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Title: Allospecific graft tolerance by induction of cytolytic CD4+ T cells
Other Titles: Allospecifieke tolerantie van transplantaten door inductie van cytolytische CD4+ T cellen
Authors: Giets, Elke
Issue Date: 28-Nov-2012
Abstract: Prior research in our laboratory (Janssens, et al., 2003)(Carlier et al. 2012) demonstrated that class II-restricted T cell epitopes including a CXXS motif, or even better a CXXC motif (Schmidt, et al., 2006), within epitope flanking residues but outside of the MHC anchoring- or T cell contact residues, elicit cytolytic CD4+ T cells. Acquisition of cytolytic properties results from an increased immune synapse formation between CXXC-peptide presenting APC and antigen-specific class II-restricted CD4+ T cells. Upon cognate interaction with nominal antigen-presenting APC, these cCD4+ T cells induce apoptosis of APC, down-sizing as such the effector T cell pool and limiting the immune response. From these data (Carlier et al. 2012) a universal strategy to avoid or overcome unwanted immune responses was developed, as in theory any class II-restricted T cell epitope containing a CXXC motif could induce cCD4+ T cells upon cognate recognition. One of many examples of such an undesired T cell-mediated immune response is the rejection of transplants. Transplantation offers a final solution for many end-stage diseased patients. Unfortunately, in the clinic, chronic rejection remains a problem despite progress in immunosuppressive treatment (Heeger & Dinavahi, 2012). Manifestation of adverse effects, i.e. cardiovascular disease, infection and malignancies, is detrimental for many patients on the long-term (Heeger & Dinavahi, 2012) (Meier-Kriesche, et al., 2003) (Euvrard, et al., 2003). Therefore, transplantation tolerance referring to a state of sustained specific non-responsiveness of the recipient’s immune system to donor alloantigens and allowing long-term allograft survival in the absence of potential harmful chronic immunosuppressive therapy (Golshayan & Pascual, 2008) remains an unreached goal for many immunologists. From a research approach, chronic rejection is considered to be caused mainly by indirect presentation of donor antigen (Game & Lechler, 2002), explaining the relevance of trying to intervene in MHC class II-restricted antigen presentation relying on the method proposed by our colleagues (Carlier et al. 2012). We hypothesized that by immunizing recipient mice with peptides derived from donor-alloantigens and containing a CXXC-motif, we would induce donor antigen-specific cytolytic CD4+ T cells. Upon cognate recognition, the presence of such cCD4+ T cells was suggested to result in induction of apoptosis specifically in APC presenting the nominal donor peptide. Specific elimination of such APC would down-size the donor-reactive effector population and would as such delay, prevent or down-regulate the rejection process against donor tissue. Models to study this demanded a well-identified class II-restricted antigen unique to the donor and a donor antigen-carrying tissue or cell population to transplant. We opted for skin graft models considering their robustness and simplicity. Because we would work in a transplant setting and chronic rejection has been associatedwith indirectly-activated donor-reactive host T cells, we hypothesized that elimination of donor antigen-presenting APC might result in long-term donor-specific transplantation tolerance.Two models to study the intervention in an immune response (i.e. rejection of donor tissue) resulting from the indirect MHC class II-restricted presentation of an antigen (i.e. donor-specific antigen) were chosen. A first model, the H-Y model, depended on the immune response against the DBY antigen in C57BL/6 mice (Simpson, et al., 2001), generated when male cells or tissue are grafted into female recipients. A second model, the GFP-model, depended on the immune reaction in C57BL/6 mice against GFP-expressing cells or tissue (Matsuo, et al., 2007). For both models CXXC-containing peptides were produced. Prolongation of skin graft survival (i.e. male and GFP-expressing respectively) in female wild type C57BL/6 mice treated with CXXC-containing peptide (i.e. ccDBY and ccGFP respectively), was observed in both experimental models and is described in Chapter 4. In the same chapter 4, in vivo experiments delivered proof-of-principle. When immunized with ccDBY peptide, female mice (n=9) showed acceptance of male full-thickness skin grafts. In contrast, untreated control mice (n=11) rejected their grafts from day 10 after transplantation. Immunization with the natural peptide, wtDBY, led to the spontaneous occurrence of acceptance in 4 out of 8 cases, though these mice were distinguishable from ccDBY-treated mice in appearance and graft infiltrate. Male grafts accepted by ccDBY peptide-treated female mice are described macroscopically as well as microscopically in this chapter. Hair growth, lack of both necrosis and contraction of the graft site, were characteristic observations. Mice were healthy and retained the capacity to mount an immune response towards unrelated antigens as demonstrated by a tetanus toxoid challenge (n=7) and by rejection of a sex-matched GFP-expressing graft (n=3). Microscopic investigation of graft biopsies showed a modest overall inflammatory infiltration in surviving grafts but a concentrated T lymphocyte population, accumulating around the epithelium of hair follicles and epidermis, and spreading throughout the graft in time. This observation was more pronounced and advanced in mice rejecting their grafts, with infiltrates correlating to the lack of hair growth, necrosis and finally rejection of the grafts. The phenotype of T lymphocytes in accepted grafts was investigated and a raise in CD8+ T cells, capable of graft destruction, and Foxp3+CD3+ T cells, capable of preventing graft destruction, was observed in such grafts (n=6). Presence