T cell autoreactivity directed toward CD1c itself rather than toward carried self lipids.

The hallmark function of αß T cell antigen receptors (TCRs) involves the highly specific co-recognition of a major histocompatibility complex molecule and its carried peptide. However, the molecular basis of the interactions of TCRs with the lipid antigen-presenting molecule CD1c is unknown. We identified frequent staining of human T cells with CD1c tetramers across numerous subjects. Whereas TCRs typically show high specificity for antigen, both tetramer binding and autoreactivity occurred with CD1c in complex with numerous, chemically diverse self lipids. Such extreme polyspecificity was attributable to binding of the TCR over the closed surface of CD1c, with the TCR covering the portal where lipids normally protrude. The TCR essentially failed to contact lipids because they were fully seated within CD1c. These data demonstrate the sequestration of lipids within CD1c as a mechanism of autoreactivity and point to small lipid size as a determinant of autoreactive T cell responses.

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.

Increasing HbA1c is associated with reduced CD8+ T cell functionality in response to influenza virus in a TCR-dependent manner in individuals with diabetes mellitus.

Diabetes mellitus is on the rise globally and is a known susceptibility factor for severe influenza virus infections. However, the mechanisms by which diabetes increases the severity of an influenza virus infection are yet to be fully defined. Diabetes mellitus is hallmarked by high glucose concentrations in the blood. We hypothesized that these high glucose concentrations affect the functionality of CD8+ T cells, which play a key role eliminating virus-infected cells and have been shown to decrease influenza disease severity. To study the effect of hyperglycemia on CD8+ T cell function, we stimulated peripheral blood mononuclear cells (PBMCs) from donors with and without diabetes with influenza A virus, anti-CD3/anti-CD28-coated beads, PMA and ionomycin (PMA/I), or an influenza viral peptide pool. After stimulation, cells were assessed for functionality [as defined by expression of IFN-γ, TNF-α, macrophage inflammatory protein (MIP)-1ß, and lysosomal-associated membrane protein-1 (CD107a)] using flow cytometry. Our results showed that increasing HbA1c correlated with a reduction in TNF-α production by CD8+ T cells in response to influenza stimulation in a TCR-specific manner. This was not associated with any changes to CD8+ T cell subsets. We conclude that hyperglycemia impairs CD8+ T cell function to influenza virus infection, which may be linked with the increased risk of severe influenza in patients with diabetes.

Determination of Intracellular Esterase Activity Using Ratiometric Raman Sensing and Spectral Phasor Analysis.

Carboxylesterases (CEs) are a class of enzymes that catalyze the hydrolysis of esters in a variety of endogenous and exogenous molecules. CEs play an important role in drug metabolism, in the onset and progression of disease, and can be harnessed for prodrug activation strategies. As such, the regulation of CEs is an important clinical and pharmaceutical consideration. Here, we report the first ratiometric sensor for CE activity using Raman spectroscopy based on a bisarylbutadiyne scaffold. The sensor was shown to be highly sensitive and specific for CE detection and had low cellular cytotoxicity. In hepatocyte cells, the ratiometric detection of esterase activity was possible, and the result was validated by multimodal imaging with standard viability stains used for fluorescence microscopy within the same cell population. In addition, we show that the detection of localized ultraviolet damage in a mixed cell population was possible using stimulated Raman scattering microscopy coupled with spectral phasor analysis. This sensor demonstrates the practical advantages of low molecular weight sensors that are detected using ratiometric Raman imaging and will have applications in drug discovery and biomedical research.

HLA-B*27:05 alters immunodominance hierarchy of universal influenza-specific CD8+ T cells.

Seasonal influenza virus infections cause 290,000-650,000 deaths annually and severe morbidity in 3-5 million people. CD8+ T-cell responses towards virus-derived peptide/human leukocyte antigen (HLA) complexes provide the broadest cross-reactive immunity against human influenza viruses. Several universally-conserved CD8+ T-cell specificities that elicit prominent responses against human influenza A viruses (IAVs) have been identified. These include HLA-A*02:01-M158-66 (A2/M158), HLA-A*03:01-NP265-273, HLA-B*08:01-NP225-233, HLA-B*18:01-NP219-226, HLA-B*27:05-NP383-391 and HLA-B*57:01-NP199-207. The immunodominance hierarchies across these universal CD8+ T-cell epitopes were however unknown. Here, we probed immunodominance status of influenza-specific universal CD8+ T-cells in HLA-I heterozygote individuals expressing two or more universal HLAs for IAV. We found that while CD8+ T-cell responses directed towards A2/M158 were generally immunodominant, A2/M158+CD8+ T-cells were markedly diminished (subdominant) in HLA-A*02:01/B*27:05-expressing donors following ex vivo and in vitro analyses. A2/M158+CD8+ T-cells in non-HLA-B*27:05 individuals were immunodominant, contained optimal public TRBV19/TRAV27 TCRαß clonotypes and displayed highly polyfunctional and proliferative capacity, while A2/M158+CD8+ T cells in HLA-B*27:05-expressing donors were subdominant, with largely distinct TCRαß clonotypes and consequently markedly reduced avidity, proliferative and polyfunctional efficacy. Our data illustrate altered immunodominance patterns and immunodomination within human influenza-specific CD8+ T-cells. Accordingly, our work highlights the importance of understanding immunodominance hierarchies within individual donors across a spectrum of prominent virus-specific CD8+ T-cell specificities prior to designing T cell-directed vaccines and immunotherapies, for influenza and other infectious diseases.

