Viewing autoimmune pathogenesis from the perspective of antigen processing and determinant hierarchy

Abstract

Autoimmunity results from the breakdown of immune tolerance to defined target self antigens. As with any foreign antigen, a self antigen is continuously processed by the antigen-presenting cells (APCs) and its epitopes are displayed by the major histocompatibility complex on the cell surface (dominant epitopes). However, this self antigen fails to induce a T cell response as the T cells against its dominant epitopes have been purged in the thymus during negative selection. On the contrary, the T cells against poorly processed (cryptic) self epitopes escape tolerance induction in the thymus and make it to the periphery. Such T cells are generally harmless as their cognate epitopes are not presented efficiently in the periphery. But under conditions of inflammation and immune activation, previously cryptic epitopes can be revealed on the APC surface for activation of the ambient T cells. This can initiate autoimmunity in individuals who are susceptible owing to their genetic and environmental constellation. Subsequent waves of enhanced processing of other epitopes on the same or different self antigen then cause ‘diversification’ or ‘spreading’ of the initial T cell response, resulting in propagation of autoimmunity. However, depending on the disease process and the self antigen involved, ‘epitope spreading’ may instead contribute to natural regression of autoimmunity. This landmark conceptual framework developed by Eli Sercarz and his team ties together determinant hierarchy, selection of epitope-specific T cells, and the induction/progression of autoimmunity. I am extremely fortunate to have worked with Eli and to be a part of this fascinating research endeavor.

This article contains a summary of the work that I, along with some of my colleagues, including several student trainees, performed when working with Prof. Eli Sercarz at UCLA and LIAI, as well as of some work done at UMB after I moved to Maryland. Together, it provides a comprehensive view of the basic mechanisms and disease-related processes in autoimmunity from the perspective of antigen processing and presentation. The text is organized to highlight our work involving two main antigens, lysozyme and heat-shock protein 65 (Hsp65). The former includes mouse lysozyme (ML) (self) and hen eggwhite lysozyme (HEL) (foreign) ^1,2, whereas the latter includes rat hsp65 (Rhsp65; self) and mycobacterial hsp65 (Bhsp65; foreign) ³. Hsp65 is involved in the pathogenesis of rat adjuvant-induced arthritis (AA), an experimental models for human rheumatoid arthritis (RA) ⁴. These studies highlight a unifying theme, namely the impact of determinant hierarchy (dominance/ crypticity) on T cell repertoire selection and activation/regulation of autoreactive T cells ^1,5–8. This variety of projects was feasible because Eli not only fully supported, but always encouraged me (and other lab members) to feel free to select interesting projects. I considered that a true privilege and a challenge at the same time, and tried to make the most of it. I am very grateful to Eli for his generosity and mentorship!

Defining the T cell determinants within self (mouse) lysozyme (ML) and examining the T cell repertoire against ML and its foreign homologue, HEL

Using ML as a model self antigen and HEL as its foreign homologue, we addressed in a series of studies few key questions relating to immune tolerance and induction of autoimmunity ^2,9,10: a) are wild type mice to fully tolerant to ML, as assessed by challenge with native (whole) ML and recall of T cell response with whole ML and synthetic peptides corresponding to different regions of ML?; b) what is the determinant specificity of the mature T cell repertoire in the periphery, which represents the residual repertoire following negative selection of potentially self-reactive T cells, as assessed by immunization of mice with peptides of ML and recall with the same peptide or whole ML?; c) how similar is the pattern of response to ML determinants of mice of different major histocompatibility complex (MHC) haplotypes?; d) is there any correlation in the molecular position of the T cell determinants that are potentially tolerogenic/ immunogenic within the homologous self/foreign lysozymes; and e) what might be the functional significance of ML-directed T cells that are crossreactive with HEL?