of both suggests that a balance between both populations dictates the outcome of graft tolerance in ccDBY-immunized mice. For immunization, ccDBY peptide was adsorbed on an adjuvant, i.e. alum. To avoid the misperception that effects were caused by the adjuvant rather than the peptide, we immunized mice with the adjuvant alone (n=3) or with ccDBY peptideadsorbed on a different adjuvant (n=8). Both experiments indicated that the adjuvant per se did not count for the observed tolerance. Important to note are the similarities between ccDBY-immunized male-‘tolerant’ mice and untreated mice tolerant for a syngeneic graft (n=8). A parallel with the natural process of self-tolerance means that ccDBY-induced graft survival is of physiological relevance and might rely on the same mechanisms. Subsequently, we extrapolated our findings from the male-to-female DBY skin grafting model to another donor/recipient mismatch setting with ease of post-operational evaluation. In a wild type C57BL/6 female mouse, immunization with ccGFP peptide (n=5) produced in a similar way as ccDBY peptide, resulted in tolerance of a sex-matched GFP-expressing skin graft, compared to rejection in untreated recipients (3 out of 4 rejected) and accelerated rejection in wtGFP-treated recipients (n=5). Not only did ccDBY peptide immunization prevent male skin graft rejection in naïve female recipients for the initial follow-up period of 21 days, it still did so after several months. These long-term tolerant female mice (n=3) were demonstrated to be capable of accepting a second identical male skin graft, without intermediate treatment. Reminiscent of the successive male skin graft acceptance in female ccDBY-treated mice, ccGFP-treated female mice tolerating a first female GFP-graft on a long-term base (>90 days) could accept a second identical skin graft without intermediate handling (4 out of 5 accepted the recall graft). In Chapter 5, it was demonstrated in vitro that ccDBY immunization, which had led to prolonged graft survival in vivo, indeed induces a population of CD4+ T cells with specific function and phenotype. To this end, CD4+ T cells were cloned. The CD4+ T cell clone (cyt1/8) was specific for DBY and capable of inducing cell death in male APC in vitro. The cytolytic capacity of this clone was confirmed in vivo with the specific elimination of male APC but not female APC in a male mouse. Since we were in possession of female transgenic Marilyn mice whose T cell population solely consists of DBY-specific CD4+ T cells, we immunized such mice with ccDBY peptide and isolated CD4+ T cells. After expansion of the DBY-specific population, such cells were evaluated for their cytolytic activity and they were shown to be capable of diminishing the living population of professional male APC, i.e. male dendritic cells and male B cells. Both CD4+ T cell lines were characterized for phenotype, which led to the common finding of those cells being terminally differentiated effector/memory CD4+ T cells. Of note, though these cells were induced by ccDBY immunization, the cells were negative for the regulatory T cell marker Foxp3. In accordance to their cytolytic function towards male APC, these cells stained positive for markers implicated in apoptosis induction, GZB and FasL. These phenotypic characteristics were established on a single T cell clone and need to be confirmedwith other clones and/or polyclonal T cells. However, the findings presented here match closely what has been observed in other investigation fields, namely multiple sclerosis and insulin dependent diabetes (unpublished data) and we therefore conclude that our present findings are relevant. In the same chapter we proposed a suggestive mechanism behind tolerance induction upon ccDBY immunization. We propose a dynamic cellular interaction between spleen, graft-draining lymph node and graft site, resulting in male skin graft acceptance. An explanation for occasionally observed tolerance in wtDBY-treated mice is proposed in this chapter. As illustrated in an ELISpot assay, neither ccDBY-immunization nor wtDBY-immunization did deplete the CD4+ effector population. However, the DBY-specific CD4+ effector T cell population in wtDBY-immunized mice was smaller than the one seen in ccDBY-treated mice. Repeated administration of the strong wtDBY peptide might have led to activation-induced cell death (AICD) in DBY-reactive CD4+ T cells, correlating with the lower number of CD4+ T cells and the spontaneous occurrence of tolerance in wtDBY-treated mice. Of course, this matter requires further investigation, but it indicates that prolonged graft survival observed in ccDBY-treated and some wtDBY-treated mice relies on distinct mechanisms. In ccDBY-treated mice an effector population remains present but is held in check by other cell populations, Treg and/or cytolytic CD4+ T cells, whereas wtDBY-treated mice show prolonged graft survival seemingly due to a loss in effector T cells which might be caused by an overactive natural process to control effector T cell populations, namely AICD. To conclude, we believe that we have obtained proof-of-principle for a new universal strategy of preventing an unwanted immune response. We demonstrated in two minor mismatch models of skin transplantation that immunization prior to transplantation with a CXXC-containing donor-specific antigen can prolong skin graft survival for >3 months, what we considered as acceptance. Recipient mice showed preserved immune competency and remained healthy. Considering the observed presence of donor antigen-specific CD4+ T cells with cytolytic function towards donor antigen presenting APC in CXXC-containing peptide treated mice and the prior work of colleagues, we see this elimination of APC which present donor antigen as a major cause for prolonged skin graft survival.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular and Vascular Biology

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