Australian researchers find LCs are preprogrammed to prevent unwanted immune responses against harmless commensal organisms.

Epidermal Langerhans cells (LCs) play a purely tolerogenic role that prevents the body from mounting an immune attack on harmless bacteria that live on epidermal surfaces, researchers claim. A team led by Barbara Fazekas de St Groth, Ph.D., at Australia’s Centenary Institute of Cancer Medicine and Cell Biology, used an animal model in which only LCs were capable of mediating immune responses to antigens, as an approach to studying the role of this cell type after antigen administration. They found that delivering antigens epicutaneously essentially resulted in LCs turning on T cells and killing them off to prevent an immune response being triggered. “No matter what we threw at them to get them to activate a long-term immune response, the Langerhans cells always induced immune tolerance,” Dr. Fazekas says.

In contrast, the team found when a pathogenic event occurs such that deeper layers of the skin are breached, the underlying dermal dendritic cells are activated, which overwhelms the tolerizing LC response, and an immune response proper is triggered. Reporting their findings in PNAS, the researchers suggest the discovery lends itself to the development of new approaches to certain immune-related disorders, such as inflammatory bowel disease. Their published paper is titled “Langerhans cells are precommitted to immune tolerance induction.”

Epidermal LCs have long been regarded as prototypic dendritic cells that are highly active in antigen uptake and rapidly acquire potent co-stimulatory capacity after in vitro culture, the researchers report. However, more recent in vivo work has questioned the immunogenicity of LCs: for example, during herpes viral infection of the skin, migrated LCs isolated from draining lymph nodes (dLN) weren’t able to induce proliferation of virus-specific CD8 T cells in vitro. “The current lack of consensus regarding LC function may relate, at least in part, to the difficulties in determining the contribution of a relatively small number of LCs to responses driven primarily by non-LC DC subsets in cutaneous LN (cLN),” the team suggests.

To test the function of LCs in vivo, the researchers used a previously described bone marrow (BM) chimeric mouse model in which only LCs (and no other dendritic cells) can present specific antigens to CD4 T cells. In this model all DC subsets express MHC class II IA molecules, but only LCs express MHC class II IE, which is an absolute requirement for the presentation of moth cytochrome C peptide (pMCC) to 5C.C7 T-cell receptor (TCR) transgenic T cells. Using this model the response of adoptively transferred and labeled 5C.C7 CD4 T cells can effectively be used as a readout for LC function.

Experiments using this model were striking, the team reports. Despite an initial recruitment of T cells into cell division following subcutaneous antigen administration, an 8.6-fold higher peak in T-cell numbers was observed in the control animals, compared with LC chimeras. Moreover, the majority of T cells in the chimeric animals didn’t survive long term, and while LC chimeras retained some IL-2-producing CD4 T cell, accounting for their initial proliferative response, there was no evidence of IL-5 and IL-10. Moreover, abundant production of IL-17 and IFNγ was seen in control animals, but not in the LC chimeras.

To test for antigen-specific memory up to 90 days post immunization, chimeras and control animals were subcutaneously challenged with antigen at a different site. The frequency of 5C.C7 cells in dLN of control chimeras increased by 15-fold within 16 hours, an increase that was largely due to redistribution to dLN. However, these responses were not seen in LC chimeras, although the animals did retain some IL-2-producing CD4 T cells, indicating that they were still capable of responding to T cell receptor stimulation.

Overall it appeared that LCs exposed to subcutaneous antigen recruited CD4 T cells into an abortive, proliferative response that resulted in tolerance instead of the generation of effector/memory function, the researchers note.

Interestingly, after subcutaneous antigen challenge, migrating LCs didn’t upregulate CD80 and CD86 expression, even though migratory dermal DCs (mDDCs) increased CD80/86 expression within 4 hours of antigen challenge and continued to do so for the next four days. This observation led the team to test epicutaneous immunization via topical administration of antigen. This resulted in rapid upregulation of both CD80 and CD86 by migratory LCs. CD69 upregulation by antigen-specific T cells occurred a day or so after the arrival of migratory LCs from the immunization site, indicating that the T-cell response was driven by migrating antigen-bearing LCs and not by free antigen presented by m-LCs already present in the LN at the time of immunization. Addition of adjuvants to the topical antigen preparation caused further activation of m-LCs, evidenced by even greater upregulation of CD80/86. Epicutaneous immunization also induced over 20% of migratory LCs in dLN of LC chimeras to express IL-12. However, the activated LCs still failed to support the generation of CD4 T-cell memory in response to antigen challenge.

