CD8+ T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope cross-react with selective seasonal coronaviruses.

Efforts are being made worldwide to understand the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the coronavirus disease 2019 (COVID-19) pandemic, including the impact of T cell immunity and cross-recognition with seasonal coronaviruses. Screening of 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 toward circulating OC43 and HKU-1 betacoronaviruses but not 229E or NL63 alphacoronaviruses because of different peptide conformations. T cell receptor (TCR) sequencing indicated that cross-reactivity was driven by private TCR repertoires with a bias for TRBV27 and a long CDR3ß loop. Our findings demonstrate the basis of selective T cell cross-reactivity for an immunodominant SARS-CoV-2 epitope and its homologs from seasonal coronaviruses, suggesting long-lasting protective immunity.

Impact of HLA-DR Antigen Binding Cleft Rigidity on T Cell Recognition.

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 "snapshot", 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.

Detection of Lymphocytic Choriomeningitis Virus-Specific Memory B Cells Using Antigen Tetramers.

Memory B cells are central to the establishment of immunological memory, providing long-term protection against specific pathogens and playing a vital role in the efficacy of vaccines. Understanding how memory B cell formation is disrupted during persistent infection is essential for new therapeutics. Lymphocytic choriomeningitis virus (LCMV) is an ideal model for investigating memory B cells in acute versus chronic infection. This protocol details techniques to isolate, enrich, and examine LCMV-specific memory B cells in both acute and chronic LCMV infection. Using an antigen tetramer enrichment system and flow cytometry, this method assesses low-frequency, polyclonal antigen-specific memory B cells.

Elevated BMI reduces the humoral response to SARS-CoV-2 infection.

Objective: Class III obesity (body mass index [BMI] ≥ 40 kg m-2) significantly impairs the immune response to SARS-CoV-2 vaccination. However, the effect of an elevated BMI (≥ 25 kg m-2) on humoral immunity to SARS-CoV-2 infection and COVID-19 vaccination remains unclear. Methods: We collected blood samples from people who recovered from SARS-CoV-2 infection approximately 3 and 13 months of post-infection (noting that these individuals were not exposed to SARS-CoV-2 or vaccinated in the interim). We also collected blood samples from people approximately 5 months of post-second dose COVID-19 vaccination (the majority of whom did not have a prior SARS-CoV-2 infection). We measured their humoral responses to SARS-CoV-2, grouping individuals based on a BMI greater or less than 25 kg m-2. Results: Here, we show that an increased BMI (≥ 25 kg m-2), when accounting for age and sex differences, is associated with reduced antibody responses after SARS-CoV-2 infection. At 3 months of post-infection, an elevated BMI was associated with reduced antibody titres. At 13 months of post-infection, an elevated BMI was associated with reduced antibody avidity and a reduced percentage of spike-positive B cells. In contrast, no significant association was noted between a BMI ≥ 25 kg m-2 and humoral immunity to SARS-CoV-2 at 5 months of post-secondary vaccination. Conclusions: Taken together, these data showed that elevated BMI is associated with an impaired humoral immune response to SARS-CoV-2 infection. The impairment of infection-induced immunity in individuals with a BMI ≥ 25 kg m-2 suggests an added impetus for vaccination rather than relying on infection-induced immunity.

Homologous peptides derived from influenza A, B and C viruses induce variable CD8+ T cell responses with cross-reactive potential.

Objective: Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally, infecting humans and causing widespread morbidity and mortality. Here, we investigate the T cell response towards an immunodominant IAV epitope, NP265-273, and its IBV and ICV homologues, presented by HLA-A*03:01 molecule expressed in ~ 4% of the global population (~ 300 million people). Methods: We assessed the magnitude (tetramer staining) and quality of the CD8+ T cell response (intracellular cytokine staining) towards NP265-IAV and described the T cell receptor (TCR) repertoire used to recognise this immunodominant epitope. We next assessed the immunogenicity of NP265-IAV homologue peptides from IBV and ICV and the ability of CD8+ T cells to cross-react towards these homologous peptides. Furthermore, we determined the structures of NP265-IAV and NP323-IBV peptides in complex with HLA-A*03:01 by X-ray crystallography. Results: Our study provides a detailed characterisation of the CD8+ T cell response towards NP265-IAV and its IBV and ICV homologues. The data revealed a diverse repertoire for NP265-IAV that is associated with superior anti-viral protection. Evidence of cross-reactivity between the three different influenza virus strain-derived epitopes was observed, indicating the discovery of a potential vaccination target that is broad enough to cover all three influenza strains. Conclusion: We show that while there is a potential to cross-protect against distinct influenza virus lineages, the T cell response was stronger against the IAV peptide than IBV or ICV, which is an important consideration when choosing targets for future vaccine design.

Lineage-restricted neoplasia driven by Myc defaults to small cell lung cancer when combined with loss of p53 and Rb in the airway epithelium.

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.

Protein purification and crystallization of HLA-A∗02:01 in complex with SARS-CoV-2 peptides.

Understanding T-cell responses requires identifying viral peptides presented by human leukocyte antigens (HLAs). 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. For complete details on the use and execution of this protocol, please refer to Szeto et al. (2021).

Molecular Basis of a Dominant SARS-CoV-2 Spike-Derived Epitope Presented by HLA-A*02:01 Recognised by a Public TCR.

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.

The presentation of SARS-CoV-2 peptides by the common HLA-A∗02:01 molecule.

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.

CD4+ T cell-mediated HLA class II cross-restriction in HIV controllers.

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.