Identifying cryptic epitopes within ML: crypticity is MHC associated

To address these questions, we tested the immune response to ML/HEL of inbred wild type mice of different MHC class II haplotypes and defined genetic backgrounds (non-MHC genes) ^2,9,10. We used ML-M, which is the major variant of ML that is present in the myeloid cells and in circulation. (The other variant, ML-P, resides in the Paneth cells of the intestine.) We employed standard T cell proliferation and cytokine assays using the draining lymph node cells (LNC) and/or splenocytes following subcutaneous immunization with antigen emulsified in an adjuvant. The T cell recall response was tested using either whole antigen or synthetic 14-15-mer peptides spanning the entire length of that antigen. We observed that mice of diverse MHC haplotypes were tolerant to native ML, as evident from a lack of recall response to ML or its peptides after a challenge with whole ML ². Interesting, however, these same mice had T cells in their mature repertoire that could be activated by immunization with certain peptides of ML, which represent processed form of native ML. Such peptide-induced T cell responses, however, were not recalled by native ML, indicating that the determinants residing within these peptides were somehow hidden (or cryptic) from the immune system. Thus, self-reactive T cells are part of the mature repertoire, but they can be activated by peptides comprising cryptic self epitopes. However, crypticity is not a pre-determined attribute of the amino acid sequence of a given determinant. This is evident from our results showing that different sets of determinants are cryptic in each strain when comparing mice of different MHC haplotype (e.g., H-2^k, H-2^b, and H-2^d) ². Furthermore, results using mice of same MHC but different genetic backgrounds further showed that crypticity is MHC associated. The presence of poorly processed ‘minor’ or ‘cryptic’ epitopes and how they escape tolerance induction has previously been shown using other antigens and model systems as well ^5,6,11–13, thus offering a broader view of such epitopes that are particularly relevant for autoimmunity.

For more insight into potential T cell determinants within ML and their functional impact on T cell tolerance, we examined the MHC binding of a set of ML peptides spanning the whole molecule ⁹. Interestingly, the peptides that bound well to MHC could be grouped into those that were immunogenic, and others that were non-immunogenic. The former group corresponded to the cryptic epitopes identified in our earlier study, whereas the latter group was hypothesized to represent dominant determinants within ML that had induced T cell tolerance during thymic selection. As cryptic epitopes are not efficiently revealed from native ML, we suggested that they failed to induce thymic T cell tolerance ⁹. Thus, the hierarchy (dominance/crypticity) of determinants within ML contributed to shaping of the T cell repertoire against this self antigen.

Mice deficient in ML provide insight into tolerogenic self epitopes

To seek evidence for the above concept, we employed mice of the H-2^k haplotype that were deficient in ML, and compared their response to whole ML and its peptides with that of syngeneic wild type mice ¹⁴. (The founder LysMcre or ML-M knockout (MLKO) mice were kindly provided by Dr. Irmgard Forster from Germany.) As anticipated, ML was immunogenic in MLKO mice but not in wild type mice. The response of the former was directed to a dominant C-terminal determinant in the region 105-119 ¹⁴. Furthermore, neonatal tolerization of MLKO mice with peptide representing this dominant determinant rendered these mice non-responsive to native ML, besides tolerance to the peptide immunogen itself, when tested later at adult stage. The dominance of that epitope within ML was further supported by the finding that APCs of wild type mice spontaneously displayed that epitope of endogenous ML on their surface in a functional form, as evident from their ability to activate peptide-induced T cells in co-culture experiments without the addition of exogenous peptide ¹⁴. As far as cryptic epitopes within ML are concerned, there was no difference in the patterns of response of MLKO and wild type mice. The same was the case for the foreign lysozyme, HEL ¹⁴. Taken together ^2,9,14, our studies validated the impact of self determinant hierarchy on shaping of the self-directed T cell repertoire.