To see whether LCs also induced tolerance in genetically unmanipulated animals, the team compared the responses of wild-type mice to epicutaneous and subcutaneous immunization, “reasoning that if epicutaneous antigen were presented mainly by LCs, then epicutaneous responses should recapitulate the tolerogenic responses we had documented in LC chimeras.” B10.BR mice were adoptively transferred with 5C.C7 cells and immunized with antigen/adjuvant either subcutaneously or epicutaneously. Significantly fewer 5C.C7 cells were recovered six days after epicutaneous immunization than after subcutaneous immunization, and again, no donor T cells could be recovered by day 70. The number of IL-17-, IFNγ-, and IL-2-producing cells were also significantly reduced. Indeed, “the effect of epicutaneous immunization in wild-type mice mirrored that seen in LC chimeras, confirming that LCs subserve a tolerogenic function in normal animals,” the authors state.

They moved on to carry out further tests on LCs to confirm tolerogenicity. This time they focused on translocation of the NF-κB transcription factor RelB to the nucleus, which is one of the most well-established markers of DC immunogenicity in vivo. Whereas a proportion of m-DCCs showed clear evidence of nuclear translocation of RelB following epicutaneous and subcutaneous administration of a sensitizor, RelB translocation was never observed in migrating m-LCs. “Thus, the activation and nuclear translocation of RelB appeared to be a reliable correlate of DC immunogenicity in vivo.”

Interestingly, migratory LCs took much longer to arrive at draining lymph nodes than DDCs, the authors note. This observation hinted at the notion that while T-cell activation in LC chimeras correlated with the arrival of migrating LCs from the immunization site, it was possible that they arrived too late to rescue a default tolerogenic response stimulated by steady-state m-LCs already in the node. To test whether migratory LCs could actively participate in ongoing responses initiated by rapidly migrating DDCs, they generated combined radiation chimeras in which both LCs and DDCs expressed MHC class II IE. In these chimeras, IE+ m-DCs in cutaneous lymph nodes (cLN) comprised a 1:1 mixture of m-LCs and m-DDCs, compared with a 1:3 mixture in wild-type mice. The number of donor T cells in the first 10 days post immunization was similar in the combined and control chimeras, but the number of effector cells (including IFNγ IL-17), was significantly reduced in the combined chimeras, even though memory cell numbers and memory function were roughly preserved. “These results indicate that LCs potently regulate the effector phase of the immune response by limiting T-cell effector function when the ratio of m-LCs to m-DDCs is sufficiently high.”

There remained the possibility that early presentation of free antigen by steady-state antigen-presenting m-LCs renders CD4 T cells unable to respond productively to a subsequent exposure to activated m-LCs. To test this possibility, the team delayed the transfer of 5C.C7 T cells for three days after LC chimera immunization to allow migration of activated m-LCs. However, T cells transferred into hosts preimmunized with topical antigen and adjuvent underwent only low-level CD69 up-regulation and proliferation, suggesting significant competition from the endogenous T-cell response. Moreover, when the host animals were treated with topical adjuvant but administration of antigen was delayed until the day after 5C.C7 T-cell transfer, significantly more proliferation was observed, but no effector cytokines were detected. “Thus, primary antigen presentation by preactivated m-LCs still failed to drive effector/memory differentiation in naïve CD4 T cells.”

The overall data demonstrate that “LCs maintain tolerogenic function under a range of conditions that are commonly believed to induce immunogenicity,” the authors conclude. “Our results indicate that naïve CD4 T cells initially proliferated strongly in response to antigen presented by LCs but then gradually disappeared without effector/memory cell differentiation, rendering the animal tolerant to subsequent challenge with specific antigen.” These observations held true whether the antigens were delivered subcutaneously or epicutaneously, or whether adjuvants were included. “Thus, LCs appear to possess an inherent commitment to tolerogenic function, even when displaying a CD80/86high phenotype associated with immunogenicity in other DC subtypes.”

The authors suggest that understanding LC function in vivo will provide insights into how DCs can mediate tolerance to TLR-expressing commensal organisms that colonize both external and internal epithelial surfaces, but switch to triggering a strong immune response to pathogens. They postulate that LCs promote tolerance to skin commensals under steady-state conditions as long as the structural integrity of the basement membrane remains intact. However, invading pathogens that breach the barrier generate a strong response that is overwhelmingly mediated by rapidly migrating DDCs, whereas “minor disturbances” are subject to a combination of immunogenic DDC signals and LC modulation of effector function but not memory generation.

The tolerogenic activity of LCs also lends itself to the development of new treatment options for immune-mediated diseases such as inflammatory bowel disease, the researchers state. “If we do manage to mimic what Langerhans cells do, then we could develop treatments that would precisely tolerize against specific antigens—just like the immune system of the skin does now,” Dr. Fazekas suggests.

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