Wiebke Hartmann

Foxp3+ Regulatory T Cells Delay Expulsion of Intestinal Nematodes by Suppression of IL-9-Driven Mast Cell Activation in BALB/c but Not in C57BL/6 Mice

Accumulating evidence suggests that IL-9-mediated immunity plays a fundamental role in control of intestinal nematode infection. Here we report a different impact of Foxp3+ regulatory T cells (Treg) in nematode-induced evasion of IL-9-mediated immunity in BALB/c and C57BL/6 mice. Infection with Strongyloides ratti induced Treg expansion with similar kinetics and phenotype in both strains. Strikingly, Treg depletion reduced parasite burden selectively in BALB/c but not in C57BL/6 mice. Treg function was apparent in both strains as Treg depletion increased nematode-specific humoral and cellular Th2 response in BALB/c and C57BL/6 mice to the same extent. Improved resistance in Treg-depleted BALB/c mice was accompanied by increased production of IL-9 and accelerated degranulation of mast cells. In contrast, IL-9 production was not significantly elevated and kinetics of mast cell degranulation were unaffected by Treg depletion in C57BL/6 mice. By in vivo neutralization, we demonstrate that increased IL-9 production during the first days of infection caused accelerated mast cell degranulation and rapid expulsion of S. ratti adults from the small intestine of Treg-depleted BALB/c mice. In genetically mast cell-deficient (Cpa3-Cre) BALB/c mice, Treg depletion still resulted in increased IL-9 production but resistance to S. ratti infection was lost, suggesting that IL-9-driven mast cell activation mediated accelerated expulsion of S. ratti in Treg-depleted BALB/c mice. This IL-9-driven mast cell degranulation is a central mechanism of S. ratti expulsion in both, BALB/c and C57BL/6 mice, because IL-9 injection reduced and IL-9 neutralization increased parasite burden in the presence of Treg in both strains. Therefore our results suggest that Foxp3+ Treg suppress sufficient IL-9 production for subsequent mast cell degranulation during S. ratti infection in a non-redundant manner in BALB/c mice, whereas additional regulatory pathways are functional in Treg-depleted C57BL/6 mice.

Pathogenic Nematodes Suppress Humoral Responses to Third-Party Antigens In Vivo by IL-10–Mediated Interference with Th Cell Function

One third of the human population is infected with helminth parasites. To promote their longevity and to limit pathology, helminths have developed several strategies to suppress the immune response of their host. As this immune suppression also acts on unrelated third-party Ags, a preexisting helminth infection may interfere with vaccination efficacy. In this study, we show that natural infection with Litomosoides sigmodontis suppressed the humoral response to thymus-dependent but not to thymus-independent model Ags in C57BL/6 mice. Thereby, we provide evidence that reduced humoral responses were mediated by interference with Th cell function rather than by direct suppression of B cells in L. sigmodontis-infected mice. We directly demonstrate suppression of Ag-specific proliferation in OVA-specific Th cells after adoptive transfer into L. sigmodontis-infected mice that led to equally reduced production of OVA-specific IgG. Transferred Th cells displayed increased frequencies of Foxp3+ after in vivo stimulation within infected but not within naive mice. Helminth-mediated suppression was induced by established L. sigmodontis infections but was completely independent of the individual worm burden. Using DEREG mice, we rule out a central role for host-derived regulatory T cells in the suppression of transferred Th cell proliferation. In contrast, we show that L. sigmodontis-induced, host-derived IL-10 mediated Foxp3 induction in transferred Th cells and significantly contributed to the observed Th cell hypoproliferation within infected mice.

Nematode-induced interference with the anti-Plasmodium CD8 T-cell response can be overcome by optimizing antigen administration

Malaria is still responsible for up to 1 million deaths per year worldwide, highlighting the need for protective malaria vaccines. Helminth infections that are prevalent in malaria endemic areas can modulate immune responses of the host. Here we show that Strongy‐Ioides ratti, a gut‐dwelling nematode that causes transient infections, did not change the efficacy of vaccination against Plasmodium berghei. An ongoing infection with Litomosoides sigmodontis, a tissue‐dwelling filaria that induces chronic infections in BALB/c mice, significantly interfered with vaccination efficacy. The induction of P. berghei circumspor‐ozoite protein (CSP)‐specific CD8+ T cells, achieved by a single immunization with a CSP fusion protein, was diminished in L. sigmodontis‐infected mice. This modulation was reflected by reduced frequencies of CSP‐specific CD8+ T cells, reduced CSP‐specific IFN‐y and TNF‐a production, reduced CSP‐specific cytotoxicity, and reduced protection against P. berghei challenge infection. Implementation of a more potent vaccine regime, by first priming with CSP‐expressing recombinant live Salmonella prior to CSP fusion protein immunization, restored induction of CSP‐specific CD8+ T cells and conferred almost sterile immunity to P. berghei challenge infection also in L. sigmodontis‐infected mice. In summary, we show that appropriate vaccination regimes can overcome helminth‐induced interference with vaccination efficacy.

