Teun Guichelaar

Heat shock proteins induce T cell regulation of chronic inflammation

The significance of immune responses to certain heat shock proteins (HSPs) that develop in virtually all inflammatory diseases is only now becoming clear. In experimental models, HSPs prevent or arrest inflammatory damage, and initial clinical trials in chronic inflammatory disease have shown HSP peptides to promote production of anti-inflammatory cytokines—indicating immunoregulatory potential. HSPs are ubiquitous self-antigens that are highly expressed in inflamed tissues. The prokaryotic homologous proteins, present in every bacterial species, are dominantly immunogenic. This is striking, especially as these proteins have large areas of sequence homologies with the host (mammalian) counterparts. In several experimental models of autoimmune diseases, immunisation with bacterial HSPs inhibited disease development, as did oral/nasal administration. Based on the experimental evidence so far, it is tempting to speculate that: firstly, exposure to homologues of these self-antigens, as present in, for instance, the bacterial intestinal flora, has a decisive impact on the regulation of self-tolerance at the level of T cells; and secondly, such proteins or their derivative peptides may have a role in an antigen specific immunotherapy approach involving modulation of relevant T cells, without the immediate necessity of defining disease specific autoantigens. Recent findings in experimental asthma and atherosclerosis have indicated that the field of application of such immunotherapy can be broader than just autoimmunity.

Oral or nasal antigen induces regulatory T cells that suppress arthritis and proliferation of arthritogenic T cells in joint draining lymph nodes.

The propagation of mucosal tolerance as a therapeutic approach in autoimmune diseases remains a difficult goal to achieve, and therefore further mechanistic studies are necessary to develop potential clinical protocols to induce mucosal regulatory T cells (Tr cells). In this study we addressed whether oral or nasal proteoglycan induced functional Tr cells in the cartilage proteoglycan-induced chronic arthritis model. Both nasal and oral application of human proteoglycan before induction of disease suppressed arthritis severity and incidence. Tolerized mice showed enhanced numbers of IL-10 producing CD4+ cells in the paw-draining lymph nodes. Furthermore, CD4+ spleen cells displayed enhanced expression of molecules associated with Tr cells, such as IL-10, Foxp3, and TGF-β. Transfer of CD4+ spleen cells from mucosally tolerized donors into proteoglycan-immunized mice abolished arthritis and reduced humoral responses, indicative of Tr cells with the capacity to inhibit already induced immune responses. Tr cells were activated upon transfer, because enhanced proliferation was observed in the joint draining lymph nodes compared with activated T cells from nontolerized donors. Upon cotransfer with naive proteoglycan-specific T cells, mucosally induced Tr cells inhibited proliferation of these arthritogenic T cells in vivo. Herein we show that both oral and nasal Ag application induced Tr cells, which had a direct inhibitory effect on already established pathogenic B and T cell responses.

Autoantigen-Specific IL-10-Transduced T Cells Suppress Chronic Arthritis by Promoting the Endogenous Regulatory IL-10 Response

Deficient T cell regulation can be mechanistically associated with development of chronic autoimmune diseases. Therefore, combining the regulatory properties of IL-10 and the specificity of autoreactive CD4+ T cells through adoptive cellular gene transfer of IL-10 via autoantigen-specific CD4+ T cells seems an attractive approach to correct such deficient T cell regulation that avoids the risks of nonspecific immunosuppressive drugs. In this study, we studied how cartilage proteoglycan-specific CD4+ T cells transduced with an active IL-10 gene (TIL-10) may contribute to the amelioration of chronic and progressive proteoglycan-induced arthritis in BALB/c mice. TCR-transgenic proteoglycan-specific TIL-10 cells ameliorated arthritis, whereas TIL-10 cells with specificity for OVA had no effect, showing the impact of Ag-specific targeting of inflammation. Furthermore, proteoglycan-specific TIL-10 cells suppressed autoreactive proinflammatory T and B cells, as TIL-10 cells caused a reduced expression of IL-2, TNF-α, and IL-17 and a diminished proteoglycan-specific IgG2a Ab response. Moreover, proteoglycan-specific TIL-10 cells promoted IL-10 expression in recipients but did not ameliorate arthritis in IL-10-deficient mice, indicating that TIL-10 cells suppress inflammation by propagating the endogenous regulatory IL-10 response in treated recipients. This is the first demonstration that such targeted suppression of proinflammatory lymphocyte responses in chronic autoimmunity by IL-10-transduced T cells specific for a natural Ag can occur via the endogenous regulatory IL-10 response.

