Bi-specific MHC Heterodimers for Characterization of Cross-reactive T Cells*

Abstract

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2K b carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify crossreactive CD8؉ T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

Vaccination and heterologous immunity: educating the immune system

“…[57][58][59][60] In a T cell repertoire, that which is common between individuals, be it TCR VA usage or a common amino acid sequence or 'motif', is a public specificity; that which is different between individuals, such as a CDR3 sequence, is a 'private' specificity. Thus, genetically identical hosts have, as a consequence of random DNA recombination events, genetically different immune systems, and this diversity of TCR usage poses a challenge when one considers whether an epitope-specific T cell response may be crossreactive with another epitope.…”

“…23,24,27,28,34,[54][55][56] Furthermore, structural studies on crossreactivity between LCMV and VACV epitopes can pinpoint the target of crossreactivity and render the OVA SIINFEKL epitope crossreactive with LCMV by an amino acid substitution. 57,58 Crossreactivity is an essential feature of TCR, and the positive selection of T cells in the thymus is mediated through self-peptides that crossreact with a substantial repertoire of foreign epitopes.…”

Identification and Validation of Targets for Cancer Immunotherapy: From the Bench-to-Bedside

“…T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen et al [74] have found that cross-reactive T cells i.e. T cells which recognise two different antigens can be identified providing an extra tool in vaccine development.…”

“…A streptavidin molecule conjugated to a fluorescent detector binds to four (tetramers) pMHCs or can used to create multimers (for example dimers, pentamers, dextramers) of these constructed MHC molecules courtesy of the biotin-avidin interaction [73]. T cell populations are added to this mixture and T cells with the specific receptor for the epitope of interest will bind and be measurable by flow cytometry [74]. Shen et al [74] have found that cross-reactive T cells i.e.…”

Heterologous Immunity and Persistent Murine Cytomegalovirus Infection

“…with 5 ϫ 10 5 PFU of MCMV or given salivary gland homogenate from naive mice as controls. Memory CD8 T cells specific for GP [33][34][35][36][37][38][39][40][41] , NP 396 -404 , and GP 118 -125 , (a to d) and memory CD4 T cells specific for GP [61][62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80] from the spleen (e) were examined at weeks 6, 12, 23, and 48 post-MCMV infection by ICS (n ϭ 5). The complete experiment was done once, but a similar observation was made in a separate experiment in cells harvested at weeks 9 and 24 post-MCMV inoculation.…”