IL-33 Augments Virus-Specific Memory T Cell Inflation and Potentiates the Efficacy of an Attenuated Cytomegalovirus-Based Vaccine.

Candidate vaccines designed to generate T cell-based immunity are typically vectored by nonpersistent viruses, which largely fail to elicit durable effector memory T cell responses. This limitation can be overcome using recombinant strains of CMV. Proof-of-principle studies have demonstrated the potential benefits of this approach, most notably in the SIV model, but safety concerns require the development of nonreplicating alternatives with comparable immunogenicity. In this study, we show that IL-33 promotes the accumulation and recall kinetics of circulating and tissue-resident memory T cells in mice infected with murine CMV. Using a replication-deficient murine CMV vector, we further show that exogenous IL-33 boosts vaccine-induced memory T cell responses, which protect against subsequent heterologous viral challenge. These data suggest that IL-33 could serve as a useful adjuvant to improve the efficacy of vaccines based on attenuated derivatives of CMV.

Genetic Bias, Diversity Indices, Physiochemical Properties and CDR3 Motifs Divide Auto-Reactive from Allo-Reactive T-Cell Repertoires.

The distinct properties of allo-reactive T-cell repertoires are not well understood. To investigate whether auto-reactive and allo-reactive T-cell repertoires encoded distinct properties, we used dextramer enumeration, enrichment, single-cell T-cell receptor (TCR) sequencing and multiparameter analysis. We found auto-reactive and allo-reactive T-cells differed in mean ex vivo frequency which was antigen dependent. Allo-reactive T-cells showed clear differences in TCR architecture, with enriched usage of specific T-cell receptor variable (TRBJ) genes and broader use of T-cell receptor variable joining (TRBJ) genes. Auto-reactive T-cell repertoires exhibited complementary determining regions three (CDR3) lengths using a Gaussian distribution whereas allo-reactive T-cell repertoires exhibited distorted patterns in CDR3 length. CDR3 loops from allo-reactive T-cells showed distinct physical-chemical properties, tending to encode loops that were more acidic in charge. Allo-reactive T-cell repertoires differed in diversity metrics, tending to show increased overall diversity and increased homogeneity between repertoires. Motif analysis of CDR3 loops showed allo-reactive T-cell repertoires differed in motif preference which included broader motif use. Collectively, these data conclude that allo-reactive T-cell repertoires are indeed different to auto-reactive repertoires and provide tangible metrics for further investigations and validation. Given that the antigens studied here are overexpressed on multiple cancers and that allo-reactive TCRs often show increased ligand affinity, this new TCR bank also has translational potential for adoptive cell therapy, soluble TCR-based therapy and rational TCR design.

The Many Faces of CD4+ T Cells: Immunological and Structural Characteristics.

As a major arm of the cellular immune response, CD4+ T cells are important in the control and clearance of infections. Primarily described as helpers, CD4+ T cells play an integral role in the development and activation of B cells and CD8+ T cells. CD4+ T cells are incredibly heterogeneous, and can be divided into six main lineages based on distinct profiles, namely T helper 1, 2, 17 and 22 (Th1, Th2, Th17, Th22), regulatory T cells (Treg) and T follicular helper cells (Tfh). Recent advances in structural biology have allowed for a detailed characterisation of the molecular mechanisms that drive CD4+ T cell recognition. In this review, we discuss the defining features of the main human CD4+ T cell lineages and their role in immunity, as well as their structural characteristics underlying their detection of pathogens.

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.

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses.

Memory CD8(+)T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A*0201-M158and the hypervariable HLA-B*3501-NP418antigens. The TCRαßs for HLA-B*3501-NP418 (+)CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19(+)TCRαß was selected in HLA-A*0201(+)donors responding to M158 This public TCR cross-recognized naturally occurring M158variants complexed with HLA-A*0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19(+)TCR specificity for the WT and mutant M158peptides, suggesting the possibility of universal CTL immunity in HLA-A*0201(+)individuals. Combined with the high population frequency of HLA-A*0201, these data potentially explain the relative conservation of M158 Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs.