Human Regulatory T Cells Do Not Suppress the Antitumor Immunity in the Bone Marrow: A Role for Bone Marrow Stromal Cells in Neutralizing Regulatory T Cells

Purpose: Regulatory T cells (Tregs) are potent tools to prevent graft-versus-host disease (GVHD) induced after allogeneic stem cell transplantation or donor lymphocyte infusions. Toward clinical application of Tregs for GVHD treatment, we investigated the impact of Tregs on the therapeutic graft-versus-tumor (GVT) effect against human multiple myeloma tumors with various immunogenicities, progression rates, and localizations in a humanized murine model. Experimental Design: Immunodeficient Rag2−/−γc−/− mice, bearing various human multiple myeloma tumors, were treated with human peripheral blood mononuclear cell (PBMC) alone or together with autologous ex vivo cultured Tregs. Mice were analyzed for the in vivo engraftment, homing of T-cell subsets, development of GVHD and GVT. In additional in vitro assays, Tregs that were cultured together with bone marrow stromal cells were analyzed for phenotype and functions. Results: Treatment with PBMC alone induced variable degrees of antitumor response, depending on the immunogenicity and the growth rate of the tumor. Coinfusion of Tregs did not impair the antitumor response against tumors residing within the bone marrow, irrespective of their immunogenicity or growth rates. In contrast, Tregs readily inhibited the antitumor effect against tumors growing outside the bone marrow. Exploring this remarkable phenomenon, we discovered that bone marrow stroma neutralizes the suppressive activity of Tregs in part via production of interleukin (IL)-1β/IL-6. We furthermore found in vitro and in vivo evidence of conversion of Tregs into IL-17–producing T cells in the bone marrow environment. Conclusions: These results provide new insights into the Treg immunobiology and indicate the conditional benefits of future Treg-based therapies. Clin Cancer Res; 19(6); 1467–75. ©2012 AACR.

Eradication of Medullary Multiple Myeloma by CD4+ Cytotoxic Human T Lymphocytes Directed at a Single Minor Histocompatibility Antigen

Purpose: The essential role of CD4+ T cells as helpers of anticancer immunity is indisputable. Little is known, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored the efficacy of immunotherapy with sole CD4+ cytotoxic human T cells directed at a hematopoietic-restricted minor histocompatibility antigen (mHag). Experimental Design: In macrophage-depleted Rag2−/−γc−/− mice, which were also devoid of T, B, and natural killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the mHag-specific T-cell receptor (TCR) were injected to treat full-blown mHag+ human multiple myeloma tumors. Results: mHag-specific antitumor responses were achieved after injection of native or mHag-TCR-transduced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, it failed to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled out mHag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell survival in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extramedullary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of long-term surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors with 3AB11 was also ineffective. Conclusions: Taken together, these results for the first time show the feasibility of immunotherapy of primary bone marrow tumors with sole CD4+ human T cells directed to a tumor-associated mHag. Extramedullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain a challenging issue towards effective cellular immunotherapy of hematologic malignancies. Clin Cancer Res; 16(22); 5481–8. ©2010 AACR.

Application of cultured human regulatory T cells requires preclinical in vivo evaluation.

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Cartilage proteoglycan-specific T cells as vectors of immunomodulatory biologicals in chronic proteoglycan-induced arthritis.

Systemic administration of agents that neutralize or antagonize Th1-mediated pro-inflammatory responses has been demonstrated to ameliorate inflammation in chronic autoimmune disease. However, systemic administration of such immunosuppressive biologicals causes serious side effects and has only limited success. To minimize these side effects, autoantigen-specific lymphocytes have been proposed as a carrier to deliver immunosuppressive agents to sites of inflammation. Here we studied the effects of primary cartilage proteoglycan-specific CD4+ T cells that were transduced using an efficient method of viral transduction with active genes encoding IL-1beta receptor antagonist, soluble TNF-alpha receptor-Ig, IL-4 or IL-10 in chronic proteoglycan-induced arthritis in mice. This is the first study describing such gene therapy using primary CD4+ T cells in a chronic arthritis. Moreover, the impact of proteoglycan-specific Th1, Th2 or naïve T cells was studied. Although proteoglycan-TCR transgenic CD4+ T cells can transfer arthritis to lymphopenic recipients, none of the proteoglycan-TCR transgenic T cell phenotypes that were tested induced worsening of arthritis in wild type hosts. Proteoglycan-specific T cells ameliorated arthritis when expressing the transduced IL-10 gene, and not when expressing the other transgenes/phenotypes. Although all of the tested biologicals can suppress in a wide range of different inflammatory disorders, especially IL-10 would therefore serve as a promising candidate to be used in cellular gene therapy for chronic arthritis.

