor T cells were tracked using the Thy 1.1 marker, and stained with other cell surface and intracellular staining Abs.
Three to five mice per group were used for each experiment. All studies were replicated in one or two repeat experiments. One-way ANOVA with Tukey’s post hoc test was performed to evaluate statistical significance, with a p value < 0.05 considered significant.
Results and Discussion
T. gondii vaccination of helminth-infected mice primes parasite Ag-reactive T cells but generates an attenuated effector CD8 T cell response
To investigate the mechanisms underlying helminth immunomodulation of T cell responses, we adopted a published model of H. polygyrus–T. gondii coinfection (Supplemental Fig. 1A), because the specific helminth and the T cell priming sporozoan parasites involved are natural and coevolved pathogens of the mouse and have well-documented immunomodulatory effects (15–17). Khan et al. (18) have previously shown that when mice are first infected with H. polygyrus 7 d prior to live T. gondii cyst challenge, polyclonal CD8 T cells fail to respond by IFN-γ production. Nevertheless, it was unclear whether this defect reflected a helminth-imposed inhibition of cytokine production or a complete blockade in the priming and activation of T. gondii–reactive CD8 T cells. To address this question, we tracked T. gondii–reactive CD8 T cells using a PE-labeled Kb tetramer containing the SVLAFRRL peptide derived from the Tgd057 Ag of T. gondii (13). Instead of using live T. gondii parasites, we challenged helminth-preinfected and control mice with a replication-defective irradiated parasites to forestall differential tachyzoite outgrowth and, thus, equalize the Ag dose in vivo. As shown in Supplemental Fig. 1B, CD44hi- tetramer–binding CD8 T cells were detected in both the spleens and peritoneal effector site of H. polygyrus-infected mice primed with the T. gondii vaccine. Nevertheless, their frequency and numbers were both significantly decreased by coinfection (Supplemental Fig. 1B–D). Numbers of activated CD44hi CD8 T cells were also elevated after vaccination of helminth-infected mice, albeit less than the levels found in normal vaccinated mice (data not shown). Taken together, these findings suggest that helminth coinfection does not block CD8 T cell priming but severely curtails their expansion.
To address how helminth coinfection alters the quality of the CD8 T cell response, we used CD62L downregulation and killer-like receptor G1 (KLRG1) upregulation as markers of effector T cell differentiation. As seen in Fig. 1A, the differentiation of effector phenotype (CD62Llo KLRG1+) CD8 T cells, which we have previously shown is dependent on IL-12 signaling (11), is decreased in both the spleen and peritoneal exudate cells of H. polygyrus-coinfected mice. Consistent with this observation, the frequency of IFN-γ positive cells among tetramer-binding CD8 T cells is markedly decreased in coinfected mice (Fig. 1B–D). In addition, the overall incidence of IFN-γ–producing CD8 T cells is also depressed by helminth coinfection (Fig. 1B), recapitulating the effects seen by Khan et al. (18).
Helminth coinfection suppresses the innate IL-12 response through STAT6 signaling and inhibits effector CD8 T cell differentiation through alternate host Th2- and IL-10–mediated pathways
The suppression of both CD8 T cell differentiation into KLRG1+ effectors and IFN-γ cytokine production suggested the inhibition of IL-12 production might be a unitary mechanism for helminth immunomodulation, because IL-12 is known to drive CTL effector differentiation and upregulate IFN-γ production. Indeed, when IL-12 production by CD8α+ DCs is assayed within 6 h after T. gondii vaccination, we observed a nearly complete abrogation of this innate cytokine response in helminth-coinfected mice (Fig. 2A, 2B). To investigate the role of helminth-induced Th2 cytokine signaling, we tested whether helminth inhibition of innate IL-12 responses and the attenuation of CD8 T cell responses will persist in the absence of Th2 responses. As seen in Fig. 2C, the ability of H. polygyrus infection to inhibit IL-12 responses was ablated in the STAT6 null background, which already exhibited a slightly lower IL-12 response when not infected with H. polygyrus (Fig. 2C). Nevertheless, helminth suppression of effector CTL differentiation and IFN-γ production persisted in the absence of STAT6 (Fig. 2D, 2E). The latter observations suggest that helminth immunomodulation involves additional mechanisms beyond the suppression of the IL-12 response itself. Consistent with this notion, exogenous supplementation with recombinant IL-12 failed to rescue the CD8 T cell response in coinfected mice (data not shown). Additionally, the persistence of helminth effects on the CD8 T cell response in STAT6 null mice pinpointed an alternative Th2-independent regulatory mechanism is still operative. To address this scenario, we investigated coinfection in mice lacking both IL-4 and IL-10 and tested whether helminth suppression of CD8 immunity remains. As shown in Fig. 3, the negative effects of helminth coinfection on both CD8 T cell differentiation (Fig. 3A) and IFN-γ cytokine production (Fig. 3B) were abrogated by the combined deficiency of IL-4 and IL-10. This reversal was not observed in mice only lacking IL-10 (data not shown). Overall, these results indicate that helminth suppression of CD8 effector differentiation and function involves alternate host Th2- and IL-10–mediated regulatory mechanisms that extend beyond the suppression of the initial innate IL-12 responsiveness of CD8α+ DCs.
