Modulation of Adjuvant Arthritis by Cellular and Humoral Immunity to Hsp65.

Heat shock proteins (Hsps) are highly conserved, and their expression is upregulated in cells by heat and other stressful stimuli. These proteins play a vital role in preserving the structural and functional integrity of cells under stress. Despite the ubiquitous expression of Hsps in an individual, the immune system is not fully tolerant to them. In fact, Hsps are highly immunogenic in nature, and immune response to these proteins is observed in various inflammatory and autoimmune diseases. Studies on the immunopathogenesis of autoimmune arthritis in the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA) as well as observations in patients with RA and juvenile idiopathic arthritis (JIA) have unraveled immunoregulatory attributes of self-Hsp65-directed immunity. Notable features of Hsp65 immunity in AA include protection rather than disease induction following immunization of Lewis rats with self (rat)-Hsp65; the diversification of T cell response to mycobacterial Hsp65 during the course of AA and its association with spontaneous induction of response to self-Hsp65; the cross-reactive T cells recognizing foreign and self homologs of Hsp65 and their role in disease suppression in rats; the suppressive effect of antibodies to Hsp65 in AA; and the use of Hsp65, its peptides, or altered peptide ligands in controlling autoimmune pathology. The results of studies in the AA model have relevance to RA and JIA. We believe that these insights into Hsp65 immunity would not only advance our understanding of the disease process in RA/JIA, but also lead to the development of novel therapeutic approaches for autoimmune arthritis.

Nanoscale artificial antigen presenting cells for T cell immunotherapy.

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. FROM THE CLINICAL EDITOR: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

Larazotide acetate regulates epithelial tight junctions in vitro and in vivo.

Tight junctions (TJs) control paracellular permeability and apical-basolateral polarity of epithelial cells, and can be regulated by exogenous and endogenous stimuli. Dysregulated permeability is associated with pathological conditions, such as celiac disease and inflammatory bowel disease. Herein we studied the mechanism by which larazotide acetate, an 8-mer peptide and TJ regulator, inhibits the cellular changes elicited by gliadin fragments, AT-1002, and cytokines. Previously, we demonstrated that AT-1002, a 6-mer peptide derived from the Vibrio cholerae zonula occludens toxin ZOT, caused several biochemical changes in IEC6 and Caco-2 cells resulting in decreased transepithelial electrical resistance (TEER) and increased TJ permeability. In this study, larazotide acetate inhibited the redistribution and rearrangement of zonula occludens-1 (ZO-1) and actin caused by AT-1002 and gliadin fragments in Caco-2 and IEC6 cells. Functionally, larazotide acetate inhibited the AT-1002-induced TEER reduction and TJ opening in Caco-2 cells. Additionally, larazotide acetate inhibited the translocation of a gliadin 13-mer peptide, which has been implicated in celiac disease, across Caco-2 cell monolayers. Further, apically applied larazotide acetate inhibited the increase in TJ permeability elicited by basolaterally applied cytokines. Finally, when tested in vivo in gliadin-sensitized HLA-HCD4/DQ8 double transgenic mice, larazotide acetate inhibited gliadin-induced macrophage accumulation in the intestine and preserved normal TJ structure. Taken together, our data suggest that larazotide acetate inhibits changes elicited by AT-1002, gliadin, and cytokines in epithelial cells and preserves TJ structure and function in vitro and in vivo.

Self heat-shock protein 65-mediated regulation of autoimmune arthritis.

Heat-shock proteins (Hsps) have been invoked in the pathogenesis of a variety of autoimmune diseases. The mycobacterial heat-shock protein 65 (Bhsp65) has been studied extensively as one of the antigenic triggers of autoimmunity in experimental models of, as well as patients with, rheumatoid arthritis. As Hsps are highly conserved and immunogenic, it is generally anticipated that self Hsps might serve as the endogenous targets of the immune response initiated by the homologous foreign Hsps. Contrary to this expectation, studies in the rat adjuvant arthritis (AA) model have revealed that priming of the self (rat) hsp65 (Rhsp65)-directed T cells in the Lewis rat leads to protection against AA instead of disease induction or aggravation. The arthritis-protective attribute of the self hsp65 is also evident following spontaneous priming of the anti-Rhsp65 T cells during the natural course of AA. Furthermore, immunization of rats with human hsp60, or with Bhsp65 peptides that are crossreactive with the corresponding self hsp65 peptides, leads to protection against AA. Importantly, high levels of T cell reactivity against self hsp60 in patients with juvenile idiopathic arthritis positively correlate with a favorable outcome of the disease. Thus, immune response against self hsp65 in autoimmune arthritis is protective rather than being pathogenic.

In vivo functional efficacy of tumor-specific T cells expanded using HLA-Ig based artificial antigen presenting cells (aAPC).