Bone marrow provides an environment that prevents suppression of therapeutic graft-vs.-tumor immunity by regulatory T cells

Regulatory T cells (Tregs) can prevent graft-vs.-host disease as induced by the infusion of donor lymphocytes to cancer patients, but often they also suppress therapeutic antitumor immunity. We discuss an exception to this phenomenon, exemplifying how the milieu provided by the bone marrow may neutralize Tregs to allow local immune responses against cancer.

Human induced CD4+CD25+FOXP3+ regulatory T cells are suppressive in vitro, but fail to suppress inflammation in vivo

Background Regulatory T cells (Treg) are important to maintain immune homeostasis. Presence of Treg correlates with a favourable disease course in juvenile idiopathic arthritis (JIA) patients. A distinction within the Treg population can be made between naturally occurring Treg (nTreg), which are derived from the thymus, and peripherally induced Treg (iTreg). Induction of Treg in the periphery is a promising way to modulate diseases like JIA, since it is easier to obtain high numbers of cells. However, the functionality of activation-induced CD4+CD25+FOXP3+ Treg has been highly debated in the last few years. Furthermore, in vivo induction of Treg by TCR or co-stimulation could induce a cytokine storm. Therefore, it is important to carefully monitor iTreg functionality. Objectives In this study, the authors tested whether human iTreg suppress immune responses in vitro, and in vivo in a humanised mouse model of xenogeneic graft versus host disease (x-GvHD). Methods CD4+CD25− T cells were isolated from human PBMC and cultured with aCD3/aCD28 with or without interleukin-2 and TGFβ to obtain iTreg. CD4+CD25high T cells were cultured with αCD3/αCD28, interleukin-2 and TGFβ to obtain nTreg. Supernatant was taken for Luminex analysis and cells were stained for Treg markers. Suppression assays were performed to determine suppressive capacity. RAG−/− γc−/− mice were sublethally irradiated and injected with clodronate liposomes to deplete phagocyting cells. Next, human PBMC were injected with or without iTreg or nTreg from the same human donor. Mice were scored for x-GvHD during 9 weeks. Results and conclusions The authors show here that induced Treg FOXP3 expression levels and suppressive capacity in vitro were comparable to nTreg. As expected, nTreg efficiently prevented acute x-GvHD. However, in contrast with nTreg, iTreg did not suppress disease. The results show that polyclonally induced Treg display no suppressive capacity in x-GvHD, due to a quick loss of FOXP3 in vivo. This underscores the importance to use humanised mouse models for validation of data regarding iTreg function obtained in in vitro assays, before proceeding to application in patients.

HSP Reactive T Cells are Anti-Inflammatory and Disease Suppressive in Arthritic Diseases

Immune responses to certain heat-shock proteins (HSP) develop in virtually all inflammatory diseases; however, the significance of such responses is only now becoming clear. In models of experimental arthritis, HSPs can prevent or arrest inflammatory damage, and in initial clinical trials in patients with chronic inflammatory diseases, including rheumatoid arthritis, HSP peptides have been shown to promote the production of anti-inflammatory cytokines, indicating immunoregulatory potential of HSP. Heat shock proteins, also called stress-proteins, are ubiquitous self-antigens that are over-expressed in inflamed tissues. For some reason, the prokaryotic homologous proteins, present in every bacterial species, are dominantly immunogenic. This is striking, especially given the fact that these proteins have large areas of sequence homologies with the host (mammalian) counterparts. Furthermore, in experimental models of arthritis, immunisation with bacterial heat shock proteins has been seen to lead to inhibition of disease development. In addition oral or nasal administration has similarly been seen to lead to disease inhibition. Based on the experimental evidence collected, it becomes attractive to suppose that the exposure to homologues of these self antigens, as present in for instance the bacterial intestinal flora, has a decisive impact on the regulation of self tolerance at the level of T cells. If so, it becomes attractive to use such proteins or their derivative peptides for modulation of inflammation relevant T cells as an antigen specific immunotherapy approach, without the immediate necessity of defining disease specific auto-antigens