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Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

The >30 mutated residues in the Omicron spike protein have led to its rapid classification as a new SARS-CoV-2 variant of concern. As a result, Omicron may escape from the immune system, decreasing the protection provided by COVID-19 vaccines. Preliminary data shows a weaker neutralizing antibody response to Omicron compared to the ancestral SARS-CoV-2 virus, which can be increased after a booster vaccine. Here, we report that CD8+ T cells can recognize Omicron variant epitopes presented by HLA-A*02:01 in both COVID-19 recovered and vaccinated individuals, even 6 months after infection or vaccination. Additionally, the T cell response was stronger for Omicron variant epitopes after the vaccine booster. Altogether, T cells can recognize Omicron variants, especially in vaccinated individuals after the vaccine booster.One-Sentence SummaryCD8+ T cells response against Omicron variant epitopes is stronger after the vaccine booster.

CD8+ T cells are crucial for anti-viral immunity; however, understanding T cell responses requires the identification of epitopes presented by human leukocyte antigens (HLA). To date, few SARS-CoV-2-specific CD8+ T cell epitopes have been described. Internal viral proteins are typically more conserved than surface proteins and are often the target of CD8+ T cells. Therefore, we have characterized eight peptides derived from the internal SARS-CoV-2 nucleocapsid protein predicted to bind HLA-A∗02:01, the most common HLA molecule in the global population. We determined not all peptides could form a complex with HLA-A∗02:01, and the six crystal structures determined revealed that some peptides adopted a mobile conformation. We therefore provide a molecular understanding of SARS-CoV-2 CD8+ T cell epitopes. Furthermore, we show that there is limited pre-existing CD8+ T cell response toward these epitopes in unexposed individuals. Together, these data show that SARS-CoV-2 nucleocapsid might not contain potent epitopes restricted to HLA-A∗02:01., Graphical abstract, Immunology; structural biology; virology

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has caused significant morbidity and mortality worldwide. With the remarkable advances in medical research, vaccines were developed to prime the human immune system and decrease disease severity. Despite these achievements, the fundamental basis of immunity to the SARS-CoV-2 virus is still largely undefined. Here, we solved the crystal structure of three spike-derived peptides presented by three different HLA molecules, and determined the stability of the overall peptide–HLA complexes formed. The peptide presentation of spike-derived peptides can influence the way in which CD8+ T cells can recognise infected cells, clear infection, and therefore, control the outcome of the disease.

Significant efforts are being made worldwide to understand the immune response to SARS-CoV-2, responsible for the COVID-19 pandemic, including the role of pre-existing T cell immunity. Understanding the mechanisms that promote cross-recognition by T cells induced by seasonal coronaviruses will be critical for future predictions on the role of pre-existing immunity in protection against severe disease. We demonstrate that the SARS-CoV-2 nucleocapsid (N) protein induces an immunodominant response in HLA-B7+ COVID-19-recovered individuals that is also readily detectable in unexposed donors. This immunodominant response is driven by a single N-encoded epitope that displays a high degree of conservation with the homologous region in circulating coronaviruses. We show that T cell-mediated cross-reactivity can be detected towards the circulating OC43/HKU-1 coronaviruses, but not the 229E or NL63 coronaviruses, due to different peptide conformations. This cross-reactivity is driven by private T cell receptor repertoires with a bias for TRBV27 and a long CDR3b loop in unexposed and COVID-19-recovered individuals. Together, our findings demonstrate the basis of pre-existing immunity to a conserved and highly immunogenic SARS-CoV-2 epitope driven by cross-reactive memory T cells, suggesting long-lived protective immunity. Funding: This work was supported by generous donations from the QIMR Berghofer COVID 19 appeal, and financial contributions from Monash University, Australian Nuclear Science and Technology Organisation (ANSTO, AISNE ECR grants), Australian Research Council (ARC), National Health and Medical Research Council (NHMRC), and the Medical Research Future Fund (MRFF). H.S. is supported by an Australian Government Research Training Program Scholarship, E.J.G. was supported by an NHMRC CJ Martin Fellowship (#1110429) and is supported by an Australian Research Council DECRA (DE210101479), K.R.S.is supported by an Australian Research Council DECRA (DE180100512), S.G. is supported by and NHMRC SRF (#1159272). Conflict of Interest: The authors declare no competing interests. Ethical Approval: This study was performed according to the principles of the Declaration of Helsinki.Ethics approval to undertake the research was obtained from the QIMR Berghofer Medical Research Institute Human Research Ethics Committee and Monash University Human Research Ethics Committee.

