Publications by authors named "Jamie Rossjohn"

417 Publications

Rational design of a hydrolysis-resistant mycobacterial phosphoglycolipid antigen presented by CD1c to T cells.

J Biol Chem 2021 Sep 15:101197. Epub 2021 Sep 15.

Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, United States; Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, 3584 CL, Netherlands. Electronic address:

Whereas proteolytic cleavage is crucial for peptide presentation by classical major histocompatibility complex (MHC) proteins to T cells, glycolipids presented by CD1 molecules are typically presented in an unmodified form. However, the mycobacterial lipid antigen mannosyl-β1-phosphomycoketide (MPM) may be processed through hydrolysis in antigen presenting cells, forming mannose and phosphomycoketide (PM). To further test the hypothesis that some lipid antigens are processed, and to generate antigens that lead to defined epitopes for future tuberculosis vaccines or diagnostic tests, we aimed to create hydrolysis-resistant MPM variants that retain their antigenicity. Here, we designed and tested three different, versatile synthetic strategies to chemically stabilize MPM analogs. Crystallographic studies of CD1c complexes with these three new MPM analogs showed anchoring of the lipid tail and phosphate group that is highly comparable to nature-identical MPM, with considerable conformational flexibility for the mannose head group. MPM-3, a difluoromethylene-modified version of MPM that is resistant to hydrolysis showed altered recognition by cells, but not by CD1c proteins, supporting the cellular antigen processing hypothesis. Furthermore, the synthetic analogs elicited T cell responses that were cross-reactive with nature-identical MPM, fulfilling important requirements for future clinical use.
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http://dx.doi.org/10.1016/j.jbc.2021.101197DOI Listing
September 2021

Our evolving understanding of the role of the γδ T cell receptor in γδ T cell mediated immunity.

Biochem Soc Trans 2021 Sep 13. Epub 2021 Sep 13.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.

The γδ T cell immune cell lineage has remained relatively enigmatic and under-characterised since their identification. Conversely, the insights we have, highlight their central importance in diverse immunological roles and homeostasis. Thus, γδ T cells are considered as potentially a new translational tool in the design of new therapeutics for cancer and infectious disease. Here we review our current understanding of γδ T cell biology viewed through a structural lens centred on the how the γδ T cell receptor mediates ligand recognition. We discuss the limited knowledge of antigens, the structural basis of such reactivities and discuss the emerging trends of γδ T cell reactivity and implications for γδ T cell biology.
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http://dx.doi.org/10.1042/BST20200890DOI Listing
September 2021

Elucidating the Motif for CpG Oligonucleotide Binding to the Dendritic Cell Receptor DEC-205 Leads to Improved Adjuvants for Liver-Resident Memory.

J Immunol 2021 Oct 3;207(7):1836-1847. Epub 2021 Sep 3.

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.

DEC-205 is a cell-surface receptor that transports bound ligands into the endocytic pathway for degradation or release within lysosomal endosomes. This receptor has been reported to bind a number of ligands, including keratin, and some classes of CpG oligodeoxynucleotides (ODN). In this study, we explore in detail the requirements for binding ODNs, revealing that DEC-205 efficiently binds single-stranded, phosphorothioated ODN of ≥14 bases, with preference for the DNA base thymidine, but with no requirement for a CpG motif. DEC-205 fails to bind double-stranded phosphodiester ODN, and thus does not bind the natural type of DNA found in mammals. The ODN binding preferences of DEC-205 result in strong binding of B class ODN, moderate binding to C class ODN, minimal binding to P class ODN, and no binding to A class ODN. Consistent with DEC-205 binding capacity, induction of serum IL-12p70 or activation of B cells by each class of ODN correlated with DEC-205 dependence in mice. Thus, the greater the DEC-205 binding capacity, the greater the dependence on DEC-205 for optimal responses. Finally, by covalently linking a B class ODN that efficiently binds DEC-205, to a P class ODN that shows poor binding, we improved DEC-205 binding and increased adjuvancy of the hybrid ODN. The hybrid ODN efficiently enhanced induction of effector CD8 T cells in a DEC-205-dependent manner. Furthermore, the hybrid ODN induced robust memory responses, and was particularly effective at promoting the development of liver tissue-resident memory T cells.
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http://dx.doi.org/10.4049/jimmunol.2001153DOI Listing
October 2021

T cell receptor recognition of hybrid insulin peptides bound to HLA-DQ8.

Nat Commun 2021 08 25;12(1):5110. Epub 2021 Aug 25.

