Cutting Edge: Helminth coinfection blocks effector differentiation of CD8 T cells through alternate host Th2- and IL-10-mediated responses

Abstract

Concurrent helminth infection potently inhibits T cell immunity; however, whether helminths prevent T cell priming or skew clonal recruitment and effector differentiation is not known. Using coinfection with two natural mouse pathogens, Heligsomosoides 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 KLRG1+ effector CD8 T cells and IFNγ production. Nevertheless, reversal of helminth suppression of the innate IL-12 response of CD8α+ DCs, 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.

Introduction

Helminth infection occurs worldwide with approximately 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 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 antigen-engaged T cells into the effector lineage. Here, we address this issue by focusing on the effects of coinfection with the helminth parasite Heligsomoides polygyrus on the mouse CD8 T response to vaccination with the protozoan parasite, Toxoplasma gondii.

Materials and Methods

Mice

Wild-type, IL-12p35−/−, STAT6−/− and IL-10−/− mice on a C57BL/6 background were purchased from JAX. IL4−/−IL-10−/− mice were a gift from Dr. T. Wynn, NIAID, NIH and provided by Taconic Farms. Mice produced by somatic cell nuclear transfer (SCNT) 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 K^b-restricted SVLAFRRL peptide were bred from stocks provided by Dr. H. Ploegh, Whitehead Institute, MIT(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 pre-infected mice and control uninfected mice were subsequently 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 days after T. gondii injection.

Analysis of CD8 T cells responses and IL-12 production by DCs

Spleen and peritoneal cell suspension were stained with the appropriate cell surface antibody and tetramer (PE-labelled SVLAFRRL-K^b) as previously detailed (12, 13). For intracellular staining, cells were restimulated ex vivo by plating 4–6 million cells with live T. gondii tachyzoites (MOI of 0.1) for 10–12 hr at 37C. To assay for innate IL-12 responsiveness, six hours 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-A^b+ CD11c+ CD8α+ DEC205+ DCs, using published methods(14).

Adoptive Transfer experiments

CD8+ T cells were enriched by negative selection from naïve Thy 1.1⁺ tgd057-specific SCNT mice, as described(12). 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 antibodies.

Statistical analysis

Three to five mice per group were used for each experiment. All studies were replicated in one or two more repeat experiments. One-way ANOVA with Tukey’s post hoc test was performed to evaluate statistical significance, with p<0.05 considered as significant.

Results and Discussion

T. gondii vaccination of helminth infected mice primes parasite antigen-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 (supplementary Figure 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 and colleagues have previously shown that when mice are first infected with H. polygyrus 7 days prior to live T. gondii cyst challenge, polyclonal CD8 T cells fail to respond by IFNγ production (18). 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-labelled K^b tetramer containing the SVLAFRRL peptide derived from the Tgd057 antigen of T. gondii(13). Instead of using live T. gondii parasites, we challenged helminth pre-infected and control mice with a replication-defective irradiated parasites to forestall differential tachyzoite outgrowth and, thus, equalize the antigen dose in vivo. As shown in supplementary Figure 1B, CD44^hi- 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 (supplementary Figure 1 B–D). Numbers of activated CD44^hi 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 KLRG1 upregulation as markers of effector T cell differentiation. As seen in Figure 1A, the differentiation of effector phenotype (CD62L^lo 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 IFNγ positive cells among tetramer-binding CD8 T cells is markedly decreased in coinfected mice (Figure 1B, C and D). In addition, the overall incidence of IFNγ-producing CD8 T cells is also depressed by helminth coinfection (Figure 1B), recapitulating the effects seen by Khan and colleagues(18).

FIGURE 1.

Helminth coinfection inhibits the differentiation of T. gondii vaccination-induced CD8 T cells and their IFNγ responses (A) Tetramer-binding CD44^hi CD8 T cells were subdivided into four subsets based on CD62L and KLRG1 expression patterns. Coinfected mice exhibit decreased F3 and F4 KLRG1+ fractions. (B) Representative flow cytometry plots showing IFN-γ responses of tetramer-binding and tetramer negative CD8 T cells are both decreased by helminth coinfection. Decreased frequency of IFNγ –positivity among tetramer-binding CD8 T cells in coinfected mice in the peritoneal effector site (C) and spleen (D). Mean ± SEM, n=5. **P<0.01 Data shown are representative of four independent experiments.