Mechanisms underlying the crypticity of antigenic determinants

Why are cryptic epitopes not revealed under normal conditions? There are many possible reasons ^1,7,8. Most of them relate to limitations imposed by antigen processing and presentation on cryptic determinants when compared with dominant determinants. For example ^1,7,8, competitive disadvantage to an epitope for binding to a particular MHC molecule, which can result either from indolent processing of that determinant/antigen or relatively weaker avidity of that epitope for MHC binding compared to adjacent determinants within the same molecule, can render it cryptic; competition with other antigens within the same APC; the presence of amino acid residues in the flanking region of that determinant that can interfere in its binding to the MHC ^15,16 or the T cell receptor (TCR) ¹⁰ (hindering residues) ¹⁷; excessive processing of that determinant resulting in its destruction; DM-mediated antagonism ¹⁸; non-availability of an appropriate processing site adjacent to the determinant region; and relatively lower frequency of specific T cells in the mature repertoire compared to that for other determinants.

Influence of the amino acid residues flanking the core determinant on tolerogenicity/ immunogenicity of that determinant

Hindrance in interaction with TCR or MHC by the flanking residues

Above-mentioned mechanisms of crypticity have been experimentally validated in different antigenic systems, including HEL. Our work specifically unraveled the role of hindering residues ¹⁷ in rendering a potentially immunogenic determinant non-immunogenic within ML¹⁰ and HEL ¹⁵. The former was a case of hindrance in interaction of the peptide-MHC complex with specific TCR, whereas the latter was a case of hindrance in the binding of the peptide to the MHC. Such hindering residues can contribute to the crypticity of certain T cell determinants. This was clear after removal or substitution of the hindering residue that effectively reversed the non-immunogenicity of the original peptide.

(a) In ML¹⁰, we observed that peptide 46-61, which binds to A^k but not E^k, was immunogenic in CBA/J (A^k, E^k) but not in B10.A(4R) (A^k) mice. Testing of a series of alanine-substituted peptides showed that a C-terminal arginine residue (R61) flanking the core determinant 51-59 interfered in the interaction of the MHC-peptide complex with the appropriate TCR. Deliberate removal or substitution of that arginine residue rendered the peptide immunogenic in B10.A(4R) mice. Using bone marrow chimeras and other mouse strains, we systemically excluded the role of a hole in the T cell repertoire and of non-MHC (background) genes in the non-immunogenicity of ML p46-61 in B10.A(4R) mice. (b) In HEL ¹⁵, we observed a similar phenomenon but of MHC hindrance of A^b with epitope region 46-61. HEL p46-61 was immunogenic in C3H.SW (A^b) mice, but not in C57BL/6 (A^b) mice. However, the same peptide but lacking an arginine residue at the C-terminus was immunogenic in both C3H.SW and C57BL/6 mice. Additional experiments showed that C3H.SW but not C57BL/6 mice could naturally process p46-61 to remove the hindering arginine residue, and thereby could mount an immune response to that determinant.

We have highlighted above the impact of amino acids flanking the core epitope on T cell response to that epitope. Extending this further to the adjacent flanking region epitopes, other investigators have demonstrated their role in influencing competitive capture by MHC, leading to pre-emption of tolerance induction to the adjacent epitope, for example, the effect of Golli on MBP Ac1-9 determinant ¹⁹. Another example of determinant capture, but involving both I-A and I-E molecules showed that certain determinants can be lost through this mechanism and possibly afford protection against autoimmunity, as in type 1 diabetes ^20,21. In another diabetes-related T cell determinant, it was observed that while the core determinant itself was not immunogenic, the N-terminal flanking residues influenced the recognition of that epitope by the MHC, and whether a response was induced to it or not ²². Similarly, earlier studies on lysozymes and Sperm Whale/Equine myoglobin elaborated upon the structural and competitive features within and/or outside of the determinants that can affect the selection and immunogenicity of specific determinants within homologous proteins ^16,23.

