Flexibility in MHC and TCR Recognition: Degenerate Specificity at the T Cell Level in the Recognition of Promiscuous Th Epitopes Exhibiting No Primary Sequence Homology Sunil K. Joshi, 1 Padma R. Suresh, and Virander S. Chauhan 2 Recognition of peptide Ags by T cells through the TCR can be highly specific. In this report we show the degeneracy of Ag recognition at both MHC and TCR levels. We present evidence that unrelated promiscuous Th cell epitopes from various protein sources exhibit sufficient structural homology, despite minimal structural identity, to elicit cross-reactive proliferative responses at the bulk T cell level. This epitopic mimicry was also observed when peptide (CS.T3 378 –395 and TT 830 – 844 )-specific CD4 1 T cell lines and T cell hybridoma clones were used in proliferation and Ag presentation assays. A scrambled CS.T3 378 –395 peptide did not show any proliferation, indicating that the specificity of the cross-reactive responses may be linked with the primary structure of the peptides. Blocking of CS.T3 378 –395 -specific CD4 1 T cell proliferation by anti-MHC class II mAb showed that recognition of promiscuous T cell epitopes is largely in association with MHC class II molecules. These findings suggest that promiscuous Th epitopes may be useful in designing peptide-based vaccine constructs. At the same time these results show that at the T cell level there may be a great deal of immunological cross-reactivity between heterologous pathogens, and because of this the host’s response to a pathogen may be modified by its previous experience with other unrelated pathogens. The Journal of Immunology, 2001, 166: 6693– 6703. O ne of the defining features of T cell activation is a high level of Ag specificity (reviewed in Ref. 1). However, several recent observations reveal that the TCR is highly flexible in its recognition, as it can recognize large number of different peptides that do not necessarily show strong sequence homology (2–12). It has become clear that a high level of degen- eracy in TCR recognition is necessary to produce an adequate TCR repertoire capable of responding to the universe of pathogens as well as for the survival of T cells (reviewed in Ref. 13). The flex- ibility of the TCR recognition can manifest itself in a range of different biological outcomes depending on the affinity of MHC/ peptide ligand to TCR, which includes antagonist and agonist ef- fects. The structure of the MHC class II molecules allows the peptide to protrude and therefore accommodate longer peptides (15- to 23-aa residues) than MHC class I molecules that bind shorter peptides, mostly ranging from 9- to 12-aa residues. While the majority of antigenic peptides are thought to be recognized in the context of only one or a few MHC class II alleles, genetically permissive recognition in the context of several class II alleles has been reported in both murine and human systems (14 –24). Al- though the molecular basis for peptide-related cross-reactivity as well as promiscuity is not yet clear, it is quite obvious that uni- versal Th epitopes should have in their sequences structural fea- tures that allow them to interact with different MHC class II mol- ecules and further, through the MHC/peptide complex, also enable them to interact with the TCR. As part of our interest in the use of universal Th epitopes in the design of peptide-based immunogens we have asked whether the peptide that can promiscuously bind various MHC class II molecules could also produce cross-reactive T cell responses? In this report we have analyzed degeneracy at the T cell level in the context of recognition of universal Th epitopes from diverse sources such as a viral, a bacterial, and a protozoan parasite protein (14 –18) with no apparent sequence homology. At the bulk T cell level, three unrelated promiscuous T cell epitopic sequences were able to induce T cell responses in the context of multiple MHC class II molecules. Furthermore, a panel of T cell hybrids expressing a limited repertoire of TCR Vb was also able to manifest a high level of flexibility in TCR recognition. How- ever, the recognition of “promiscuous T cells” was highly specific, as none of the hybridomas was able to recognize the MHC class II-restricted (I-A d /I-E d ) peptide SWM 106 –118 . Our findings clearly demonstrate a high level of TCR plasticity, which somehow ap- pears to be restricted to the recognition of universal MHC binders. Our findings imply that unsuspected cross-reactivities may play an important role in the generation of T cell memory, the patho- genesis of autoimmune diseases, and possibly in a wide range of postimmune responses to infectious pathogens. The ability of structurally dissimilar promiscuous peptides to mimic each other when bound to class II MHC molecules may also be important in understanding the development of the TCR repertoire during thy- mic selection. Materials and Methods Experimental animals Six- to 8-wk-old female mice of various strains, viz., BALB/c (I-A d I-E d ), DBA/2 (I-A d I-E d ), C57BL/6 (I-A b I-E o ), and C3H (I-A k I-E k ) were pur- chased from the small animal facility, National Institute of Immunology Malaria Research Group, International Center for Genetic Engineering and Biotech- nology, Aruna Asaf Ali Marg, New Delhi, India Received for publication August 23, 2000. Accepted for publication March 22, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Current address: Program in Molecular Immunology, Institute of Molecular Medi- cine and Genetics, Medical College of Georgia, Augusta, GA 30912. 2 Address correspondence and reprint requests to Prof. Virander S. Chauhan, Inter- national Center for Genetic Engineering and Biotechnology, P.O. Box 10504, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail address: virander@icgeb.res.in Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00