Adoptive immunotherapy for treatment of cancers and infectious diseases is often hampered by a high degree of variability in the final T cell product and in the limited in vivo function and survival of ex vivo expanded antigen-specific cytotoxic T cells (CTL). This has stimulated interest in development of standardized artificial antigen presenting cells (aAPC) to reliably expand antigen specific CTL. However, for successful immunotherapy the aAPC ex vivo generated CTL must have anti-tumor activity in vivo. Here, we demonstrate that HLA-Ig based aAPC stimulated tumor-specific CTL from human peripheral blood T lymphocytes showed robust expansion and functional activity in a human/SCID mouse melanoma model. HLA-Ig based aAPC expanded CTL were detected in the peripheral blood up to 15 days after transfer. Non-invasive bioluminescence imaging of tumor bearing mice demonstrated antigen dependent localization of transferred CTL to the tumor site. Moreover, adoptive transfer of HLA-Ig based aAPC generated CTL inhibited the tumor growth both in prevention and treatment modes of therapy and was comparable to that achieved by dendritic cell expanded CTL. Thus, our data demonstrate potential therapeutic in vivo activity of HLA-Ig based aAPC expanded CTL to control tumor growth.

Regulation of autoimmune arthritis by self-heat-shock proteins.

Heat-shock proteins (hsps) are highly conserved and immunogenic, and they are generally perceived to be attractive initiators or targets of a pathogenic immune response, and as such, have been implicated in the pathogenesis of autoimmune arthritis. However, studies in animal models and arthritis patients have unraveled the disease-regulating attributes of self-hsp65. We propose that the self-hsp65 induces a protective and beneficial immune response because of its ubiquitous distribution, stress inducibility and participation in tolerogenic processes. By contrast, the foreign hsp65 that does not influence the above processes and that resides admixed with microbial ligands for innate receptors generates an inflammatory pathogenic response. The regulatory properties of self-hsps need be fully explored and might be used for therapeutic purposes.

Antigen-specific tolerogenic and immunomodulatory strategies for the treatment of autoimmune arthritis.

OBJECTIVES: To review various antigen-specific tolerogenic and immunomodulatory approaches for arthritis in animal models and patients in regard to their efficacy, mechanisms of action, and limitations. METHODS: We reviewed the published literature in Medline (PubMed) on the induction of antigen-specific tolerance and its effect on autoimmune arthritis, as well as the recent work on B-cell-mediated tolerance from our laboratory. The prominent key words used in different combinations included arthritis, autoimmunity, immunotherapy, innate immunity, tolerance, treatment, and rheumatoid arthritis (RA). Although this search spanned the years 1975 to 2007, the majority of the short-listed articles belonged to the period 1990 to 2007. The relevant primary as well as cross-referenced articles were then collected from links within PubMed and reviewed. RESULTS: Antigen-specific tolerance has been successful in the prevention and/or treatment of arthritis in animal models. The administration of soluble native antigen or an altered peptide ligand intravenously, orally, or nasally, and the delivery of the DNA encoding a particular antigen by gene therapy have been the mainstay of immunomodulation. Recently, the methods for in vitro expansion of CD4+CD25+ regulatory T-cells have been optimized. Furthermore, interleukin-17 has emerged as a promising new therapeutic target in arthritis. However, in RA patients, non-antigen-specific therapeutic approaches have been much more successful than antigen-specific tolerogenic regimens. CONCLUSION: An antigen-specific treatment against autoimmune arthritis is still elusive. However, insights into newly emerging mechanisms of disease pathogenesis provide hope for the development of effective and safe immunotherapeutic strategies in the near future.

T cells against the pathogenic and protective epitopes of heat-shock protein 65 are crossreactive and display functional similarity: novel aspect of regulation of autoimmune arthritis.

OBJECTIVE: In autoimmune situations, the outcome of immune response against a disease-related antigen is typically viewed in terms of the balance between the pathogenic versus the protective subsets of antigen-reactive T cells. Using the rat adjuvant arthritis (AA) model of human rheumatoid arthritis (RA), we examined the antigen specificity and the functional attributes of the T cell repertoire directed against defined pathogenic versus protective epitopes of heat-shock protein 65 (hsp65), and determined the immunologic basis of the AA-protective effect of subsets of T cells primed by the pathogenic determinant. METHODS: Lewis (RT.1l) rats were pretreated subcutaneously with the pathogenic epitope 177-191 of mycobacterial hsp65 (B177) in adjuvant (incomplete Freund's adjuvant/complete Freund's adjuvant/CpG) and then immunized with heat-killed M. tuberculosis H37Ra for disease induction. The antigen specificity/crossreactivity of the T cells primed by B177 or the AA-protective determinant 465-479 of the homologous rat hsp65 (R465) was tested by using proliferation assay, cytokine ELISA, tolerance induction, and adoptive transfer. RESULTS: Pretreatment of Lewis rats with the arthritogenic determinant B177 using an immunogenic rather than a tolerogenic regimen affords protection against AA instead of initiation or aggravation of AA. This protective effect of B177 is mediated in part by activation of T cells that are crossreactive with R465. CONCLUSION: Downmodulation of AA by a pathogenic foreign epitope involving T cells crossreactive with a distant, protective self-determinant represents a novel aspect of immune regulation, and suggests further exploration of the use of pathogenic epitopes for the treatment of autoimmune arthritis.

