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We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as "variants of concern," was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants.

The repertoire of human αβ T-cell receptors (TCRs) is generated via somatic recombination of germline gene segments. Despite this enormous variation, certain epitopes can be immunodominant, associated with high frequencies of antigen-specific T cells and/or exhibit bias toward a TCR gene segment. Here, we studied the TCR repertoire of the HLA-A*0201-restricted epitope LLWNGPMAV (hereafter, A2/LLW) from Yellow Fever virus, which generates an immunodominant CD8 + T cell response to the highly effective YF-17D vaccine. We discover that these A2/LLW-specific CD8 + T cells are highly biased for the TCR α chain TRAV12-2. This bias is already present in A2/LLW-specific naïve T cells before vaccination with YF-17D. Using CD8 + T cell clones, we show that TRAV12-2 does not confer a functional advantage on a per cell basis. Molecular modeling indicated that the germline-encoded complementarity determining region (CDR) 1α loop of TRAV12-2 critically contributes to A2/LLW binding, in contrast to the conventional dominant dependence on somatically rearranged CDR3 loops. This germline component of antigen recognition may explain the unusually high precursor frequency, prevalence and immunodominance of T-cell responses specific for the A2/LLW epitope.

Heat stress (HS) negatively impacts livestock performance and carcass traits while beta-adrenergic agonist (βAA) supplementation improves animal production and efficiency; both can stimulate lipolysis in adipose. The objective of this study was to understand the independent and interacting effects of HS and βAA on the subcutaneous adipose transcriptome in lambs and on visceral adipose fatty acid mobilization in steers. For study 1, 24 wethers were assigned to thermal neutral (TN THI = 65) or HS (THI = 80) conditions and supplemented without (NS) or with ractopamine hydrochloride (RH) for 30d in a 2 x 2 factorial. TN lambs were pair-fed the average intake of HS. RNA collected from subcutaneous fat at harvest was sequenced. Differential expression (DE) analyses identified 71 (Padj < 0.05) loci altered due to the interaction of environment and supplement. No DE genes were observed for the main effect of supplement, but HS reduced expression (P < 0.05) of RBM3 and increased expression (P < 0.05) of ATXN7L1. HS was predicted by pathway analyses to impair adipogenesis and fatty acid mobilization. In study 2, 24 steers were assigned to TN (THI = 65) or HS (THI = 83) and NS or zilpaterol hydrochloride (ZH) treatments for 21d in a 2 x 2 factorial. TN steers were pair-fed the intake of their HS cohort. Ex vivo fatty acid mobilization from visceral adipose in response to 0 or 1µM epinephrine was quantified at harvest. Fatty acid mobilization did not differ among groups when unstimulated, but epinephrine-stimulated mobilization was less (P = 0.05) in HS and greater (P < 0.01) in ZH steers. These findings indicate that heat stress-induced reduction of fatty acid mobilization from adipose may be associated with increased expression of stress-responsive genes. Moreover, the stimulatory effect of ZH on epinephrine-induced fatty acid mobilization was present after chronic treatment. Our data have yet to identify an interaction between HS and βAA supplementation that demonstrably impacts well-being.

The HLA‐A*02:01‐restricted decapeptide EAAGIGILTV, derived from melanoma antigen recognized by T‐cells‐1 (MART‐1) protein, represents one of the best‐studied tumor associated T‐cell epitopes, but clinical results targeting this peptide have been disappointing. This limitation may reflect the dominance of the nonapeptide, AAGIGILTV, at the melanoma cell surface. The decapeptide and nonapeptide are presented in distinct conformations by HLA‐A*02:01 and TCRs from clinically relevant T‐cell clones recognize the nonapeptide poorly. Here, we studied the MEL5 TCR that potently recognizes the nonapeptide. The structure of the MEL5‐HLA‐A*02:01‐AAGIGILTV complex revealed an induced fit mechanism of antigen recognition involving altered peptide–MHC anchoring. This “flexing” at the TCR–peptide–MHC interface to accommodate the peptide antigen explains previously observed incongruences in this well‐studied system and has important implications for future therapeutic approaches. Finally, this study expands upon the mechanisms by which molecular plasticity can influence antigen recognition by T cells.