Non-availability of a proteolytic cleavage site adjacent to a potential determinant

In another study ²⁴, we described a different mechanism by which certain determinants might not be processed well from native ML to be displayed by APCs to activate specific T cells; it involved the absence of a potential enzymatic cleavage site adjacent to a T cell determinant. We examined 3 known cryptic determinants within ML, one each for mice of H-2^k, H-2^b, and H-2^d haplotype. We deliberately mutated/grafted an amino acid residue to create an antigen processing site (the ‘dibasic’ motif) ²¹ adjacent to the above-mentioned cryptic determinants, one epitope in each molecule ²⁴. The dibasic motif contained paired arginine-arginine (RR) or arginine-lysine (RK), which is known to be cleaved by certain enzymes within APCs. Interestingly, two of the 3 mutated ML proteins when used as immunogens induced vigorous T cell response to previously cryptic determinants, which now were revealed as dominant determinants in that particular MHC haplotype-bearing mice, but not in unrelated haplotype-bearing mice ²⁴. By comparison, wild type ML remained non-immunogenic in the same mouse strain.

These results have important implications not only in explaining the crypticity of certain self determinants, but also in the deliberate reversal of T cell tolerance by altering the antigen processing events. The latter approach can be of value in facilitating the display of a previously cryptic determinant that is disease-regulating in autoimmunity. An example of such epitopes can be found in adjuvant arthritis, where cryptic C-terminal determinants of Bhsp65 are regulatory in nature ^3,25, as described below. It would also be applicable to cryptic determinants of a tumor antigen whose upregulated display can help induce an anti-tumor response in the face of tolerance to native tumor antigens ^1,26,27. Previous work on introducing a dibasic motif adjacent to a determinant of HEL showing enhancement in T cell response to that epitope also supports the application of this approach to vaccine design ²¹.

The involvement of cryptic self determinants in the induction and propagation of autoimmunity

What might be the functional significance of cryptic epitopes in the context of immune tolerance and autoimmunity? Based on our work and that of others, we have proposed that the T cell repertoire against cryptic self determinants can be engaged for the initiation and propagation of autoimmunity. Two of the mechanisms by which this repertoire can be tapped include ^1,7,8: a) activation of the T cell against cryptic determinants by crossreactive foreign homologues of the self protein; and b) alteration of the local milieu that now can facilitate the processing and presentation of previously cryptic determinants. However, cryptic self determinants may not always be pathogenic. As shown in our work in an experimental model of arthritis, cryptic determinants may also be disease-regulating in nature ^3,25. In that situation, the same two conditions mentioned above for the induction and progression of autoimmunity would instead result in regression of autoimmunity and resistance to autoimmunity. These features of cryptic epitopes are discussed below in more detail.

Activation of self-reactive T cells by the cross-reactive foreign antigenic determinants

We described above that ML harbors cryptic determinants. Further examination of the T cell repertoire against its foreign homologue, HEL, in the same mouse strains revealed an interesting correlation among the two proteins ². The regions within ML that were cryptic, roughly overlapped in position with the dominant determinants within HEL. Accordingly, the corresponding determinant regions in this pair of self and foreign lysozyme showed an opposite profile. We proposed that this outcome is attributable to differential antigen processing and presentation of the two related proteins ². An implication of this observation is that crypticity of ML determinants might allow self-reactive T cells to escape tolerance induction during thymic development. These T cells then form the mature repertoire, where they can be activated by homologous determinants of the foreign lysozyme ². In this sense, the T cell response to HEL can be shaped in part by crypticity of determinants within ML. Similarly, HEL-primed T cells can be engaged by peptides of ML bearing cryptic epitope. In fact, our work offered support for such a cross-reactive T cell repertoire ^2,9. We showed that the T cells primed by HEL also give a recall response to peptides representing cryptic ML determinants, and vice versa. Furthermore, repeated priming by HEL with the purpose of expanding the T cell pool resulted in higher T cell response to ML peptides than that in mice injected once with HEL ⁹. As with ML and HEL, the influence of differential antigen processing on epitope-directed T cell responses has previously been reported for another pair of homologous lysozymes as well ²⁸.

