HSP Immune Responses and Lack of Self Tolerance

Besides their immunodominance as microbial antigens, under various circumstances HSP do elicit immune responses also when (over-)expressed as self antigens by cells or tissues. And this seems to be a peculiar feature of HSP, especially because in many cases immune responses to this self antigen are not associated with pathogenic autoimmunity. Evidently, healthy individuals have a broad repertoire of T and B cells with specificity for mammalian or self HSP. Self HSP specific immunity has been seen to exist in mice, rats, humans and other species studied so far. Apparently, thymic selection ensures the selection of a repertoire of cells with cognate receptors that can recognize such proteins, despite the fact that they are omni-present in almost every cell of our body.

The mechanism of the lack of tolerance for self HSP is unclear. A low level of constitutive expression or a restricted tissue representation would be an obvious explanation. However, in the normal thymus HSP are relative abundantly expressed in the medullary epithelium (Anderson et al., 2002; Birk et al., 1996; Ostberg et al., 2002) and yet fail to induce tolerance by negative selection. And also the transgenic overexpression of mouse Hsp60 in the thymic cortical epithelium and bone marrow derived cells of NOD mice, through the mouse class II Ea promoter, did not eliminate T cell responses to a panel of self Hsp60 peptides, although an epitope shift with disease suppressing potential was noted (Birk et al., 1996). Possibly, a peculiar subcellular localization characteristic for HSP creates a threshold effect, somehow allowing for an efficient expansion of cells with low affinity receptors that thereby escape deletion.

Whatever the mechanisms behind this lack of tolerance may be, it needs to be compensated efficiently by immune regulation in the periphery in order to control self HSP specific immunity. And indeed, as we will discuss later, immune regulatory mechanisms have been shown to be a prominent feature of HSP specific immune responses. And here we seem to have arrived at one of the most intriguing aspects of immunity for HSP. Their evolutionary conservation, their omni-presence in cells and tissues and their inducible nature seems to have made HSP into very dependable self-antigens for the immune system to target regulation. For the same reason Cohen has mentioned HSP as prime examples of his so-called homuncular antigens (Cohen et al., 2003). And along similar lines of reasoning, the findings that HSP such as HSP90 are part of a subset of highly conserved and immunodominant self antigens that are target for natural autoantibodies in normal human IgG also indicate that HSP may be essential for selection of natural B and T cells repertoires and for the maintenance of self-tolerance (Pashov et al., 2002).

Cell Stress Leads to HSP Expression and Induction of Immune Responses

The inducible nature of HSP is particularly evident in inflammation. Many features of inflammation, such as production of inflammatory mediators, cytokines such as TNF-a and IL1, cell debris, reactive oxygen intermediates and raised temperature are perfect inducers of heat shock protein expression. In fact raised temperature due to fever and inflammation are key components of virtually all immune responses. The effects of raised temperature in the form of experimental hyperthermia on HSP expression and its facilitation of many distinct forms of specific immunity, has been the subject of many studies already (Repasky and Issels, 2002).

HSP have been claimed to become expressed on the cell surface, serving as a target for NK cells and for antibodies leading to ADCC (Multhoff and Hightower, 1996), despite the fact that these proteins are lacking the molecular features of cell surface antigens. In patients with active RA, and not during remission, peripheral blood and synovial fluid derived T cells were shown to present cell surface HSP (Sato et al., 1996). Expression of HSP in T cells has been reported earlier and it seems that HSP are expressed in T cells at lower levels of stress as compared to other cells (such as B cells), which may indicate that T cells do need overexpressed HSP in order to resist the harsh environment of the site of inflammation (Gothard et al., 2003). Also rheumatoid joint DCs were shown to have cell surface expression of Hsp70 (Martin et al., 2003).

Class I Restricted T Cell Responses

HSP are intracellular cytosolic proteins. Therefore HSP fragment are presented in the context of MHC class I molecules, and induction of HSP specific Class I restricted CTL responses has been documented in many different situations of cell stress.

One of the first observations was made with CTL raised against mycobacterial Hsp65, as these CD8+ class I restricted T cells recognized macrophages subjected to various forms of cells stress. This was a first demonstration of the fact that HSP are processed in stressed host cells and can be presented in the context of class I molecules (Koga et al., 1989).

Hsp60 peptide specific CTLs were found to lyse IFN-7 stressed macrophages and such lysis was specifically inhibited by Hsp60-specific antisense oligonucleotides (Zugel et al., 1995). In measles virus-infected patients CTLs were detected with specificity for a Hsp90 derived peptide in the context of HLA-A2 (Herberts et al.,

2003). In cancer patients, tumor infiltrating lymphocytes were found to harbor CTLs with specificity for Hsp70 derived peptides (Azuma et al., 2003; Faure et al.,

2004). In other words, CTL perceive the altered levels of HSP peptides in the class I molecules after infection and tumorigenesis.