Nematode-derived proteins suppress proliferation and cytokine production of antigen-specific T cells via induction of cell death.

In order to establish long-lasting infections in their mammalian host, filarial nematodes have developed sophisticated strategies to dampen their host’s immune response. Proteins that are actively secreted by the parasites have been shown to induce the expansion of regulatory T cells and to directly interfere with effector T cell function. Here, we analyze the suppressive capacity of Onchocerca volvulus -derived excreted/secreted proteins. Addition of two recombinant O . volvulus proteins, abundant larval transcript-2 (OvALT-2) and novel larval transcript-1 (OvNLT-1) to cell cultures of T cell receptor transgenic CD4+ and CD8+ T cells suppressed antigen-specific stimulation in vitro. Ovalbumin-specific CD4+ DO11.10 and OT-II T cells that had been stimulated with their cognate antigen in the presence of OvALT-2 or OvNLT-1 displayed reduced DNA synthesis quantified by 3H-thymidine incorporation and reduced cell division quantified by CFSE dilution. Furthermore, the IL-2 and IFN-γ response of ovalbumin-specific CD8+ OT-I T cells was suppressed by OvALT-2 and OvNLT-1. In contrast, another recombinant O . volvulus protein, microfilariae surface-associated antigen (Ov103), did not modulate T cell activation, thus serving as internal control for non-ESP-mediated artifacts. Suppressive capacity of the identified ESP was associated with induction of apoptosis in T cells demonstrated by increased exposure of phosphatidylserine on the plasma membrane. Of note, the digestion of recombinant proteins with proteinase K did not abolish the suppression of antigen-specific proliferation although the suppressive capacity of the identified excreted/secreted products was not mediated by low molecular weight contaminants in the undigested preparations. In summary, we identified two suppressive excreted/secreted products from O . volvulus , which interfere with the function of antigen-specific T cells in vitro.

T-cell-derived, but not B-cell-derived, IL-10 suppresses antigen-specific T-cell responses in Litomosoides sigmodontis-infected mice

IL‐10, a cytokine with pleiotropic functions is produced by many different cells. Although IL‐10 may be crucial for initiating protective Th2 responses to helminth infection, it may also function as a suppressive cytokine preventing immune pathology or even contributing to helminth‐induced immune evasion. Here, we show that B cells and T cells produce IL‐10 during murine Litomosoides sigmodontis infection. IL‐10‐deficient mice produced increased amounts of L. sigmodontis‐specific IFN‐γ and IL‐13 suggesting a suppressive role for IL‐10 in the initiation of the T‐cell response to infection. Using cell type‐specific IL‐10‐deficient mice, we dissected different functions of T‐cell‐ and B‐cell‐derived IL‐10. Litomosoides sigmodontis‐specific IFN‐γ, IL‐5, and IL‐13 production increased in the absence of T‐cell‐derived IL‐10 at early and late time points of infection. In contrast, B‐cell‐specific IL‐10 deficiency did not lead to significant changes in L. sigmodontis‐specific cytokine production compared to WT mice. Our results suggest that the initiation of Ag‐specific cellular responses during L. sigmodontis infection is suppressed by T‐cell‐derived IL‐10 and not by B‐cell‐derived IL‐10.

Strongyloides ratti infection modulates B and T cell responses to third party antigens.

It is estimated that over one third of the world population is infected with helminths, Strongyloides ssp. accounting for approximately 30-100 million cases. As helminth infections often result in a modulation of the hosts immune system, infected people may display impaired responses to concurrent infections and to third party antigens. Here, we employ the experimental system of murine Strongyloides ratti infection to investigate the impact of helminth infections on experimental vaccinations. We demonstrate that concurrent infection with S. ratti strongly affected the humoral response to a thymus dependent model antigen, whereby predominantly Th1 associated IgG2b production was suppressed. We provide evidence that this suppression was due to modulation of T helper cell and not B cell function as the responses to a thymus independent model antigen remained unchanged in S. ratti infected mice. Moreover, using an adoptive transfer system, we show that infection with S. ratti directly interfered with antigen-specific proliferation of T cell receptor transgenic CD4(+) T helper cells in vivo. Finally, using IL-10 deficient mice and mice that selectively lack T helper cell derived IL-10 we rule out a role for host-derived IL-10 in mediating the suppression of thymus dependent model antigen response in S. ratti infected mice.