Above findings highlight the importance of foreign/self cross-reactive repertoire in the disease process in autoimmunity. In this regard, it is well known that infectious agents can act as a trigger for the initiation of autoimmunity. Furthermore, the induction of certain experimental autoimmune diseases in animal models involves immunization with a foreign homologue of a self protein. For example, the use of bovine or guinea pig myelin basic protein (MBP) for the induction of experimental autoimmune encephalomyelitis (EAE) ^29,30; the use of bovine type II collagen for the induction of collagen-induced arthritis (CIA) ³¹; and the use of mycobacteria containing bacterial heat-shock protein 65 (Bhsp65) for the induction of adjuvant arthritis (AA) ³. Similarly, patients with many different autoimmune diseases frequently give a history of past infections that preceded the onset of autoimmunity and/or a relapse of disease symptoms. Molecular mimicry between antigens of an infectious agent and a target self antigen is one of the mechanisms that has been invoked in diseases such as rheumatoid arthritis ³², multiple sclerosis ^33,34, type 1diabetes ³⁵, and myocarditis ³⁶, and it involves activation of the cross-reactive T cell repertoire.

Activation of self-reactive T cells via upregulation of cryptic epitopes under inflammatory and immune-activating conditions

Another important aspect of the role of cryptic determinants in autoimmunity involves upregulation of their display from the native antigen under certain conditions, particularly those in an inflammatory environment ^1,7,8,29. The presence of pro-inflammatory cytokines combined with enhanced costimulatory activity of APCs may enhance the processing and presentation of cryptic epitopes, which under normal conditions are processed inefficiently, or not at all, from the native antigen. As the T cells against cryptic determinants are available in the mature repertoire of healthy individuals, the display of previously cryptic epitopes can initiate autoimmune reactivity. This in turn would promote further inflammation and tissue damage, resulting in the release of additional tissue antigens (self antigens) along with their enhanced processing and presentation. These repeated waves of events can diversify or broaden the original autoreactivity to target additional new determinants within the same self antigen (intramolecular epitope spreading) ^1,29 or a different antigen (inter-molecular epitope spreading) ^1,37. This may account for chronicity of the original autoimmune manifestations or relapses/flares in some diseases.

Diversification of response to cryptic self determinants during the course of autoimmunity

Epitope spreading to cryptic determinants contributes to disease progression in autoimmunity

The first experimental demonstration of epitope spreading involving cryptic self epitopes was reported from Eli’s laboratory ²⁹. Here, EAE was induced in (SJL x B10.PL) F1 mice by immunization with MBP in adjuvant. The initial T cell response in these mice was confined to the dominant MBP peptide Ac1-11, but in the later chronic stage, new T cell responses appeared that targeted 3 other epitopes of MBP (epitopes 35-47, 81-100, and 121-140) ²⁹. These 3 regions of MBP harbor cryptic epitopes, and the T cells primed by peptides comprising these cryptic epitopes are pathogenic, thereby validating the functional significance of the newly arising T cell responses. Importantly, a similar diversification of response to the same 3 cryptic epitopes also occurred when EAE was induced by Ac1-11 peptide instead of whole MBP ²⁹. This was explained by the enhanced processing and presentation of endogenous MBP in the inflammatory milieu of EAE. Subsequently, other investigators reported similar phenomenon in another model of EAE, proteolipid peptide (PLP) peptide 139-151-induced relapsing EAE in SJL mice³⁸, as well as in patients with multiple sclerosis ³⁹. The role of cryptic epitopes as well as reciprocal T-B cell epitope crossreactivity in epitope spreading has also been described in lupus ^40,41 and some other diseases. A practical application of these findings is that interventions such as peptide- or protein-induced tolerance for controlling autoimmunity is likely to succeed only when given early before epitope spreading has occurred ^1,29. Subsequently, it may be difficult to control T cell responses to multiple epitopes.