Helminth suppression of the CD8 T cell effector response can be recapitulated in a monoclonal T cell population
Although tetramer-positive CD8 T cells were evidently activated and expanded in helminth-coinfected mice, it is not clear that the clonal composition of the T. gondii–reactive, tetramer binding cells remains conserved. By suppressing the innate activation of DCs, helminth infection may not only disrupt IL-12 production, but could also result in dysregulated Ag-presentation, ultimately leading to altered clonal recruitment to the polyclonal T cell response (19). Thus, the defective effector differentiation and function of Ag-specific cells may simply result from the skewed recruitment of T cell clones with inferior Ag binding and signaling capabilities. To query whether helminth effects the required differential clonal recruitment, we adoptively transferred Thy 1.1+ monoclonal T cells reactive to Tgd057 into control and coinfected recipients and tracked not only their resultant phenotypes but also their clonal contribution relative to the entire tetramer-binding population. As shown in Fig. 4A, when transferred into coinfected mice, descendants of the Thy 1.1+ Tgd057-reactive T cell clone underwent the same decreases in effector (CD62LloKLRG1hi) differentiation mirroring the changes seen among endogenous polyclonal tetramer binding CD8 T cells. Importantly, helminth coinfection did not significant alter the relative contribution of the transferred monoclonal T cells (Fig. 4B). Finally, IFN-γ production was similarly inhibited by coinfection, regardless of the clonal origin and heterogeneity of the tetramer binding cells (Fig. 4C). Thus, the full spectrum of defects in the polyclonal CD8 T cell response to Tgd057, that is induced helminth coinfection, can be recapitulated in a monoclonal population of Ag-reactive T cells, arguing against a strict requirement for clonal skewing in this helminth-induced process.
Using an H. polygyrus–T. gondii coinfection model, we have demonstrated a profound negative impact of helminth coinfection on the effector differentiation and cytokine production of CD8 T cells elicited by vaccination. Our analysis of the Tgd057-directed CD8 T cell response to T. gondii indicated that helminth coinfection not only quantitatively dampens the vaccine-elicited response, but that it also alters the quality of the resulting T cell response. Interestingly, differential clonal recruitment did not appear to be required for the effect of helminth coinfection. Nevertheless, it is noteworthy that helminth immunomodulation occurs at multiple levels affecting innate and adaptive immunity. In addition to the effects we described in this study, i.e., suppression of innate IL-12 production by DCs, attenuation of effector differentiation into KLRG1+ effectors and the inhibition of IFN-γ production by CD8 T cells, we have also observed decreased NK cell activation, decreased CD4 T cell responses and deficiencies in chemokine responsiveness of red pulp macrophages and monocytes following T. gondii vaccination of H. polygyrus infected mice (data not shown). The emerging consensus view from several publications, including our recent study of the CD8 effector differentiation during vaccination with T. gondii (12, 20–22) indicates that the efficient generation of effector T cells occurs through a multistep process that relies upon the optimal functioning of an interrelated network of innate Ag-presenting cells, helper cells, and accessory cells that provide costimulatory signals and cytokines. By each targeting multiple players in this integrated network, 1) helminth-induced Th2 responses and 2) regulatory mechanisms mediated by IL-10 (and perhaps TGFβ) may represent distinct pathways that may be individually sufficient to stall the development and dampen the function of effector T cells. Our current and previous observation (10) that simultaneous blockade of both Th2 and IL-10 responses is required to abrogate helminth immunomodulation of anti-pathogen (T. gondii) and autoimmune (diabetes) effector immunity lends credence to this notion. Individually, these two major arms of helminth immunomodulation provide a high level of functional redundancy and, in the intact host, together, they confer a great degree of robustness to this evolutionarily conserved mechanism for host-pathogen detente (5).
We thank Hidde Ploegh (Massachusetts Institute of Technology) and Thomas Wynn (National Institutes of Health) for generously providing mouse strains. Tetramers were produced by Gijs Grotenbreg (National University of Singapore).
This work was supported by National Institutes of Health/National Institutes of Allergy and Infectious Diseases Grants AI083405 and AI124691.
The online version of this article contains supplemental material.
Abbreviations used in this article:
killer-like receptor G1.
The authors have no financial conflicts of interest.