A Novel and Divergent Role of Granzyme A and B in Resistance to Helminth Infection

Granzyme (gzm) A and B, proteases of NK cells and T killer cells, mediate cell death, but also cleave extracellular matrices, inactivate intracellular pathogens, and induce cytokines. Moreover, macrophages, Th2 cells, regulatory T cells, mast cells, and B cells can express gzms. We recently reported gzm induction in human filarial infection. In this study, we show that in rodent filarial infection with Litomosoides sigmodontis, worm loads were significantly reduced in gzmA×B and gzmB knockout mice during the whole course of infection, but enhanced only early in gzmA knockout compared with wild-type mice. GzmA/B deficiency was associated with a defense-promoting Th2 cytokine and Ab shift, enhanced early inflammatory gene expression, and a trend of reduced alternatively activated macrophage induction, whereas gzmA deficiency was linked with reduced inflammation and a trend toward increased alternatively activated macrophages. This suggests a novel and divergent role for gzms in helminth infection, with gzmA contributing to resistance and gzmB promoting susceptibility.

Cutting Edge: The BTLA–HVEM Regulatory Pathway Interferes with Protective Immunity to Intestinal Helminth Infection

Helminths exploit intrinsic regulatory pathways of the mammalian immune system to dampen the immune response directed against them. In this article, we show that infection with the parasitic nematode Strongyloides ratti induced upregulation of the coinhibitory receptor B and T lymphocyte attenuator (BTLA) predominantly on CD4+ T cells but also on a small fraction of innate leukocytes. Deficiency of either BTLA or its ligand herpes virus entry mediator (HVEM) resulted in reduced numbers of parasitic adults in the small intestine and reduced larval output throughout infection. Reduced parasite burden in BTLA- and HVEM-deficient mice was accompanied by accelerated degranulation of mucosal mast cells and increased Ag-specific production of the mast cell–activating cytokine IL-9. Our combined results support a model whereby BTLA on CD4+ T cells and additional innate leukocytes is triggered by HVEM and delivers negative signals into BTLA+ cells, thereby interfering with the protective immune response to this intestinal parasite.

Nematode-induced interference with vaccination efficacy targets follicular T helper cell induction and is preserved after termination of infection.

One-third of the human population is infected with parasitic worms. To avoid being eliminated, these parasites actively dampen the immune response of their hosts. This immune modulation also suppresses immune responses to third-party antigens such as vaccines. Here, we used Litomosoides sigmodontis-infected BALB/c mice to analyse nematode-induced interference with vaccination. Chronic nematode infection led to complete suppression of the humoral response to thymus-dependent vaccination. Thereby the numbers of antigen-specific B cells as well as the serum immunoglobulin (Ig) G titres were reduced. TH2-associated IgG1 and TH1-associated IgG2 responses were both suppressed. Thus, nematode infection did not bias responses towards a TH2 response, but interfered with Ig responses in general. We provide evidence that this suppression indirectly targeted B cells via accessory T cells as number and frequency of vaccine-induced follicular B helper T cells were reduced. Moreover, vaccination using model antigens that stimulate Ig response independently of T helper cells was functional in nematode-infected mice. Using depletion experiments, we show that CD4+Foxp3+ regulatory T cells did not mediate the suppression of Ig response during chronic nematode infection. Suppression was induced by fourth stage larvae, immature adults and mature adults, and increased with the duration of the infection. By contrast, isolated microfilariae increased IgG2a responses to vaccination. This pro-inflammatory effect of microfilariae was overruled by the simultaneous presence of adults. Strikingly, a reduced humoral response was still observed if vaccination was performed more than 16 weeks after termination of L. sigmodontis infection. In summary, our results suggest that vaccination may not only fail in helminth-infected individuals, but also in individuals with a history of previous helminth infections.

Litomosoides sigmodontis induces TGF‐β receptor responsive, IL‐10‐producing T cells that suppress bystander T‐cell proliferation in mice

Helminth parasites suppress immune responses to prolong their survival within the mammalian host. Thereby not only helminth‐specific but also nonhelminth‐specific bystander immune responses are suppressed. Here, we use the murine model of Litomosoides sigmodontis infection to elucidate the underlying mechanisms leading to this bystander T‐cell suppression. When OT‐II T cells specific for the third‐party antigen ovalbumin are transferred into helminth‐infected mice, these cells respond to antigen‐specific stimulation with reduced proliferation compared to activation within non‐infected mice. Thus, the presence of parasitic worms in the thoracic cavity translates to suppression of T cells with a different specificity at a different site. By eliminating regulatory receptors, cytokines, and cell populations from this system, we provide evidence for a two‐staged process. Parasite products first engage the TGF‐β receptor on host‐derived T cells that are central to suppression. In a second step, host‐derived T cells produce IL‐10 and subsequently suppress the adoptively transferred OT‐II T cells. Terminal suppression was IL‐10‐dependant but independent of intrinsic TGF‐β receptor‐ or PD‐1‐mediated signaling in the suppressed OT‐II T cells. Blockade of the same key suppression mediators, i.e. TGF‐β‐ and IL‐10 receptor, also ameliorated the suppression of IgG response to bystander antigen vaccination in L. sigmodontis‐infected mice.