Diversification of response to Hsp65 during the course of AA is disease-regulating rather than being pathogenic

Above examples demonstrate the functional significance of cryptic self determinants in disease induction and propagation. However, our work in the rat adjuvant arthritis (AA) model of human RA revealed a related, but opposite outcome of diversification/spreading of response, namely regulation of autoimmunity ³. AA was induced in Lewis rats by immunization with heat-killed M. tuberculosis H37Ra (Mtb). The mycobacterial Hsp65 (Bhsp65) is one of the major target antigens in this disease model. The region 180-188 and its longer stretch 177-191 harbors the pathogenic epitope. In the early phase of AA, the T cell responses were directed mostly to the determinants in the N-terminal region and middle region of Bhsp65 ³. Interestingly, in the late phase of AA, the T cell responses to N-terminal region were reduced, the responses to the middle region determinants were unaffected, but new T cell responses developed against Bhsp65 C-terminal determinants (BCTD), namely 417-431, 441-455, 465-479, 513-527, and 521-535. Importantly, immunization of naïve Lewis rats with peptides comprising BCTD afforded protection against subsequent induction of AA by Mtb injection ³. A similar protective effect was also observed following adoptive transfer of BCTD-primed T cells into naïve rats and then challenge with Mtb ²⁵. Thus, our observations unraveled the mechanism by which arthritic rats spontaneously recover from active arthritis.

The role of self Hsp65 in diversification of the T cell response in AA

Examination of the mechanism underlying the diversification of response in AA revealed the role of endogenous self (rat) hsp65 (Rhsp65), the self homologue of the foreign Bhsp65 ³. We observed that arthritic rats in the late phase of AA develop not only T cell responses to BCTD, but also to the corresponding Rhsp65 C-terminal determinants (RCTD) ³. This occurred without any challenge with exogenous Rhsp65, indicating that endogenous Rhsp65 might have contributed to the priming of these T cells, with additional contribution of activation of the cross-reactive T cells. In fact, the T cells primed with BCTD were shown to be crossreactive with RCTD, and vice versa ³. This crossreactivity between RCTD and BCTD is reminiscent of the above-mentioned crossreactivity between T cell determinants of ML and HEL. Similar crossreactivity but targeted to the determinant region 256-270 in arthritic rats, and the role of T cells reactive against this epitope in disease regulation has previously been reported by other investigators ^42,43. Furthermore, ‘spreading’ regulatory control involving anergic T cells and defined hsp65 epitopes was also proposed to explain disease regulation via tolerization and diversification of T cell response to Hsp65 ⁴⁴.

Differential processing of the C-terminal determinants of self and foreign Hsp65

We further examined the determinant hierarchy of the C-terminal determinants of the homologous Rhsp65 and Bhsp65. Immunization of Lewis rats with native Rhsp65 induced T cell responses to RCTD, indicating that tolerance to Rhsp65 is incomplete, and that RCTD represent dominant self determinants within Rhsp65 ⁴⁵. On the contrary, challenge of rats with native Bhsp65 failed to induce T cell response to BCTD, although responses to other epitopes were measurable. However, BCTD peptides could prime T cell responses, showing that BCTD are cryptic epitopes ²⁵. Additionally, BCTD primed T cells are crossreactive with RCTD and vice versa. Taken together all these results ^25,45, we proposed that during the course of arthritic inflammation induced by Mtb injection, the expression of endogenous Rhsp65 is upregulated and then efficiently processed and presented to display its dominant determinants. At the same time, the processing and presentation of previously cryptic BCTD are upregulated during inflammation, and these determinants can then engage RCTD-crossreactive T cells in addition to priming BCTD-specific T cells. These events can explain the observed diversification of T cell response in AA ³.

Induction of regulatory T cell responses by self Hsp65

Further support for the functional significance of Rhsp65-reactive T cells is gained from the findings that challenge of naïve rats with this self antigen affords protection against subsequent induction of AA by Mtb challenge ⁴⁵. Similarly, immunization of naïve rats with RCTD peptides or adoptive transfer of T cells primed by RCTD can induce protection against AA ⁴⁵. These results with Rhsp65/RCTD further validate the disease-regulating attribute of the epitope spreading to cryptic and crossreactive BCTD observed in AA. Studies by other investigators have also emphasized upon disease regulation by rat/human hsp65 in the AA model ^43,46. The significance of protective effect of self hsp65 (Rhsp65)-directed T cells has also been validated in patients with juvenile idiopathic arthritis (JIA) (or juvenile chronic arthritis, JCA). These patients develop a remitting-relapsing disease symptoms. Higher levels of T cell response to self hsp65 (human Hsp65) develop in these patients just before remission from active disease, and it correlates with a favorable prognosis ^47,48. Thus, under certain disease conditions, as in JIA, self antigen-directed T cell responses can be regulatory in nature instead of being pathogenic.