A Photoaffinity Displacement Assay and Probes to Study the Cyclin-Dependent Kinase Family.

The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe-based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest.

Lack of Heterologous Cross-reactivity toward HLA-A*02:01 Restricted Viral Epitopes Is Underpinned by Distinct αßT Cell Receptor Signatures.

αßT cell receptor (TCR) genetic diversity is outnumbered by the quantity of pathogenic epitopes to be recognized. To provide efficient protective anti-viral immunity, a single TCR ideally needs to cross-react with a multitude of pathogenic epitopes. However, the frequency, extent, and mechanisms of TCR cross-reactivity remain unclear, with conflicting results on anti-viral T cell cross-reactivity observed in humans. Namely, both the presence and lack of T cell cross-reactivity have been reported with HLA-A*02:01-restricted epitopes from the Epstein-Barr and influenza viruses (BMLF-1 and M158, respectively) or with the hepatitis C and influenza viruses (NS31073 and NA231, respectively). Given the high sequence similarity of these paired viral epitopes (56 and 88%, respectively), the ubiquitous nature of the three viruses, and the high frequency of the HLA-A*02:01 allele, we selected these epitopes to establish the extent of T cell cross-reactivity. We combined ex vivo and in vitro functional assays, single-cell αßTCR repertoire sequencing, and structural analysis of these four epitopes in complex with HLA-A*02:01 to determine whether they could lead to heterologous T cell cross-reactivity. Our data show that sequence similarity does not translate to structural mimicry of the paired epitopes in complexes with HLA-A*02:01, resulting in induction of distinct αßTCR repertoires. The differences in epitope architecture might be an obstacle for TCR recognition, explaining the lack of T cell cross-reactivity observed. In conclusion, sequence similarity does not necessarily result in structural mimicry, and despite the need for cross-reactivity, antigen-specific TCR repertoires can remain highly specific.

Towards identification of immune and genetic correlates of severe influenza disease in Indigenous Australians.

This corrects the article DOI: 10.1038/icb.2015.93.

Maintenance of the EBV-specific CD8+ TCRαß repertoire in immunosuppressed lung transplant recipients.

Epstein-Barr virus (EBV) is one of the most common viruses in humans, capable of causing life-threatening infections and cancers in immunocompromised individuals. Although CD8+ T cells provide key protection against EBV, the persistence and dynamics of specific T-cell receptor (TCR) clones during immunosuppression in transplant patients is largely unknown. For the first time, we used a novel single-cell TCRαß multiplex-nested reverse transcriptase PCR to dissect TCRαß clonal diversity within GLCTLVAML (GLC)-specific CD8+ T cells in healthy individuals and immunocompromised lung transplant recipients. The GLC peptide presented by HLA-A*02:01 is one of the most immunogenic T-cell targets from the EBV proteome. We found that the GLC-specific TCRαß repertoire was heavily biased toward TRAV5 and encompassed five classes of public TCRαßs, suggesting that these clonotypes are preferentially utilized following infection. We identified that a common TRAV5 was diversely paired with different TRAJ and TRBV/TRBJ genes, in both immunocompetent and immunocompromised individuals, with an average of 12 different TCRαß clonotypes/donor. Moreover, pre-transplant GLC-specific TCRαß repertoires were relatively stable over 1 year post transplant under immunosuppression in the absence or presence of EBV reactivation. In addition, we provide the first evidence of early GLC-specific CD8+ T cells at 87 days post transplant, which preceded clinical EBV detection at 242 days in an EBV-seronegative patient receiving a lung allograft from an EBV-seropositive donor. This was associated with a relatively stable TCRαß repertoire after CD8+ T-cell expansion. Our findings provide insights into the composition and temporal dynamics of the EBV-specific TCRαß repertoire in immunocompromised transplant patients and suggest that the early detection of EBV-specific T cells might be a predictor of ensuing EBV blood viremia.

Molecular challenges imposed by MHC-I restricted long epitopes on T cell immunity.

It has widely been accepted that major histocompatibility complex class I molecules (MHC-I) are limited to binding small peptides of 8-10 residues in length. However, this consensus has recently been challenged with the identification of longer peptides (≥11 residues) that can also elicit cytotoxic CD8+ T cell responses. Indeed, a growing number of studies demonstrate that these non-canonical epitopes are important targets for the immune system. As long epitopes represent up to 10% of the peptide repertoire bound to MHC-I molecules, here we review their impact on antigen presentation by MHC-I, TCR recognition, and T cell immunity.