Infection and Immunity Program & Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

HLA-DQ8, a genetic risk factor in type I diabetes (T1D), presents hybrid insulin peptides (HIPs) to autoreactive CD4+ T cells. The abundance of spliced peptides binding to HLA-DQ8 and how they are subsequently recognised by the autoreactive T cell repertoire is unknown. Here we report, the HIP (GQVELGGGNAVEVLK), derived from splicing of insulin and islet amyloid polypeptides, generates a preferred peptide-binding motif for HLA-DQ8. HLA-DQ8-HIP tetramer T cells from the peripheral blood of a T1D patient are characterised by repeated TRBV5 usage, which matches the TCR bias of CD4+ T cells reactive to the HIP peptide isolated from the pancreatic islets of a patient with T1D. The crystal structure of three TRBV5+ TCR-HLA-DQ8-HIP complexes shows that the TRBV5-encoded TCR β-chain forms a common landing pad on the HLA-DQ8 molecule. The N- and C-termini of the HIP is recognised predominantly by the TCR α-chain and TCR β-chain, respectively, in all three TCR ternary complexes. Accordingly, TRBV5 + TCR recognition of HIP peptides might occur via a 'polarised' mechanism, whereby each chain within the αβTCR heterodimer recognises distinct origins of the spliced peptide presented by HLA-DQ8.
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http://dx.doi.org/10.1038/s41467-021-25404-xDOI Listing
August 2021

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Proc Natl Acad Sci U S A 2021 Aug;118(34)

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC 3000, Australia;

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.
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http://dx.doi.org/10.1073/pnas.2104420118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403964PMC
August 2021

A high-affinity human TCR-like antibody detects celiac disease gluten peptide-MHC complexes and inhibits T cell activation.

Sci Immunol 2021 Aug;6(62)

Centre for Immune Regulation and Department of Immunology, University of Oslo and Oslo University Hospital-Rikshospitalet, Oslo, Norway.

Antibodies specific for peptides bound to human leukocyte antigen (HLA) molecules are valuable tools for studies of antigen presentation and may have therapeutic potential. Here, we generated human T cell receptor (TCR)-like antibodies toward the immunodominant signature gluten epitope DQ2.5-glia-α2 in celiac disease (CeD). Phage display selection combined with secondary targeted engineering was used to obtain highly specific antibodies with picomolar affinity. The crystal structure of a Fab fragment of the lead antibody 3.C11 in complex with HLA-DQ2.5:DQ2.5-glia-α2 revealed a binding geometry and interaction mode highly similar to prototypic TCRs specific for the same complex. Assessment of CeD biopsy material confirmed disease specificity and reinforced the notion that abundant plasma cells present antigen in the inflamed CeD gut. Furthermore, 3.C11 specifically inhibited activation and proliferation of gluten-specific CD4 T cells in vitro and in HLA-DQ2.5 humanized mice, suggesting a potential for targeted intervention without compromising systemic immunity.
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http://dx.doi.org/10.1126/sciimmunol.abg4925DOI Listing
August 2021

Structural basis of biased T cell receptor recognition of an immunodominant HLA-A2 epitope of the SARS-CoV-2 spike protein.

J Biol Chem 2021 Aug 10;297(3):101065. Epub 2021 Aug 10.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia; Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia. Electronic address:

CD8 T cells play an important role in vaccination and immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Although numerous SARS-CoV-2 CD8 T cell epitopes have been identified, the molecular basis underpinning T cell receptor (TCR) recognition of SARS-CoV-2-specific T cells remains unknown. The T cell response directed toward SARS-CoV-2 spike protein-derived S peptide presented by the human leukocyte antigen (HLA)-A∗02:01 allomorph (hereafter the HLA-A2 epitope) is, to date, the most immunodominant SARS-CoV-2 epitope found in individuals bearing this allele. As HLA-A2-specific CD8 T cells utilize biased TRAV12 gene usage within the TCR α-chain, we sought to understand the molecular basis underpinning this TRAV12 dominance. We expressed four TRAV12 TCRs which bound the HLA-A2 complex with low micromolar affinity and determined the crystal structure of the HLA-A2 binary complex, and subsequently a ternary structure of the TRAV12 TCR complexed to HLA-A2. We found that the TCR made extensive contacts along the entire length of the S peptide, suggesting that the TRAV12 TCRs would be sensitive to sequence variation within this epitope. To examine this, we investigated cross-reactivity toward analogous peptides from existing SARS-CoV-2 variants and closely related coronaviruses. We show via surface plasmon resonance and tetramer studies that the TRAV12 T cell repertoire cross-reacts poorly with these analogous epitopes. Overall, we defined the structural basis underpinning biased TCR recognition of CD8 T cells directed at an immunodominant epitope and provide a framework for understanding TCR cross-reactivity toward viral variants within the S peptide.
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http://dx.doi.org/10.1016/j.jbc.2021.101065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8352664PMC
August 2021

Binding of a pyrimidine RNA base-mimic to SARS-CoV-2 nonstructural protein 9.