Immune response to BCTD in WKY rats (arthritis-resistant) and Fischer rats (acquired resistance).

We described above that arthritic Lewis rats developed T cell responses to BCTD during the course of AA. Evidence from two different rat strains provides additional support to the disease-regulating attributes of these determinants ^3,49.

(a) First, Wistar Kyoto (WKY) rats have the same MHC class II haplotype as the Lewis rat, but are resistant to induction of AA after Mtb challenge ³. However, Mtb-immunized WKY rats raised robust early T cell responses to BCTD in the period that corresponds to the incubation phase of arthritis in the AA-susceptible Lewis rats. The latter develop T cell responses to BCTD only in the late phase of AA. Thus, apparently the timing of development of BCTD-directed T cell responses following Mtb challenge can influence susceptibility versus resistance to AA, such that early response prevents development of AA, whereas late response helps in spontaneous regression of inflammation ³. Study of the cytokine profile of WKY and Lewis rats revealed a similar temporal difference in the timing of expression of IFN-γ and TNF-α, both appearing early after Mtb challenge in WKY, but in late stage of disease in Lewis rats. Counter-intuitively, both cytokines had a protective effect against AA. No Th2 deviation was observed. A Th1 cytokine milieu has also been shown to be protective in other disease models, although precise regulatory pathways may differ from that in the AA model ^50,51. What we observed in AA was different from that in the same pair of rats tested for EAE. While Lewis are susceptible, WKY are resistant to EAE ⁵². The main difference between the two strains here was in the cytokine profile, not in T cell response or TCR Vβ usage. The response of WKY was biased towards Th2 cytokines compared to Lewis rats ⁵².

(b) Second, Fischer F344 (F344) rats are susceptible to AA, but to slightly lower extent than Lewis rats. We observed that F344 rats raised in a barrier facility (BF-F344) are susceptible to AA induction by Mtb challenge, whereas F344 rats raised in a conventional facility (CV-F344) acquire resistance to AA ⁴⁹. This acquisition of resistance can be prevented if BF-F344 rats are given neomycin or acidified water following their transfer to the conventional facility. Interestingly, the acquired resistance to AA can be adoptively transferred to naïve BF-F344 using splenocytes of CV-F344 rats ⁴⁹. Furthermore, CV-F344 but not BF-F344 rats spontaneously raise T cell responses to BCTD of Bhsp65, whose levels keep on increasing with the duration of housing in the conventional facility. We attribute the induction of these T cell responses to exposure of CV-F344 rats to environmental microbes that possess Hsp65, which are highly conserved proteins. Taken together, results from the studies on WKY and F344 rats demonstrate the disease-regulating attributes of BCTD, which further support the original finding in arthritic Lewis rats.

Concluding remarks

Summary

The above studies performed on two pairs of antigens, each having a self antigen and its foreign homologue, elaborate the important role of various factors affecting antigen processing in establishing determinant hierarchy (dominance/crypticity). The latter in turn has a significant influence not only in shaping of the T cell repertoire in the thymus, but also in activation, or the lack of it, of potentially autoreactive T cells in the periphery. Furthermore, the impact of inflammation and immune-stimulating conditions on altering the display of cryptic epitopes leading to activation of previously silent T cells is one of the most critical steps in the pathogenesis of autoimmunity. To this can be added the differential processing of homologous antigens, the impact of the flanking residues on determinant display, and the activation of the crossreactive T cell repertoire by foreign antigens, particularly those belonging to infectious agents, which can trigger autoimmunity via molecular mimicry. However, not all outcomes are pathogenic. In the case of certain self antigens and diseases/animal models, the very same factors can activate disease-regulating T cell responses directed against protective cryptic epitopes instead of pathogenic cryptic epitopes. This is evident from examples of two types of diversification or epitope spreading, a pathogenic one in EAE, but a regulatory one in AA. These complex variables need to be taken into consideration when developing preventive or therapeutic approaches for various autoimmune disorders.

