TCR-signaling strength generally correlates with peptide-MHC binding affinity; however, exceptions exist. We find high-affinity, yet non-stimulatory, interactions occur with high frequency in the human T cell repertoire. Here, we studied human TCRs that are refractory to activation by pMHC ligands despite robust binding. Analysis of 3D affinity, 2D dwell time, and crystal structures of stimulatory versus non-stimulatory TCR-pMHC interactions failed to account for their different signaling outcomes. Using yeast pMHC display, we identified peptide agonists of a formerly non-responsive TCR. Single-molecule force measurements demonstrated the emergence of catch bonds in the activating TCR-pMHC interactions, correlating with exclusion of CD45 from the TCR-APC contact site. Molecular dynamics simulations of TCR-pMHC disengagement distinguished agonist from non-agonist ligands based on the acquisition of catch bonds within the TCR-pMHC interface. The isolation of catch bonds as a parameter mediating the coupling of TCR binding and signaling has important implications for TCR and antigen engineering for immunotherapy.

Summary The immune system can mount T cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of ‘orphan’ T cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A*02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens, which enables the facile identification of tumor antigens through unbiased screening.

The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K ؉ channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na ؉ wasting, consistent with the role of this channel in apical K ؉ recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K ؉ wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with ϳ25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(؉/؉), heterozygous ROMK(؉/؊), and null ROMK(؊/؊) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (؉/؉ and ؉/؊) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(؉/؉); about half that of the wild-type in ROMK(؉/؊), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(؊/؊) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K ؉ absorption/secretion in the nephron.

Chheda et al. have identified an HLA-A2–restricted CD8+ T cell epitope encompassing the H3.3K27M mutation and a corresponding TCR that specifically recognizes the H3.3K27M epitope in glioma cells. These data establish a preclinical basis for T cell–based therapy for HLA-A2+ patients with H3.3K27M+ glioma.

SUMMARY A diverse T cell receptor (TCR) repertoire is essential for controlling viral infections. However, information about TCR repertoires to defined viral antigens is limited. We performed a comprehensive analysis of CD8+ TCR repertoires for two dominant viral epitopes: pp65495–503 (NLV) of cytomegalovirus and M158–66 (GIL) of influenza A virus. The highly individualized repertoires (87–5,533 α or β clonotypes per subject) comprised thousands of unique TCRα and TCRβ sequences and dozens of distinct complementary determining region (CDR)3α and CDR3β motifs. However, diversity is effectively restricted by preferential V-J combinations, CDR3 lengths, and CDR3α/CDR3β pairings. Structures of two GIL-specific TCRs bound to GIL–HLA-A2 provided a potential explanation for the lower diversity of GIL-specific versus NLV-specific repertoires. These anti-viral TCRs occupied up to 3.4% of the CD8+ TCRβ repertoire, ensuring broad T cell responses to single epitopes. Our portrait of two anti-viral TCR repertoires may inform the development of predictors of immune protection.