The data currently available on how the immune system recognises the SARS-CoV-2 virus is growing rapidly. While there are structures of some SARS-CoV-2 proteins in complex with antibodies, which helps us understand how the immune system is able to recognise this new virus, however, we lack data on how T cells are able to recognise this virus. T cells, especially the cytotoxic CD8+ T cells, are critical for viral recognition and clearance. Here we report the X-ray crystallography structure of a T cell receptor, shared among unrelated individuals (public TCR) in complex with a dominant spike-derived CD8+ T cell epitope (YLQ peptide). We show that YLQ activates a polyfunctional CD8+ T cell response in COVID-19 recovered patients. We detail the molecular basis for the shared TCR gene usage observed in HLA-A*02:01+ individuals, providing an understanding of TCR recognition towards a SARS-CoV-2 epitope. Interestingly, the YLQ peptide conformation did not change upon TCR binding, facilitating the high-affinity interaction observed.

Small cell lung cancer (SCLC) is an aggressive neuroendocrine cancer characterized by loss of function TP53 and RB1 mutations in addition to mutations in other oncogenes including MYC. Overexpression of MYC together with Trp53 and Rb1 loss in pulmonary neuroendocrine cells of the mouse lung drives an aggressive neuroendocrine low variant subtype of SCLC. However, the transforming potential of MYC amplification alone on airway epithelium is unclear. Therefore, we selectively and conditionally overexpressed MYC stochastically throughout the airway or specifically in neuroendocrine, club, or alveolar type II cells in the adult mouse lung. We observed that MYC overexpression induced carcinoma in situ which did not progress to invasive disease. The formation of adenoma or SCLC carcinoma in situ was dependent on the cell of origin. In contrast, MYC overexpression combined with conditional deletion of both Trp53 and Rb1 exclusively gave rise to SCLC, irrespective of the cell lineage of origin. However, cell of origin influenced disease latency, metastatic potential, and the transcriptional profile of the SCLC phenotype. Together this reveals that MYC overexpression alone provides a proliferative advantage but when combined with deletion of Trp53 and Rb1 it facilitates the formation of aggressive SCLC from multiple cell lineages.

Understanding T cell responses requires identifying viral peptides presented by Human Leukocyte Antigens (HLA). X-ray crystallography can be used to visualize their presentation. This protocol describes the expression, purification, and crystallization of HLA-A*02:01, one of the most frequent HLA in the global population in complex with peptides derived from the SARS-CoV-2 Nucleocapsid protein. This protocol can be applied to different HLA-class I molecules bound to other peptides., Graphical Abstract

Efforts are being made worldwide to understand the immune response to SARS-CoV-2, the virus responsible for the COVID-19 pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening SARS-CoV-2 peptide pools revealed that the nucleocapsid (N) protein induced an immunodominant response in HLA-B7+ COVID-19-recovered individuals that was also detectable in unexposed donors. A single N-encoded epitope that was highly conserved across circulating coronaviruses drove this immunodominant response. In vitro peptide stimulation and crystal structure analyses revealed T cell-mediated cross-reactivity towards circulating OC43 and HKU-1 beta coronaviruses, but not 229E or NL63 alpha coronaviruses, due to different peptide conformations. TCR sequencing indicated cross-reactivity was driven by private T cell receptor repertoires with a bias for TRBV27 and a long CDR3β loop. Together, our findings demonstrate the basis of selective T cell cross-reactivity towards an immunodominant SARS-CoV-2 epitope and its homologues from seasonal coronaviruses, suggesting long-lived protective immunity., Graphical Abstract, The impact of seasonal coronaviruses on immune responses to SARS-CoV-2 is an active area of research. Here, Lineburg et al identify CD8+ T cells specific for a conserved and immunodominant SARS-CoV-2 epitope in HLA-B7+ individuals. Furthermore, SARS-CoV-2 epitope-specific CD8+ T cells display cross-reactivity to beta but not alpha coronaviruses due to distinct peptide–HLA conformations.