Personal reflections

It was a great privilege to work with Eli, both as a member of his research group at UCLA and LIAI, and later after I moved to UMB and continued some collaborative work. Besides being an extremely productive training period, it was also very enjoyable for me in many ways. His research group always had trainees from different countries and it provided for a wonderful cultural exchange as well. Eli’s energy and enthusiasm for science and life in general were unparalleled, and always inspiring and infectious, though not easy to imitate. As I settled down in the lab at UCLA upon arrival, it did not take much time to experience the combination of creativity and some chaos, which became easier to manage after some tips from senior lab members. Eli’s lab was known for its pioneering work on HEL, but because of my interest in autoimmunity, I proposed working on ML. Eli was very supportive, but also gently cautioned me that I would be on my own for reagents. I didn’t realize it till I spent several months to prepare ML from ascites of cell line-injected mice, and did manual synthesis of ML peptides by the ‘tea-bag’ method (under supervision of our Lab Co-director, Alex Miller, who was very knowledgeable, patient, and loved to work out the details; I am very thankful to him for his guidance and help.). In retrospect, on many occasions I happily recall that period of intensive work and hands-on training at the Chemical Hood. The opportunities to present and discuss the results of that project and of subsequent ones in weekly “Data Circus” was fun. Occasionally, we had our lab meetings at the beautiful beachside, which was a sample of a fine work-life balance.

Eli’s pioneering work on the structure-function aspects of T cell determinants and autoimmunity, with which I am most familiar, has been appreciated worldwide over the decades. Many of the terms and phenomena discovered by his lab can be found in immunology textbooks. In addition to his creative genius, he was a great mentor that a trainee could ask for, and I am very lucky in that regard. In my transition period, he encouraged me to develop a project that I could develop further in my independent position. With my background in mycobacterial infections during my graduate and postdoctoral work (with Prof. G.P. Talwar at AIIMS, New Delhi, India, and Dr. Thomas P. Gillis at GWL Hansen’s Disease, Baton Rouge, LA, respectively; my other two mentors for whom I have great respect and admiration), I picked an autoimmune disease that involved immune aspects of mycobacteria. That was the beginning of my research work in the rat adjuvant arthritis model.

Eli was very inclusive of his trainees when it came to interactions with the outside scientists, whether visiting the lab/department or when attending Workshops/Conferences locally or internationally. Besides scientific discussions, such interactions also involved participation in social and cultural activities. Through that participation, we could appreciate and enjoy Eli’s friendly and warm personality, his knowledge and taste for a variety of cuisines, and his love for music and dance. I felt so fortunate to meet and learn from many distinguished researchers from within the US and all over the world during my stay in Eli’s lab. It was a great honor for us all when Eli was recognized with the “AAI Mentoring Award, 2007.”

Eli’s home at Topanga was a place of warmth and joy, where we had many of our lab gatherings. He, his wife Rabyn and all other family members were generous and caring hosts. My family has a special memory of one such occasion. Rabyn effortlessly drew a beautiful sketch of my daughter Arushi and gave it to her; we still cherish that sketch to this day.

After settling down at UMB, I invited Eli for a seminar at UMB. He was so happy to visit my lab, to meet with my lab members and colleagues, and to enjoy an evening at the Orioles’ baseball game. Before the game, we exchanged some notes on how similar, or not, baseball is to cricket. Once the game began, Eli’s demeanor was no different from that of the little kids sitting next to us. I fondly remember that ballgame now whenever I am in the Oriole’s Park.