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Background: Autologous tolerogenic dendritic cells (tolDC) are a promising therapeutic strategy for inflammatory arthritis (IA) as they can regulate autoantigen-specific T cell responses. Here, we investigated two outstanding priorities for clinical development: (i) the suitability of using heat-shock proteins (HSP), abundant in inflamed synovia, as surrogate autoantigens to be presented by tolDC and (ii) identification of functional biomarkers that confirm tolDC regulatory activity., Methods: Cell proliferation dye-labelled human peripheral blood mononuclear cells of IA (rheumatoid arthritis (RA) and psoriatic arthritis (PsA)) patients or healthy donors were cultured with HSP40-, HSP60- and HSP70-derived peptides or recall antigens (e.g. tuberculin purified protein derivative (PPD)) in the presence or absence of tolDC or control DC for 9 days. Functional characteristics of proliferated antigen-specific T-cells were measured using flow cytometry, gene expression profiling and cytokine secretion immunoassays. Repeated measures analysis of variance (ANOVA) with Bonferroni correction for comparisons between multiple groups and paired Student t test for comparisons between two groups were used to determine significance., Results: All groups showed robust CD4 + T-cell responses towards one or more HSP-derived peptide(s) as assessed by a stimulation index > 2 (healthy donors: 78%, RA: 73%, PsA: 90%) and production of the cytokines IFNγ, IL-17A and GM-CSF. Addition of tolDC but not control DC induced a type 1 regulatory (Tr1) phenotype in the antigen-specific CD4 + T-cell population, as identified by high expression of LAG3, CD49b and secretion of IL-10. Furthermore, tolDC inhibited bystander natural killer (NK) cell activation in a TGFβ dependent manner., Conclusions: HSP-specific CD4 + T-cells are detectable in the majority of RA and PsA patients and can be converted into Tr1 cells by tolDC. HSP-loaded tolDC may therefore be suitable for directing T regulatory responses to antigens in inflamed synovia of IA patients. Tr1 markers LAG3, CD49b and IL-10 are suitable biomarkers for future tolDC clinical trials.

Technologies that enable induction of therapeutic tolerance may revolutionize the treatment of autoimmune diseases by their supposed potential to induce drug-free and lasting disease remission. In combination with diagnostic tests that screen for individuals at risk, these approaches may offer chances to halt disease before serious damage in the tissues can occur. In fact, for healthy individuals at risk, this could lead to a preventive form of vaccination. For therapeutic tolerance to re-instate natural self-tolerance it seems essential to induce tolerance for the critical autoantigens involved in disease. However, for most autoimmune diseases such antigens are poorly defined. This is the case for both disease inciting autoantigens and antigens that become involved through epitope spreading. A possible source of surrogate auto-antigens expressed in tissues during inflammation are heat shock proteins (HSP) or stress proteins. In this mini-review we discuss unique characteristics of HSP which provide them with the capacity to inhibit inflammatory processes. Various studies have shown that epitopes of HSP60 and HSP70 molecules can function as vaccines to downregulate a variety of autoimmune inflammatory diseases. Currently, several research groups are developing cell therapies with the intention to reach therapeutic tolerance. In this review, in which we are proposing to ex vivo load tolerant dendritic cells with a Treg inducing HSP70 derived peptide called B29, we are discussing the chances to develop this as an autologous tolDC therapeutic tolerance therapy for rheumatoid arthritis.

Tolerogenic dendritic cells (tolDCs) are a promising therapeutic tool to restore immune tolerance in autoimmune diseases. The rationale of using tolDCs is that they can specifically target the pathogenic T-cell response while leaving other, protective, T-cell responses intact. Several ways of generating therapeutic tolDCs have been described, but whether these tolDCs should be loaded with autoantigen(s), and if so, with which autoantigen(s), remains unclear. Autoimmune diseases, such as rheumatoid arthritis, are not commonly defined by a single, universal, autoantigen. A possible solution is to use surrogate autoantigens for loading of tolDCs. We propose that heat-shock proteins may be a relevant surrogate antigen, as they are evolutionarily conserved between species, ubiquitously expressed in inflamed tissues and have been shown to induce regulatory T cells, ameliorating disease in various arthritis mouse models. In this review, we provide an overview on how immune tolerance may be restored by tolDCs, the problem of selecting relevant autoantigens for loading of tolDCs, and why heat-shock proteins could be used as surrogate autoantigens., (© 2017 John Wiley & Sons Ltd.)

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterised by excessive immune responses resulting in inflammation of the joints. Although current therapies can be successful in dampening inflammation, a long-lived state of tolerance is seldom achieved. Therefore, novel therapies are needed that restore and maintain tolerance in patients with RA. Targeting regulatory T cells (Tregs) is a successful strategy to achieve tolerance, as was shown in studies performed in animal models and in human clinical trials. The antigen-specificity of Tregs is crucial for their effectiveness and allows for very specific targeting of these cells. However, which antigen is suitable for autoimmune diseases such as RA, for which the autoantigens are largely unknown? Heat shock proteins (HSPs) are ubiquitously expressed and can be up-regulated during inflammation. Additionally, HSPs, or HSP-derived peptides are immunogenic and can be recognised by a variety of immune cells, including Tregs. Therefore, this review highlights the potential of HSP-specific Tregs to control inflammatory immune responses. Targeting HSP-specific Tregs in RA can be achieved via the administration of HSPs (derived peptides), thereby controlling inflammatory responses. This makes HSPs attractive candidates for therapeutic intervention in chronic autoimmune diseases, with the ultimate goal of inducing long-lasting tolerance.

