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T cells play a crucial role in clearing SARS-CoV-2 and in forming long-term memory responses to that coronavirus. The highly immunogenic nucleocapsid (N) protein of SARS-CoV-2 is much more conserved than the spike (S) protein across variants of concern, making it an attractive vaccine target for activating cytotoxic CD8 + T cells. Of particular interest are the immunodominant N epitopes LLL and SPR. Whereas LLL elicits a clonally restricted T cell response, the response to SPR is highly diverse. To understand the basis for this difference, here we determine structures of T cell receptors (TCRs) bound to LLL–HLA-A2 and SPR–HLA-B7, revealing the structural underpinnings of highly restricted Vα gene usage by LLL-specific TCRs, as well as multiple structural solutions to recognizing SPR and thereby generating a clonally diverse T cell response to that epitope. These structures also provide frameworks for understanding T cell recognition of SARS-CoV-2 variants and other coronaviruses. Finally, we compare the X-ray structures of TCR–LLL–HLA-A2 and TCR–SPR–HLA-B7 complexes with models predicted by multiple versions of AlphaFold, highlighting some success while showing room for improvement. Overall, our findings expand understanding of coronavirus T cell recognition, informing vaccine design and advances in computational modeling approaches. Previous structural studies of T cell recognition of SARS-CoV-2 have been confined to spike epitopes. Here the authors assess T cell recognition of SARS-CoV-2 nucleocapsid epitopes, which are more conserved than spike epitopes, providing structural insights into recognition of two epitopes.

Human leukocyte antigen (HLA)-I molecules generally bind short peptides (8–10 amino acids), although extended HLA-I restricted peptides (>10 amino acids) can be presented to T cells. However, the function of such extended HLA-I epitopes in tumour immunity, and how they would be recognised by T-cell receptors (TCR) remains unclear. Here we show that the structures of two distinct TCRs (TRAV4 + TRAJ21 + -TRBV28 + TRBJ2-3 + and TRAV4 + TRAJ8 + -TRBV9 + TRBJ2 - 1 + ), originating from a polyclonal T-cell repertoire, bind to HLA-B*07:02, presenting a 13-amino-acid-long tumour-associated peptide, NY-ESO-1 60–72 . Comparison of the structures reveals that the two TCRs differentially binds NY-ESO-1 60–72 –HLA-B*07:02 complex, and induces differing extent of conformational change of the NY-ESO-1 60–72 epitope. Accordingly, polyclonal TCR usage towards an extended HLA-I restricted tumour epitope translates to differing TCR recognition modes, whereby extensive flexibility at the TCR–pHLA-I interface engenders recognition. Human leukocyte antigen (HLA) presents peptides to activate T cells, but many aspects in the T cell receptor (TCR)/HLA interaction remain unclear. Here the authors show, via structural data, that two TCRs differentially recognize the same tumour peptide/HLA complex and induce contrasting conformation changes of the peptide.

Purpose Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses. Experimental design This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide ® ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy. Results In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event. Conclusions Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.

Ubiquitination controls a broad range of cellular functions. The last step of the ubiquitination pathway is regulated by enzyme type 3 (E3) ubiquitin ligases. E3 enzymes are responsible for substrate specificity and catalyze the formation of an isopeptide bond between a lysine residue of the substrate (or the N terminus of the substrate) and ubiquitin. MIR1 and MIR2 are two E3 ubiquitin ligases encoded by Kaposi's sarcoma-associated herpesvirus that mediate the ubiquitination of major histocompatibility complex class I (MHC I) molecules and subsequent internalization. Here, we found that MIR1, but not MIR2, promoted down-regulation of MHC I molecules lacking lysine residues in their intracytoplasmic domain. In the presence of MIR1, these MHC I molecules were ubiquitinated, and their association with ubiquitin was sensitive to {szligbeta}₂-mercaptoethanol, unlike lysine-ubiquitin bonds. This form of ubiquitination required a cysteine residue in the intracytoplasmic tail of MHC I molecules. An MHC I molecule containing a single cysteine residue in an artificial glycine and alanine intracytoplasmic domain was endocytosed and degraded in the presence of MIR1. Thus, ubiquitination can occur on proteins lacking accessible lysines or an accessible N terminus.

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses. Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of-B7 (P = 0.002). Transfection experiments with full-length HLA-A2 and -B8 encoding plasmids confirmed this (54,031 molecules per cell vs. 2,466, respectively, P = 0.001) independently of transcript levels suggesting a post-transcriptional regulation. Using chimeric constructs we found that the cytoplasmic tail and the transmembrane region had no impact on the differential cell surface expression. In contrast, ~65% of the difference could be mapped to the six C-terminal amino acids of the alpha 2 domain and the alpha 3 domain (amino acids 176-284), i.e. amino acids not previously shown to be of importance for differential expression levels of HLA class I molecules. We suggest that the differential cell surface expression of two common HLA-A and-B alleles is regulated by a post-translational mechanism that may involve hitherto unrecognized molecules.