The interaction between T cell receptor (TCR) and peptide (p)-Human Leukocyte Antigen (HLA) complexes is the critical first step in determining T cell responses. X-ray crystallographic studies of pHLA in TCR-bound and free states provide a structural perspective that can help understand T cell activation. These structures represent a static &ldquo, snapshot&rdquo, yet the nature of pHLAs and their interactions with TCRs are highly dynamic. This has been demonstrated for HLA class I molecules with in silico techniques showing that some interactions, thought to stabilise pHLA-I, are only transient and prone to high flexibility. Here, we investigated the dynamics of HLA class II molecules by focusing on three allomorphs (HLA-DR1, -DR11 and -DR15) that are able to present the same epitope and activate CD4+ T cells. A single TCR (F24) has been shown to recognise all three HLA-DR molecules, albeit with different affinities. Using molecular dynamics and crystallographic ensemble refinement, we investigate the molecular basis of these different affinities and uncover hidden roles for HLA polymorphic residues. These polymorphisms were responsible for the widening of the antigen binding cleft and disruption of pHLA-TCR interactions, underpinning the hierarchy of F24 TCR binding affinity, and ultimately T cell activation. We expanded this approach to all available pHLA-DR structures and discovered that all HLA-DR molecules were inherently rigid. Together with in vitro protein stability and peptide affinity measurements, our results suggest that HLA-DR1 possesses inherently high protein stability, and low HLA-DM susceptibility.

In 2018 the World Health Organisation listed influenza pandemics as one of the top threats to global health. Influenza pandemics occur as a result of the emergence of antigenically distinct strains, which arise due to genomic re-assortment or through the acquisition of mutations. The resulting strains have different antigenic make-ups and implicate the ability of the immune system to respond quickly following infection. Particular HLA class II allomorphs including HLA-DRB1*07:01 have been linked with the occurrence of severe influenza infections and non-response to the influenza vaccine. However, other allomorphs such as HLA-DRB1*11:01 have been shown to be protective in viral diseases such as HIV and HCV, and might be important for influenza infection. The mechanism surrounding these associations of influenza infection and HLA class II molecules expression is unclear. As some individuals expressing particular HLA allomorphs are at a greater risk of developing severe influenza infection, further investigation into the mechanism of this association is warranted. Through stimulating T cells from donors expressing HLA-DRB1*11:01 or HLA-DRB1*07:01 with an influenza epitope (HA) derived from pandemic and epidemic influenza strains, we determined the level of cross-reactivity, phenotype and polyfunctionality of the responding CD4+ specific T cells. We have compared different allomorphs and provide the first insight into the molecular basis of influenza epitope recognition by different HLA class II allomorphs, and its functional impact.

Rare individuals, termed HIV controllers, spontaneously control HIV infection by mounting efficient T cell responses against the virus. Protective CD4

International audience; Rare individuals, termed HIV controllers, spontaneously control HIV infection by mounting efficient T cell responses against the virus. Protective CD4+ T cell responses from HIV controllers involve high-affinity public T cell receptors (TCRs) recognizing an immunodominant capsid epitope (Gag293) presented by a remarkably broad array of human leukocyte antigen (HLA) class II molecules. Here, we determine the structures of a prototypical public TCR bound to HLA-DR1, HLA-DR11, and HLA-DR15 molecules presenting the Gag293 epitope. TCR recognition was driven by contacts with the Gag293 epitope, a feature that underpinned the extensive HLA cross-restriction. These high-affinity TCRs promoted mature immunological synapse formation and cytotoxic capacity in both CD4+ and CD8+ T cells. The public TCRs suppressed HIV replication in multiple genetic backgrounds ex vivo, emphasizing the functional advantage conferred by broad HLA class II cross-restriction.