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Parsing digital or analogue TCR performance through piconewton forces

ABSTRACT: T lymphocytes leverage T-cell receptor (TCR) recognition of aberrant peptides bound to major histocompatibility complex molecules (pMHCs) on altered cells to protect the mammalian host from infectious pathogens and cancerous transformations. While adaptive immunity requires efficient TCR performance, comparison metrics are widely limited to force-free assays. Recent data reveals, however, that mechanical load on the TCR-pMHC bond resulting from cellular motions between a T cell-antigen presenting cell conjugate formed during immune surveillance tunes TCR specificity and sensitivity. Here, we cloned TCRs from lung resident CD8 T cells following influenza A virus (IAV) infection directed against two immunodominant epitopes, the luminous nucleoprotein NP366-374 /Db and sparse acid polymerase PA224-233/Db pMHCs, arrayed at >1000 vs <10 copies/cell, respectively. Using optical tweezers-based methods to apply biologically relevant loads, we observe that certain TCRs belonging to the NP repertoire require many ligands for activation while others only few, functioning in analogue or digital modes, correspondingly. In contrast, prevalent TCRs tested in the PA repertoire all perform digitally. When held at a critical force, moreover, even digital TCRs are distinguishable, exhibiting dramatic increases in bond lifetime and performance relating to their structural transition rate and distance. Digital performance is best for both specificities in vitro and in vivo, but undiscernible using conventional functional avidity or TCR sequence distance metrics. Correlates of optimal biophysical parameters include magnitudes of ERK phosphorylation, CD3 surface loss, and activation marker upregulation. Our findings underscore the requirement to match TCR performance quality with pMHC copy number, as nature does. Given tumor antigen array paucity, engaging choice digital TCRs appears critical for T-cell adoptive cancer therapies and vaccines.

ORGANISM(S): Mus musculus

PROVIDER: GSE240414 | GEO | 2024/08/21

REPOSITORIES: GEO

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Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRÎ±/Î²-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRÎ±/Î²-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.

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Parsing digital or analogue TCR performance through piconewton forces

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