Since the initial discovery of the protective role of heat shock protein (HSP) 60 in arthritis, T cell recognition of endogenous HSP was found to be one of the possible underlying mechanisms. Recently we have uncovered potent disease-suppressive Tregs (anti-inflammatory immunosuppressive T cells) recognizing HSP70 self-antigens, and enabling selective targeting of such Tregs to inflamed tissues. HSP70 is a major contributor to the major histocompatibility complex (MHC) Class II ligandome and we have shown that a conserved HSP70-epitope (B29) is abundantly present in murine MHC Class II. Upon transfer, B29-induced CD4+CD25+Foxp3+T cells suppressed established proteoglycan-induced arthritis (PGIA) in mice. These self-antigen specific Tregs were activated in vivo and as little as 4.000 cells sufficed to fully inhibit arthritis. Furthermore, in vivo depletion of transferred Tregs abrogated disease suppression. Given that B29 can be presented by most human MHC class II molecules and that B29 inhibited arthritis in HLA-DQ8 (human MHC) transgenic mice, we feel that therapeutic vaccination with selected HSP peptides can be an effective route for induction of anti-inflammatory Tregs as a novel intervention in chronic inflammatory diseases., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Introduction: Exploitation of antigen-specific regulatory T cells (Tregs) as critical regulators in the control of chronic inflammatory diseases is hampered by the obscure nature of most disease-relevant autoantigens. Heat shock proteins (Hsp) are possible targets for Tregs due to their enhanced expression in inflamed (stressed) tissues and there is evidence that Hsp can induce anti-inflammatory immunoregulatory T-cell responses., Areas Covered: Recent publications showing that exogenous administration of stress proteins has induced immunoregulation in various models of inflammatory disease have also been shown to be effective in first clinical trials in humans. Now, in the light of a growing interest in T-cell regulation, it is of interest to further explore the mechanisms through which Hsp can be utilized to trigger immunoregulatory pathways, capable of suppressing such a wide and diversified spectrum of inflammatory diseases., Expert Opinion: Therapeutic approaches via exploitation of antigen-specific Tregs will benefit from tailor-made combination therapies. Combining current therapeutic approaches with Hsp-specific therapies thereby enhancing natural immune regulation might expedite the entry of antigen-specific regulatory T cells into the therapeutic arsenal of the anti-inflammatory therapeutics.

Until recently, the immune system was seen solely as a defense system with its primary task being the elimination of unwanted microbial invaders. Currently, however, the functional significance of the immune system has obtained a much wider perspective, to include among others the maintenance and restoration of homeostasis following tissue damage. In this latter aspect, there is a growing interest in the identification of molecules involved, such as the so-called danger or damage-associated molecular patterns (DAMPs), also called alarmins. Since heat shock proteins are archetypical molecules produced under stressful conditions, such as tissue damage or inflammation, they are frequently mentioned as prime examples of DAMPs (Bianchi, J Leukoc Biol 81:1-5, 2007; Kono and Rock, Nat Rev Immunol 8:279-289, 2008; Martin-Murphy et al., Toxicol Lett 192:387-394, 2010). See for instance also a recent review (Chen and Nunez, Science 298:1395-1401, 2010). Contrary to this description, we recently presented some of the arguments against a role of heat shock protein as DAMPs (Broere et al., Nat Rev Immunol 11:565-c1, 2011). With this perspective and reflection article, we hope to elaborate on this debate and provide additional thoughts to further ignite this discussion on this critical and evolving issue.

T cell responses to heat shock proteins (HSP) have disease suppressive activities through production of anti-inflammatory cytokines in patients and in models of inflammatory diseases. There is evidence that the anti-inflammatory activity of HSP-specific T cells depends on their recognition of endogenous HSP epitopes as expressed by stressed cells at sites of inflammation. Previously, we have demonstrated that such T cells can be induced by conserved sequences of microbial HSP. Now we propose that drug induced up-regulation of endogenous HSP can contribute to anti-inflammatory T cell regulation.

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.

Immunization with microbial or mammalian stress proteins or heat-shock proteins in models of experimental autoimmunity has been observed to lead to increased disease resistance. Furthermore, such immunization has been proposed to result in the induction and expansion of T cells that suppress disease upon transfer. Comparisons of microbial heat-shock proteins with other conserved immunogenic proteins of bacterial origin have indicated a unique capacity for heat-shock proteins to induce a regulatory phenotype in T cells, such as reflected by the production of IL10. Also, studies in children with chronic arthritis have indicated that T-cell responses to heat-shock proteins are associated with a benign course of the disease and with remission. Furthermore, in patients, heat-shock-protein-(HSP-) activated T cells were shown to display regulatory phenotypes consistent with CD4+ CD25+ T regulatory cells.