Preexisting CD8+ T-cell immunity to the H7N9 influenza A virus varies across ethnicities.

The absence of preexisting neutralizing antibodies specific for the novel A (H7N9) influenza virus indicates a lack of prior human exposure. As influenza A virus-specific CD8(+) T lymphocytes (CTLs) can be broadly cross-reactive, we tested whether immunogenic peptides derived from H7N9 might be recognized by memory CTLs established following infection with other influenza strains. Probing across multiple ethnicities, we identified 32 conserved epitopes derived from the nucleoprotein (NP) and matrix-1 (M1) proteins. These NP and M1 peptides are presented by HLAs prevalent in 16-57% of individuals. Remarkably, some HLA alleles (A*0201, A*0301, B*5701, B*1801, and B*0801) elicit robust CTL responses against any human influenza A virus, including H7N9, whereas ethnicities where HLA-A*0101, A*6801, B*1501, and A*2402 are prominent, show limited CTL response profiles. By this criterion, some groups, especially the Alaskan and Australian Indigenous peoples, would be particularly vulnerable to H7N9 infection. This dissection of CTL-mediated immunity to H7N9 thus suggests strategies for both vaccine delivery and development.

Towards identification of immune and genetic correlates of severe influenza disease in Indigenous Australians.

Indigenous populations, including Indigenous Australians, are highly susceptible to severe influenza disease and the underlying mechanisms are unknown. We studied immune and genetic factors that could predicate severe influenza disease in Indigenous Australians enrolled in the LIFT study: looking into influenza T-cell immunity. To examine CD8(+) T-cell immunity, we characterised human leukocyte antigen (HLA) profiles. HLA typing confirmed previous studies showing predominant usage of HLA-A*02:01, 11:01, 24:02, 34:01 and HLA-B*13:01, 15:21, 40:01/02, 56:01/02 in Indigenous Australians. We identified two new HLA alleles (HLA-A*02:new and HLA-B*56:new). Modelling suggests that variations within HLA-A*02:new (but not HLA-B56:new) could affect peptide binding. There is a relative lack of known influenza epitopes for the majority of these HLAs, with the exception of a universal HLA-A*02:01-M158 epitope and proposed epitopes presented by HLA-A*11:01/HLA-A*24:02. To dissect universal CD8(+) T-cell responses, we analysed the magnitude, function and T-cell receptor (TCR) clonality of HLA-A*02:01-M158(+)CD8(+) T cells. We found comparable IFN-γ, TNF and CD107a and TCRαß characteristics in Indigenous and non-Indigenous Australians, suggesting that the ~15% of Indigenous people that express HLA-A*02:01 have universal influenza-specific CD8(+) T-cell immunity. Furthermore, the frequency of an influenza host risk factor, IFITM3-C/C, was comparable between Indigenous Australians and Europeans, suggesting that expression of this allele does not explain increased disease severity at a population level. Our study indicates a need to identify novel influenza-specific CD8(+) T-cell epitopes restricted by HLA-A and HLA-B alleles prevalent in Indigenous populations for the rational design of universal T-cell vaccines.

T-cell immunity to influenza A viruses.

Influenza infection remains a global threat to human health. Influenza viruses are normally controlled by antibodies specific for the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). Standard influenza vaccines are aimed at inducing these antibodies, but they must be administered annually and can be rendered ineffective since different strains circulate from year to year and vary considerably in their individual HA and NA profiles. Influenza-specific T cells have been shown to be protective in animal models and typically recognize the more conserved internal influenza proteins. Improving our understanding of influenza-specific T-cell responses, including immunodominance, specific epitope sequences, strain-related epitope variation, host/virus interaction, and the balance between immunity versus immunopathology, will be important to improve future T-cell-based vaccines, which promise broader strain coverage and longer-lasting protection than current standard vaccines.

Fighting flu: novel CD8+ T-cell targets are required for future influenza vaccines.

Seasonal influenza viruses continue to cause severe medical and financial complications annually. Although there are many licenced influenza vaccines, there are billions of cases of influenza infection every year, resulting in the death of over half a million individuals. Furthermore, these figures can rise in the event of a pandemic, as seen throughout history, like the 1918 Spanish influenza pandemic (50 million deaths) and the 1968 Hong Kong influenza pandemic (~4 million deaths). In this review, we have summarised many of the currently licenced influenza vaccines available across the world and current vaccines in clinical trials. We then briefly discuss the important role of CD8+ T cells during influenza infection and why future influenza vaccines should consider targeting CD8+ T cells. Finally, we assess the current landscape of known immunogenic CD8+ T-cell epitopes and highlight the knowledge gaps required to be filled for the design of rational future influenza vaccines that incorporate CD8+ T cells.