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The nonclassical HLA molecule MHC-related protein 1 (MR1) presents metabolites of the vitamin B synthesis pathways to mucosal-associated invariant T (MAIT) cells and other MR1-restricted T cells. This new class of Ags represents a variation on the classical paradigm of self/non-self discrimination because these T cells are activated through their TCR by small organic compounds generated during microbial vitamin B2 synthesis. Beyond the fundamental significance, the invariant nature of MR1 across the human population is a tantalizing feature for the potential development of universal immune therapeutic and diagnostic tools. However, many aspects of MR1 Ag presentation and MR1-restricted T cell biology remain unknown, and the ubiquitous expression of MR1 across tissues and cell lines can be a confounding factor for experimental purposes. In this study, we report the development of a novel CRISPR/Cas9 genome editing lentiviral system and its use to efficiently disrupt MR1 expression in A459, THP-1, and K562 cell lines. We generated isogenic MR1(-/-) clonal derivatives of the A549 lung carcinoma and THP-1 monocytic cell lines and used these to study T cell responses to intracellular pathogens. We confirmed that MAIT cell clones were unable to respond to MR1(-/-) clones infected with bacteria whereas Ag presentation by classical and other nonclassical HLAs was unaffected. This system represents a robust and efficient method to disrupt the expression of MR1 and should facilitate investigations into the processing and presentation of MR1 Ags as well as into the biology of MAIT cells.

T-cell cross-reactivity is essential for effective immune surveillance, but has also been implicated as a pathway to autoimmunity. Previous studies have demonstrated that T-cell receptors (TCRs) that focus on a minimal motif within the peptide are able to facilitate a high level of T-cell cross-reactivity. However, the structural database shows that most TCRs exhibit less focussed antigen binding involving contact with more peptide residues. To further explore the structural features that allow the clonally expressed TCR to functionally engage with multiple peptide-major histocompatibility complexes (pMHCs), we examined the ILA1 CD8+ T-cell clone that responds to a peptide sequence derived from human telomerase reverse transcriptase (hTERT). The ILA1 TCR contacted its pMHC with a broad peptide-binding footprint encompassing spatially distant peptide residues. Despite the lack of focused TCR-peptide binding , the ILA1 T-cell clone was still cross-reactive. Overall, the TCR-peptide contacts apparent in the structure correlated well with the level of degeneracy at different peptide positions. Thus, the ILA1 TCR was less tolerant of changes at peptide residues that were at, or adjacent to, key contact sites. This study provides new insights into the molecular mechanisms that control T-cell cross-reactivity, with important implications for pathogen surveillance, autoimmunity and transplant rejection.

Human CD8+ cytotoxic T lymphocytes can mediate tumor regression in melanoma through the specific recognition of HLA-restricted peptides. Because of the relatively weak affinity of most anti-cancer T-cell receptors (TCRs), there is growing emphasis on immunizing melanoma patients with altered peptide ligands in order to induce strong anti-tumor immunity capable of breaking tolerance toward these self-antigens. However, previous studies have shown that these immunogenic designer peptides are not always effective. The melanocyte differentiation protein, glycoprotein 100 (gp100), encodes a naturally processed epitope that is an attractive target for melanoma immunotherapies, in particular peptide-based vaccines. Previous studies have shown that substitutions at peptide residue Glu3 have a broad negative impact on polyclonal T-cell responses. Here, we describe the first atomic structure of a natural cognate TCR in complex with this gp100 epitope and highlight the relatively high affinity of the interaction. Alanine scan mutagenesis performed across the gp100280–288 peptide showed that Glu3 was critically important for TCR binding. Unexpectedly, structural analysis demonstrated that the Glu3 → Ala substitution resulted in a molecular switch that was transmitted to adjacent residues, abrogating TCR binding and T-cell recognition. These findings help to clarify the mechanism of T-cell recognition of gp100 during melanoma responses and could direct the development of altered peptides for vaccination.

Isolation of peptide-specific T-cell clones is highly desirable for determining the role of T-cells in human disease, as well as for the development of therapies and diagnostics. However, generation of monoclonal T-cells with the required specificity is challenging and time-consuming. Here we describe a library-based strategy for the simple parallel detection and isolation of multiple peptide-specific human T-cell clones from CD8+ or CD4+ polyclonal T-cell populations. T-cells were first amplified by CD3/CD28 microbeads in a 96U-well library format, prior to screening for desired peptide recognition. T-cells from peptide-reactive wells were then subjected to cytokine-mediated enrichment followed by single-cell cloning, with the entire process from sample to validated clone taking as little as 6weeks. Overall, T-cell libraries represent an efficient and relatively rapid tool for the generation of peptide-specific T-cell clones, with applications shown here in infectious disease (Epstein–Barr virus, influenza A, and Ebola virus), autoimmunity (type 1 diabetes) and cancer.

Recent years have witnessed a rapid proliferation of gene editing in mammalian cells due to the increasing ease and reduced cost of targeted gene knockout. There has been much excitement about the prospect of engineering T-cells by gene editing in order to provide these cells with optimal attributes prior to adoptive cell therapy for cancer and autoimmune disease. I began by attempting to compare short hairpin RNA (shRNA) and zinc finger nuclease (ZFN) approaches using the CD8A gene as a target for proof of concept of gene editing in Molt3 cells. During the course of my studies the clustered regularly interspaced short palindromic repeats (CRISPR) mechanism for gene editing was discovered so I also included CRISPR/Cas9 in my studies. A direct comparison of the three gene editing tools indicated that the CRISPR/Cas9 system was superior in terms of ease, efficiency of knockout and cost.\ud As the use of gene editing tools increases there are concerns about the inherent risks associated with the use of nuclease based gene editing tools prior to cellular therapy. Expression of nucleases can lead to off target mutagenesis and malignancy. To circumvent this problem I generated a non-nuclease based gene silencing system using the CD8A zinc finger (ZF) fused to a Krüppel associated box (KRAB) repressor domain. The ZF-KRAB fusion resulted in effective silencing of the CD8A gene in both the Molt3 cell line and in primary CD8+ T-cells. Importantly, unlike CRISPR/Cas9 based gene editing, the ZF-KRAB fusion was small enough to be transferred in a single lentiviral vector with a TCR allowing simultaneous redirection of patient T-cell specificity and alteration of T-cell function in a single construct.\ud To improve the efficiency of gene editing with CRISPR/Cas9 I developed an ‘all in one’ CRISPR/Cas9 system which incorporated all elements of the CRISPR/Cas9 gene editing system in a single plasmid. The ‘all in one’ system was utilised to derive MHC-related protein 1 (MR1) deficient clones from the A549 lung carcinoma and THP-1 monocytic cell lines in order to study MR1 biology. Mucosal-associated invariant T-cell (MAIT) clones were not activated by MR1 deficient A549 or THP-1 clones infected with bacteria.