J Biol Chem 2021 Jul 28;297(3):101018. Epub 2021 Jul 28.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia; Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, United Kingdom. Electronic address:

The coronaviral nonstructural protein 9 (Nsp9) is essential for viral replication; it is the primary substrate of Nsp12's pseudokinase domain within the viral replication transcription complex, an association that also recruits other components during different stages of RNA reproduction. In the unmodified state, Nsp9 forms an obligate homodimer via an essential GxxxG protein-interaction motif, but its ssRNA-binding mechanism remains unknown. Using structural biological techniques, here we show that a base-mimicking compound identified from a small molecule fragment screen engages Nsp9 via a tetrameric Pi-Pi stacking interaction that induces the formation of a parallel trimer-of-dimers. This oligomerization mechanism allows an interchange of "latching" N-termini, the charges of which contribute to a series of electropositive channels that suggests a potential interface for viral RNA. The identified pyrrolo-pyrimidine compound may also serve as a potential starting point for the development of compounds seeking to probe Nsp9's role within SARS-CoV-2 replication.
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http://dx.doi.org/10.1016/j.jbc.2021.101018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8317483PMC
July 2021

A multilayered immune system through the lens of unconventional T cells.

Nature 2021 Jul 21;595(7868):501-510. Epub 2021 Jul 21.

Committee on Immunology, University of Chicago, Chicago, IL, USA.

The unconventional T cell compartment encompasses a variety of cell subsets that straddle the line between innate and adaptive immunity, often reside at mucosal surfaces and can recognize a wide range of non-polymorphic ligands. Recent advances have highlighted the role of unconventional T cells in tissue homeostasis and disease. In this Review, we recast unconventional T cell subsets according to the class of ligand that they recognize; their expression of semi-invariant or diverse T cell receptors; the structural features that underlie ligand recognition; their acquisition of effector functions in the thymus or periphery; and their distinct functional properties. Unconventional T cells follow specific selection rules and are poised to recognize self or evolutionarily conserved microbial antigens. We discuss these features from an evolutionary perspective to provide insights into the development and function of unconventional T cells. Finally, we elaborate on the functional redundancy of unconventional T cells and their relationship to subsets of innate and adaptive lymphoid cells, and propose that the unconventional T cell compartment has a critical role in our survival by expanding and complementing the role of the conventional T cell compartment in protective immunity, tissue healing and barrier function.
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http://dx.doi.org/10.1038/s41586-021-03578-0DOI Listing
July 2021

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection.

Nat Commun 2021 07 16;12(1):4355. Epub 2021 Jul 16.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, VIC, Australia.

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.
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http://dx.doi.org/10.1038/s41467-021-24570-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8285429PMC
July 2021

SARS-CoV-2-specific CD8 T-cell responses and TCR signatures in the context of a prominent HLA-A*24:02 allomorph.

Immunol Cell Biol 2021 Jun 4. Epub 2021 Jun 4.

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia.

In-depth understanding of human T-cell-mediated immunity in coronavirus disease 2019 (COVID-19) is needed if we are to optimize vaccine strategies and immunotherapies. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell epitopes and generation of peptide-human leukocyte antigen (peptide-HLA) tetramers facilitate direct ex vivo analyses of SARS-CoV-2-specific T cells and their T-cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS-CoV-2 epitopes restricted by HLA-A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike-derived peptides generated CD8 IFNγ responses above background, S (QYIKWPWYI), S (NYNYLYRLF) and S (VFKNIDGYF), with S generating immunodominant CD8 IFNγ responses. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for HLA-A*24:02-restricted CD8 T cells in COVID-19 patients and prepandemic controls. The precursor frequencies for HLA-A*24:02-restricted epitopes were within the range previously observed for other SARS-CoV-2 epitopes for both COVID-19 patients and prepandemic individuals. Naïve A24/SARS-CoV-2-specific CD8 T cells increased nearly 7.5-fold above the average precursor frequency during COVID-19, gaining effector and memory phenotypes. Ex vivo single-cell analyses of TCRαβ repertoires found that the A24/S CD8 T-cell TCRαβ repertoire was driven by a common TCRβ chain motif, whereas the A24/S CD8 TCRαβ repertoire was diverse across COVID-19 patients. Our study provides an in depth characterization and important insights into SARS-CoV-2-specific CD8 T-cell responses associated with a prominent HLA-A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID-19 and could be exploited in vaccine or immunotherapeutic approaches.
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http://dx.doi.org/10.1111/imcb.12482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8242669PMC
June 2021

Canonical T cell receptor docking on peptide-MHC is essential for T cell signaling.