Heat shock protein 65-reactive T cells are involved in the pathogenesis of non-antigenic dimethyl dioctadecyl ammonium bromide-induced arthritis.

Dimethyl dioctadecyl ammonium bromide (DDA) (C(38)H(80)NBr) is a nonantigenic lipoid material. DDA-induced arthritis (DIA) in the Lewis (LEW) (RT.1(l)) rat is a new experimental model for human rheumatoid arthritis (RA). DIA is a T cell-mediated autoimmune disease. However, the precise self/foreign Ags associated with the disease process in DIA are not yet known. We observed that LEW rats with DIA spontaneously raised a vigorous T cell response both to 65-kDa self (rat) heat shock protein (Rhsp65) and mycobacterial hsp65 (Bhsp65), but not to another arthritis-related Ag, bovine collagen type II. The T cell response to Rhsp65 was focused predominantly on determinant regions 120-134 and 213-227 of the self protein. Interestingly, pretreatment of adult LEW rats using either a mixture of peptides 120-134 and 213-227 of Rhsp65 or a low nonarthritogenic dose of DDA induced protection against subsequent DIA. Intriguingly, the protection induced by the latter was associated with spontaneous priming of T cells specific for peptide 213-227 of Rhsp65. Similarly, LEW rats neonatally tolerized against either Rhsp65 or Bhsp65 were significantly protected from subsequently induced DIA at adult stage, showing the disease-modulating attribute of the hsp65-specific T cells. Taken together, the above findings demonstrate that the hsp65-directed T cell repertoire is of significance in the pathogenesis of autoimmune arthritis induced by nonantigenic DDA. Like other animal models of RA involving hsp65, these first insights into the disease-associated Ags in the DIA model would pave the way for further understanding of the immunological aspects of induction and regulation of RA.

The regulatory C-terminal determinants within mycobacterial heat shock protein 65 are cryptic and cross-reactive with the dominant self homologs: implications for the pathogenesis of autoimmune arthritis.

The 65-kDa mycobacterial heat shock protein (Bhsp65) has been invoked in the pathogenesis of both adjuvant arthritis (AA) in the Lewis rat (RT.1(l)) and human rheumatoid arthritis. Arthritic Lewis rats in the late phase of AA show diversification of the T cell response to Bhsp65 C-terminal determinants (BCTD), and pretreatment of naive Lewis rats with a mixture of peptides representing these neoepitopes affords protection against AA. However, the fine specificity and physiologic significance of the BCTD-directed T cell repertoire, and the role of homologous self (rat) hsp65 (Rhsp65), if any, in spreading of the T cell response to Bhsp65 have not yet been examined. We observed that T cells primed by peptides comprising BCTD can adoptively transfer protection against AA to the recipient Lewis rats. However, these T cells can be activated by preprocessed (peptide) form of BCTD, but not native Bhsp65, showing that BCTD are cryptic epitopes. The BCTD-reactive T cells can be activated by the naturally generated (dominant) C-terminal epitopes of both exogenous and endogenous Rhsp65 and vice versa. Furthermore, certain individual peptides constituting BCTD and their self homologs can also induce protection against AA. These results support a model for the diversification of T cell response to Bhsp65 during the course of AA involving up-regulation of the display of cryptic BCTD coupled with spontaneous induction of T cell response to the cross-reactive dominant C-terminal epitopes of Rhsp65. The identification of disease-regulating cryptic determinants in Ags implicated in arthritis provides a novel approach for immunotherapy of rheumatoid arthritis.

The T cells specific for the carboxyl-terminal determinants of self (rat) heat-shock protein 65 escape tolerance induction and are involved in regulation of autoimmune arthritis.

Immunization of Lewis rats with heat-killed Mycobacterium tuberculosis H37Ra leads to development of polyarthritis (adjuvant-induced arthritis; AA) that shares several features with human rheumatoid arthritis (RA). Immune response to the 65-kDa mycobacterial heat-shock protein (Bhsp65) is believed to be involved in induction of AA as well as in experimental modulation of this disease. However, the understanding of several critical aspects of the pathogenesis of AA in the Lewis rat has severely been hampered by the lack of information both regarding the level as well as epitope specificity of tolerance to the mammalian self (rat) homologue of Bhsp65, 65-kDa rat heat-shock protein (Rhsp65), and about the functional attributes of the T cell repertoire specific for this self protein. In this study, we established that tolerance to Rhsp65 in the Lewis rat is incomplete, and that the residual T cells primed upon challenge with this self hsp65 are disease regulating in nature. We also have defined the T cell epitopes in the C-terminal region within Rhsp65 that contribute predominantly to the immune reactivity as well as the AA-protective effect of this self protein. Furthermore, the T cells primed by peptides comprising these C-terminal determinants can be efficiently restimulated by the naturally generated epitopes from endogenous Rhsp65, suggesting that self hsp65 might also be involved in natural remission from acute AA. These novel first experimental insights into the self hsp65-directed regulatory T cell repertoire in AA would help develop better immunotherapeutic approaches for autoimmune arthritis.