T-cell immunity is controlled by T cell receptor (TCR) binding to peptide major histocompatibility complexes (pMHCs). The nature of the interaction between these two proteins has been the subject of many investigations because of its central role in immunity against pathogens, cancer, in autoimmunity, and during organ transplant rejection. Crystal structures comparing unbound and pMHC-bound TCRs have revealed flexibility at the interaction interface, particularly from the perspective of the TCR. However, crystal structures represent only a snapshot of protein conformation that could be influenced through biologically irrelevant crystal lattice contacts and other factors. Here, we solved the structures of three unbound TCRs from multiple crystals. Superposition of identical TCR structures from different crystals revealed some conformation differences of up to 5 Å in individual complementarity determining region (CDR) loops that are similar to those that have previously been attributed to antigen engagement. We then used a combination of rigidity analysis and simulations of protein motion to reveal the theoretical potential of TCR CDR loop flexibility in unbound state. These simulations of protein motion support the notion that crystal structures may only offer an artifactual indication of TCR flexibility, influenced by crystallization conditions and crystal packing that is inconsistent with the theoretical potential of intrinsic TCR motions.

Figure S3. Plot of -2logLikelihood values computed for 198 purported Akaushi cattle, based on the admixture model with 1Â K versus 16Â K SNP panels. (DOCX 25 kb)

Table S1. Correlations of -2logLikelihood values computed for 198 purported Akaushi cattle using the five SNP panels. Table S2. Genomic breed characterization of 198 purported Akaushi animals based on the admixture model using each of the five SNP panels. Table S3. Genomic breed characterization of 1424 purported Santa Gertrudis cattle based on the admixture model using the 1Â K SNP panel. (DOCX 251 kb)

Figure S6. Plots of -2logLikelihood for 291 Santa Gertrudis cattle after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from right to left) are Santa Gertrudis, Beef Master, Brangus, Red Angus, Angus, Hereford, Holstein, Wagyu, Akaushi, and Jersey. (DOCX 39 kb)

Figure S5. Plots of -2logLikelihood for 583 BeefMaster cattle after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from right to left) are Beef Master, Santa Gertrudis, Brangus, Hereford, Angus, Red Angus, Holstein, Jersey, Wagyu, and Akaushi. (DOCX 39 kb)

Figure S4. Plots of -2logLikelihood for 1770 reference Brangus animals (green circles) after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from left to right) are Brangus, Angus, Red Angus, Santa Gertrudis, Beef Master, Hereford, Holstein, Jersey, Wagyu, and Akaushi. (DOCX 37 kb)

Table S1. Correlations of -2logLikelihood values computed for 198 purported Akaushi cattle using the five SNP panels. Table S2. Genomic breed characterization of 198 purported Akaushi animals based on the admixture model using each of the five SNP panels. Table S3. Genomic breed characterization of 1424 purported Santa Gertrudis cattle based on the admixture model using the 1Â K SNP panel. (DOCX 251 kb)

Figure S4. Plots of -2logLikelihood for 1770 reference Brangus animals (green circles) after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from left to right) are Brangus, Angus, Red Angus, Santa Gertrudis, Beef Master, Hereford, Holstein, Jersey, Wagyu, and Akaushi. (DOCX 37 kb)

Figure S1. Density plot of genomic breed composition of 1424 animals putatively presented as Santa Gertrudis cattle. (DOCX 23 kb)

Figure S2. Map view of 1 K (A), 3 K (B), 5 K (C), 10 K (D) and 16 K (E) SNP panels, where 1 K, 3 K, 5 K and 10 L denotes 1000, 3000, 5000 and 10,000 SNP panels, respectively. The 1-10 K panels were obtained by maximizing average Euclidean distance of SNP allele frequencies among ten cattle breeds. The 16 K consisted of 15,708 common SNPs across five currently used bovine SNP chips. (DOCX 290 kb)

Figure S2. Map view of 1 K (A), 3 K (B), 5 K (C), 10 K (D) and 16 K (E) SNP panels, where 1 K, 3 K, 5 K and 10 L denotes 1000, 3000, 5000 and 10,000 SNP panels, respectively. The 1-10 K panels were obtained by maximizing average Euclidean distance of SNP allele frequencies among ten cattle breeds. The 16 K consisted of 15,708 common SNPs across five currently used bovine SNP chips. (DOCX 290 kb)

Figure S5. Plots of -2logLikelihood for 583 BeefMaster cattle after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from right to left) are Beef Master, Santa Gertrudis, Brangus, Hereford, Angus, Red Angus, Holstein, Jersey, Wagyu, and Akaushi. (DOCX 39 kb)