Frontal fibrosing alopecia (FFA) is a recently described inflammatory and scarring type of hair loss affecting almost exclusively women. Despite a dramatic recent increase in incidence the aetiopathogenesis of FFA remains unknown. We undertake genome-wide association studies in females from a UK cohort, comprising 844 cases and 3,760 controls, a Spanish cohort of 172 cases and 385 controls, and perform statistical meta-analysis. We observe genome-wide significant association with FFA at four genomic loci: 2p22.2, 6p21.1, 8q24.22 and 15q2.1. Within the 6p21.1 locus, fine-mapping indicates that the association is driven by the HLA-B*07: 02 allele. At 2p22.1, we implicate a putative causal missense variant in CYP1B1 , encoding the homonymous xenobiotic- and hormone-processing enzyme. Transcriptomic analysis of affected scalp tissue highlights overrepresentation of transcripts encoding components of innate and adaptive immune response pathways. These findings provide insight into disease pathogenesis and characterise FFA as a genetically predisposed immuno-inflammatory disorder driven by HLA-B*07: 02. Frontal fibrosing alopecia (FFA) features lichenoid cutaneous inflammation and scarring hair loss. Here, Tziotzios et al. identify four genetic loci associated with FFA by GWAS followed by Bayesian fine-mapping, co-localisation and HLA imputation which highlights HLA-B*07:02 as a risk factor.

Vaccination with vaccinia virus (VACV) elicits heterotypic immunity to smallpox, monkeypox, and mousepox, the mechanistic basis for which is poorly understood. It is generally assumed that heterotypic immunity arises from the presentation of a wide array of VACV-derived, CD8 + T cell epitopes that share homology with other poxviruses. Herein this assumption was tested using a large panel of VACV-derived peptides presented by HLA-B*07:02 (B7.2) molecules in a mousepox/ectromelia virus (ECTV)-infection, B7.2 transgenic mouse model. Most dominant epitopes recognized by ECTV- and VACV-reactive CD8 + T cells overlapped significantly without altering immunodominance hierarchy. Further, several epitopes recognized by ECTV-reactive CD8 + T cells were not recognized by VACV-reactive CD8 + T cells, and vice versa. In one instance, the lack of recognition owed to a N72K variation in the ECTV C4R 70–78 variant of the dominant VACV B8R 70–78 epitope. C4R 70–78 does not bind to B7.2 and, hence, it was neither immunogenic nor antigenic. These findings provide a mechanistic basis for VACV vaccination-induced heterotypic immunity which can protect against Variola and Monkeypox disease. The understanding of how cross-reactive responses develop is essential for the rational design of a subunit-based vaccine that would be safe, and effectively protect against heterologous infection.

ObjectiveAssociation of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.MethodsFlow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B*7/HLA-B*51 P97 mutants was also studied.ResultsMutation of HLA-B*27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.ConclusionsThe nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B*27 FHC cell surface expression.

Objectives Vaccine‐induced protective immunity against SARS‐CoV‐2 has proved difficult to sustain. Robust T‐cell responses are thought to play an important role, but T‐cell responses against the SARS‐CoV‐2 spike protein (S‐protein), the core vaccine antigen, following vaccination or natural infection are incompletely understood. Methods Herein, the reactivity of 170 putative SARS‐CoV‐2 S‐protein CD8+ and CD4+ T‐cell peptide epitopes in the same individuals prior to vaccination, after COVID‐19 vaccination, and again following subsequent natural infection was assayed using a high‐throughput reverse transcription‐quantitative PCR (HTS‐RT‐qPCR) assay. Results The profile of immunoreactive SARS‐CoV‐2 S‐protein epitopes differed between vaccination and natural infection. Vaccine‐induced immunoreactive epitopes were localised primarily into two extra‐domanial regions. In contrast, epitopes recognised following natural infection were spread across the antigen. Furthermore, T‐cell epitopes in naïve individuals were primarily recognised in association with HLA‐A, while natural infection shifted epitope associations towards HLA‐B, particularly the B7 supertype. Conclusion This study provides insight into T‐cell responses against the SARS‐CoV‐2 S‐protein following vaccination and subsequent natural infection. We investigated the immunoreactivity of SARS‐CoV‐2 spike protein T‐cell epitopes in individuals who were SARS‐CoV‐2 naive, vaccinated with the AstraZeneca vaccine, and those who became naturally infected after vaccination. While the number of immunoreactive epitopes remained consistent across all groups, we observed a shift in T‐cell responses: naive individuals predominantly recognised HLA‐A‐associated epitopes, vaccination clustered immunoreactive epitopes within two specific regions of the spike protein, and natural infection shifted immunoreactivity towards HLA‐B‐associated epitopes, particularly the B7 supertype. These findings suggest that vaccination and natural infection elicit different T‐cell responses, providing insights that could inform future vaccine design to enhance long‐term immunity against COVID‐19.

T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA- B*07:02 -(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo . In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.