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 IFNγ cytokine production suggested the inhibition of IL-12 production might be a unitary mechanism for helminth immomodulation, because IL-12 is known to drive CTL effector differentiation and upregulate IFNγ production. Indeed, when IL-12 production by CD8α+ DCs is assayed within 6 hrs after T. gondii vaccination, we observed a nearly complete abrogation of this innate cytokine response in helminth-coinfected mice (Figure 2A and B). 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 Figure 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 (Figure 2C). Nevertheless, helminth suppression of effector CTL differentiation and IFNγ production persisted in the absence of STAT6 (Figure 2D and E). 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 (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 Figure 3, the negative effects of helminth coinfection on both CD8 T cell differentiation (Figure 3A) and IFNγ cytokine production (Figure 3B) were abrogated by the combined deficiency of IL-4 and IL-10. This reversal was not observed in mice lacking only IL-10 (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.

FIGURE 2.

Helminth suppression of the innate IL-12 response to T. gondii vaccination is dependent on STAT6, but inhibition of CD8 T cell responses persists in the absence of host Th2 signaling. (A) Representative plots of IL-12 staining of I-A^b+ CD11c+ CD8α+ DEC205+ DCs (B) Decreased frequency of IL-12–positivity among I-A^b+ CD11c+ CD8α+ DEC205+ DCs in coinfected mice. (C) Helminth inhibition of IL-12 response in the I-A^b+ CD11c+ CD8α+ DEC205+ DC compartment is reversed by STAT6 deficiency. (D). Helminth effects on the differentiation of KLRG1⁺ CD8 T cells persist in the absence of host STAT6. Similarly, Inhibition of IFNγ production by tetramer-binding CD8 T cells is inhibited in absence of STAT6. Data for peritoneal cells and spleens are shown separately in (E) and (F). Mean ± SEM, n=5. *P<0.05, **P<0.01, ***P<0.001. Data shown are representative of three independent experiments.

FIGURE 3.

Reversal of helminth inhibition of CD8 T cell responses in the absence of host IL-4 and IL-10. (A) Tetramer-binding CD44^hi CD8 T cells were subdivided into four subsets based on CD62L and KLRG1 expression patterns. Coinfected mice exhibit decreased F3 and F4 KLRG1+ fractions in both wildtype and IL-4−/−IL-10−/− (DKO) mice. Restoration of IFN-γ responses of tetramer-binding CD8 T cells in the peritoneal cavity (B) and spleens (C) of DKO mice. Mean ± SEM, n=5. **P<0.01 Data shown are representative of three independent experiments.

Helminth suppression of the CD8 T cell effector response can be recapitulated in a monoclonal T cell population

While 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 dendritic cells, helminth infection may not only disrupt IL-12 production, but could also result in dysregulated antigen-presentation, ultimately leading to altered clonal recruitment to the polyclonal T cell response (19). Thus, the defective effector differentiation and function of antigen-specific cells may simply result from the skewed recruitment of T cell clones with inferior antigen-binding and signaling capabilities. To query whether helminth effects 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 Figure 4A, when transferred into coinfected mice, descendants of the Thy 1.1⁺ Tgd057-reactive T cell clone underwent the same decreases in effector (CD62L^loKLRG1^hi) differentiation mirroring the changes seen amongst endogenous polyclonal tetramer binding CD8 T cells. Importantly, helminth coinfection did not significant alter the relative contribution of the transferred monoclonal T cells (Figure 4B). Finally, IFNγ production was similarly inhibited by coinfection, regardless of the clonal origin and heterogeneity of the tetramer binding cells (Figure 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 antigen-reactive T cells, arguing against a strict requirement for clonal skewing in this helminth-induced process.

FIGURE 4.

Recapitulation of helminth effects on CD8 T cell responses in a monoclonal CD8 T cell population. (A) Tetramer-binding CD44^hi CD8 T cells were subdivided into four subsets based on CD62L and KLRG1 expression patterns. Coinfected mice exhibit decreased F3 and F4 KLRG1+ fractions in both the transferred monoclonal Thy 1.1+ and endogenous polyclonal Thy 1.1- fractions. The top four pie charts present splenic data and the bottom four charts are from data obtained from PECs. (B) The relative (%) contribution of monoclonal Thy 1.1+ and polyclonal Thy 1.1- cells to the total tetramer-binding population was calculated for individual mice. No significant change was observed in the clonal contribution of the transferred Thy 1.1+ cells was observed. (C) Decreased frequency of IFNγ –positivity among tetramer-binding CD8 T cells in coinfected mice in both Thy 1.1+ and Thy 1.1- fractions. Mean ± SEM, n=10–11. Data shown are pooled from two independent adoptive transfer experiments.

Concluding remarks

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 here: ie., suppression of innate IL-12 production by DCs, attenuation of effector differentiation into KLRG1+ effectors and the inhibition of IFNγ 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 multi-step process that relies upon the optimal functioning of an interrelated network of innate antigen 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).

Abbreviations used in this article

KLRG1

killer like receptor G1

SCNT

somatic cell nuclear transfer