Figure S6. Plots of -2logLikelihood for 291 Santa Gertrudis cattle after removing outliers. The likelihood values were computed assuming the true allele frequencies of SNPs were equal to those of each of the 10 breeds, respectively. The assumed breeds (from right to left) are Santa Gertrudis, Beef Master, Brangus, Red Angus, Angus, Hereford, Holstein, Wagyu, Akaushi, and Jersey. (DOCX 39 kb)

Serial accumulation of mutations to fixation in the SLYNTVATL (SL9) immunodominant, HIV p17 Gag-derived, HLA A2-restricted CTL epitope produce the SLFNTIAVL triple mutant ‘ultimate’ escape variant. These mutations in solvent-exposed residues are believed to interfere with TCR recognition, although confirmation has awaited structural verification. Here, we solved a TCR co-complex structure with SL9 and the triple escape mutant to determine the mechanism of immune escape in this eminent system. We show that, in contrast to prevailing hypotheses, the main TCR contact residue is 4N and the dominant mechanism of escape is not via lack of TCR engagement. Instead, mutation of solvent exposed residues in the peptide destabilize the peptide-HLA and reduce peptide density at the cell surface. These results highlight the extraordinary lengths that HIV employs to evade detection by high-affinity TCRs with a broad peptide-binding footprint and necessitate reevaluation of this exemplar model of HIV TCR escape.

In order to test therapeutics, functional assessments are required. In pre-clinical stroke research, there is little consensus regarding the most appropriate behavioural tasks to assess deficits, especially when testing over extended times in milder models with short occlusion times and small lesion volumes. In this study, we comprehensively assessed 16 different behavioural tests, with the aim of identifying those that show robust, reliable and stable deficits for up to two months. These tasks are regularly used in stroke research, as well as being useful for examining striatal dysfunction in models of Huntington's and Parkinson's disease. Two cohorts of male Wistar rats underwent the intraluminal filament model of middle cerebral artery occlusion (30 min) and were imaged 24 h later. This resulted in primarily subcortical infarcts, with a small amount of cortical damage. Animals were tested, along with sham and naïve groups at 24 h, seven days, and one and two months. Following behavioural testing, brains were processed and striatal neuronal counts were performed alongside measurements of total brain and white matter atrophy. The staircase, adjusting steps, rotarod and apomorphine-induced rotations were the most reliable for assessing long-term deficits in the 30 min transient middle cerebral artery occlusion model of stroke.

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.

In order to test therapeutics, functional assessments are required. In pre-clinical stroke research, there is little consensus regarding the most appropriate behavioural tasks to assess deficits; especially when testing over extended times in milder models with short occlusion times and small lesion volumes. In this study we comprehensively assessed 16 different behavioural tests, with the aim of identifying those that show robust, reliable and stable deficits for up to 2 months. These tasks are regularly used in stroke research, as well as being useful for examining striatal dysfunction in models of Huntington’s and Parkinson’s disease. Two cohorts of male Wistar rats underwent the intraluminal filament model of MCAO (30min) and were imaged 24hrs later. This resulted in primarily subcortical infarcts, with a small amount of cortical damage. Animals were tested, along with sham and naïve groups at 24hrs, 7 days, and 1 and 2 months. Following behavioural testing, brains were processed and striatal neuronal counts were performed alongside measurements of total brain and white matter atrophy. The staircase, adjusting steps, rotarod and apomorphine induced rotations were the most reliable for assessing long-term deficits in the 30 min transient MCAO model of stroke.

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.

αβ T-cell receptors (TCRs) engage antigens using complementarity-determining region (CDR) loops that are either germ line-encoded (CDR1 and CDR2) or somatically rearranged (CDR3). TCR ligands compose a presentation platform (major histocompatibility complex (MHC)) and a variable antigenic component consisting of a short “foreign” peptide. The sequence of events when the TCR engages its peptide-MHC (pMHC) ligand remains unclear. Some studies suggest that the germ line elements of the TCR engage the MHC prior to peptide scanning, but this order of binding is difficult to reconcile with some TCR-pMHC structures. Here, we used TCRs that exhibited enhanced pMHC binding as a result of mutations in either CDR2 and/or CDR3 loops, that bound to the MHC or peptide, respectively, to dissect the roles of these loops in stabilizing TCR-pMHC interactions. Our data show that TCR-peptide interactions play a strongly dominant energetic role providing a binding mode that is both temporally and energetically complementary with a system requiring positive selection by self-pMHC in the thymus and rapid recognition of non-self-pMHC in the periphery.