Class II Restricted Responses

Besides class I, also class II molecules are loaded with HSP peptides. In fact, Hsp70 peptides were prominently represented in the RP-HPLC profile of the content of class II molecules of a human lymphoblastoid cell line (Newcomb and Cresswell, 1993). Many studies have shown the production of HSP specific antibodies, as a reflection of MHC II driven antigen presentation of stress induced HSP. Especially in chronic inflammatory diseases the presence of HSP specific autoantibodies has been widely documented (see hereunder). Also in infectious diseases HSP antibodies have been shown, such as higher titers of IgG antibodies specific for Hsp70 in sera of patients with HIV infection (Kocsis et al., 2003). In experimental heart transplantation in the rat, chronic rejection of the cardiac allograft was seen to go along with graft-infiltrating auto-reactive T cells reactive to HSP (Duquesnoy et al., 1999). In human renal transplant patients an increasing percentage of IL10 producing Hsp60 specific T cells was documented in the late post-transplant period (Caldas et al., 2004). Also the production of IL4 by Hsp60 specific T cells was seen to be associated with absence of rejection (Granja et al., 2004). Birk et al. showed in a Hsp60 transgenic mouse skin transplant model that the stress of rejection led to Hsp60 over-expression in the graft and that anti-graft responses were modulated with Hsp60 antibodies or peptides (Birk et al., 1999). In the low dose streptozotocin model of insulin dependent diabetes in the mouse, it was shown that the beta-cell toxin led to cell stress leading to the production of increased levels of antibodies and T cells reactive to Hsp60 (Elias et al., 1994).

Class Ib Restricted Responses

Besides the adaptive immune responses to HSP also innate responses are involved in the recognition of over-expressed HSP. And this makes sense, HSP being an evolutionary ancient collection of proteins that has been into existence before the philogenetic development of the mediators of adaptive immunity. Most likely, HSP have fulfilled and still fulfill a primitive task in signaling cellular stress and therefore there is logic in the recognition of these proteins by cells of the innate immune system.

Molecules presenting the stress signals of cells are non-classical MHC class I molecules, known as Qa-1b in the mouse and HLA-E in humans. It had been shown that Qa-1b was up-regulated during cellular stress (Imani and Soloski, 1991). Now, Michaelsson et al., have shown that HLA-E molecules on human cell lines become loaded with a Hsp60 signal peptide under cell stress, which replaces other peptides in the cleft of HLA-E, which normally are leader sequences of other class I MHC molecules. The introduction of the Hsp60 signal peptide into HLA-E was shown to lead to interference with CD94/NKG2A recognition, which is an inhibitory NK receptor. In this manner stress would enable NK cells to become activated by the detection of stressed cells (Michaelsson et al., 2002); (Soderstrom, 2003).

In an earlier study a Hsp60 peptide in Qa-1b was reported to be a target for CD8+ CTL. These CTL were induced by Salmonella typhimurium infected macrophages, were found to be specific for a Hsp65 specific peptide and to cross-recognize IFN-7 or LPS stressed macrophages (Lo et al., 2000). Most likely, a similar phenomenon has been underlying earlier studies where MHC-unrestricted recognition was claimed to exist for human CD8+ CTL that killed infected target cells and not heat-killed bacteria loaded target cells (Hermann et al., 1992). A recent study has shown that the Hsp60 peptide 216-224 (homologous to the 192-200 sequence in bacterial Hsp65), in the absence of Qdm, the leader sequence of many class 1a molecules, is the dominant peptide bound by Qa-1b (Davies et al., 2003). These findings are especially intriguing with respect to the resurrected interest in suppressor T cells (Chess and Jiang, 2004). Originally, after its serological detection, Qa-1 was found to be expressed on lymphocytes that preferentially induced CD8+ suppressor T cells. More recently, Hu at al. showed in an in vivo transgenic model that Qa-1 restricted CD8+ T cells do control CD4+ T cell responses, thereby suppressing the generation of experimental EAE. In the absence of Qa-1 no such suppressive effect was mounted (Hu et al., 2004). Along similar lines, a critical role for Qa-1 was claimed in T cell vaccination. Originally T cell vaccination was described as the phenomenon that attenuated auto-reactive T cells conferred resistance to subsequent active disease induction (Ben-Nun et al., 1981). Now in two murine models of autoimmunity it was shown that T cell vaccination depended on the activation of suppressive CD8+ T cells that specifically recognize Qa-1 and self peptides expressed by activated autoreactive CD4+ T cells (Panoutsakopoulou et al., 2004). As HSP, like Qa-1, are up-regulated in activated T cells, and as we now know that HSP sequences are major antigens for presentation through Qa-1, it is possible that herewith we see one of the mechanistic pathways through which HSP immunizations lead to regulatory activities. That such a pathway may be clinically relevant is suggested by the data that in humans HLA-E is needed for CD8+ T cells to differentiate into regulatory cells (Li et al., 2001).

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