362 Allelic variants of MR1 drive cancer and allo-reactivity by MR1-restricted T cells

Background Major histocompatibility complex class-1-related protein (MR1), unlike human leucocyte antigen (HLA) class-1, was until recently considered to be essentially monomorphic. MR1 presents metabolites in the context of host responses to bacterial infection. MR1-restricted TCRs with specificity for tumor cells have been described, raising interest in their potential therapeutic application for cancer treatment.1 2 The diversity of MR1-ligand biology has broadened with the observation that single nucleotide variants (SNVs) exist within MR1 and that allelic variants can impact host immunity.3 4 Methods The MR1-restricted T cell clone, MC.7.G5 (7G5.TCRT), with reported cancer specificity and pan-cancer activity,1 was cloned and expressed via lentiviral transduction in Jurkat E6.1 TCRαβ- β2M- CD8+ NF-κB:CFP NFAT:eGFP AP-1:mCherry cells or in human donor T cells (with or without CD8 or after knockout of the TRBC1 and 2 genes with CRISPR Cas9). Other reported cancer reactive TCRs were expressed as TCRT for comparison. Functional activity of 7G5.TCRT was demonstrated by cytotoxicity utilizing the xCELLigence analyzer and by ELISA for cytokine release after co-culture with cancer cell lines with or without loading of known MR1 ligands. MR1 allele sequencing was undertaken after amplification of the MR1 gene region by PCR. In vivo studies were undertaken at Labcorp Drug Development (Ann Arbor, MI) or Epistem Ltd (Manchester, UK). Results The TCR cloned from MC.7.G5 retains MR1 restricted functional cytotoxicity as 7G5.TCRT. However, activity is not pan-cancer, as initially reported with the clone MC.7.G5.1 Recognition is restricted to cells expressing a SNV of MR1 (MR1*04) and is not cancer specific. 7G5.TCRT and 7G5-like TCRT cells react to both cancer and healthy cells endogenously expressing MR1*04 SNVs, which encode R9H and H17R substitutions. This allelic specificity can be overcome by expressing supraphysiologic levels of the wild type MR1 (MR1*01) in cell lines. Conclusions These data demonstrate that healthy individuals can harbor T cells reactive to MR1 variants displaying self-ligands expressed in cancer and benign tissues. Described ‘cancer specific’ MR1-restricted TCRs therefore need further validation, covering conserved allelic variants of MR1. Ligands require identification to ensure targeting MR1 is restricted to expressed ligand(s) derived from metabolic aberrations in cancer and not normal tissues. For the wider field of immunology and transplant biology the observation that MR1*04 may behave as an alloantigen warrants further study. Acknowledgements Professor Andrew Sewell and Dr. Garry Dolton, University of Cardiff, Division of Infection and Immunity, Systems Immunity Research Institute. Professor David Lewinsohn, M.D., Ph.D., Division of Pulmonary and Critical Care Medicine, School of Medicine. Molecular Microbiology and Immunology Graduate Program, School of Medicine. Program in Molecular and Cellular Biosciences, Oregon Health, and Science University School of Medicine. References 1. Crowther MD, et al . Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nat. Immunol. 2020 Feb; 21 (2):178–185. 2. Seneviratna R, et al . Differential antigenic requirements by diverse MR1-restricted T cells. Immunol. Cell. Biol. 2022 Feb; 100 (2):112–126. 3. Rozemuller E, et al . MR1 encompasses at least six allele groups with coding region alterations. HLA 2021 Dec; 98 (6):509–5164. 4. Howson LJ, et al . Absence of mucosal-associated invariant T cells in a person with a homozygous point mutation in MR1. Sci. Immunol. 2020 Jul 24; 5 (49):eabc9492. Ethics Approval Ethical approval for work using human blood derived cells was received from NHS London-Bromley Research Ethics Committee REC reference 22/PR/1176. Commercial blood sources were supplied with informed consent and with certification from local research ethics committees.