Science 2021 06;372(6546)

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

T cell receptor (TCR) recognition of peptide-major histocompatibility complexes (pMHCs) is characterized by a highly conserved docking polarity. Whether this polarity is driven by recognition or signaling constraints remains unclear. Using "reversed-docking" TCRβ-variable (TRBV) 17 TCRs from the naïve mouse CD8 T cell repertoire that recognizes the H-2D-NP epitope, we demonstrate that their inability to support T cell activation and in vivo recruitment is a direct consequence of reversed docking polarity and not TCR-pMHCI binding or clustering characteristics. Canonical TCR-pMHCI docking optimally localizes CD8/Lck to the CD3 complex, which is prevented by reversed TCR-pMHCI polarity. The requirement for canonical docking was circumvented by dissociating Lck from CD8. Thus, the consensus TCR-pMHC docking topology is mandated by T cell signaling constraints.
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http://dx.doi.org/10.1126/science.abe9124DOI Listing
June 2021

CD8 T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Nat Commun 2021 05 18;12(1):2931. Epub 2021 May 18.

Department of Biochemistry and Molecular Biology & Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8 T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8 T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2-specific CD8 T cells being cross-reactive between IAV and IBV. Memory CD8 T cells towards these specificities are present in blood (CD27CD45RA phenotype) and tissues (CD103CD69 phenotype) of healthy individuals, and effector CD27CD45RAPD-1CD38CD8 T cells in IAV/IBV patients. Our data show influenza-specific CD8 T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8 T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.
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http://dx.doi.org/10.1038/s41467-021-23212-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132304PMC
May 2021

Immune cellular networks underlying recovery from influenza virus infection in acute hospitalized patients.

Nat Commun 2021 05 11;12(1):2691. Epub 2021 May 11.

Department of Biochemistry and Genetics, La Trobe Institute For Molecular Science, La Trobe University, Bundoora, VIC, Australia.

How innate and adaptive immune responses work in concert to resolve influenza disease is yet to be fully investigated in one single study. Here, we utilize longitudinal samples from patients hospitalized with acute influenza to understand these immune responses. We report the dynamics of 18 important immune parameters, related to clinical, genetic and virological factors, in influenza patients across different severity levels. Influenza disease correlates with increases in IL-6/IL-8/MIP-1α/β cytokines and lower antibody responses. Robust activation of circulating T follicular helper cells correlates with peak antibody-secreting cells and influenza heamaglutinin-specific memory B-cell numbers, which phenotypically differs from vaccination-induced B-cell responses. Numbers of influenza-specific CD8 or CD4 T cells increase early in disease and retain an activated phenotype during patient recovery. We report the characterisation of immune cellular networks underlying recovery from influenza infection which are highly relevant to other infectious diseases.
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http://dx.doi.org/10.1038/s41467-021-23018-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113517PMC
May 2021

CD1a selectively captures endogenous cellular lipids that broadly block T cell response.

J Exp Med 2021 Jul 7;218(7). Epub 2021 May 7.

Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, MA.

We optimized lipidomics methods to broadly detect endogenous lipids bound to cellular CD1a proteins. Whereas membrane phospholipids dominate in cells, CD1a preferentially captured sphingolipids, especially a C42, doubly unsaturated sphingomyelin (42:2 SM). The natural 42:2 SM but not the more common 34:1 SM blocked CD1a tetramer binding to T cells in all human subjects tested. Thus, cellular CD1a selectively captures a particular endogenous lipid that broadly blocks its binding to TCRs. Crystal structures show that the short cellular SMs stabilized a triad of surface residues to remain flush with CD1a, but the longer lipids forced the phosphocholine group to ride above the display platform to hinder TCR approach. Whereas nearly all models emphasize antigen-mediated T cell activation, we propose that the CD1a system has intrinsic autoreactivity and is negatively regulated by natural endogenous inhibitors selectively bound in its cleft. Further, the detailed chemical structures of natural blockers could guide future design of therapeutic blockers of CD1a response.
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http://dx.doi.org/10.1084/jem.20202699DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111460PMC
July 2021

CD8 T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

Immunity 2021 05 15;54(5):1066-1082.e5. Epub 2021 Apr 15.

Department of Infectious Diseases, Austin Hospital, Heidelberg, VIC 3084, Australia; Department of Medicine and Radiology, The University of Melbourne, Parkville, VIC 3000, Australia; Data Analytics Research and Evaluation (DARE) Centre, Austin Health and The University of Melbourne, Heidelberg, VIC 3084, Australia.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8 T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 T cells directed toward subdominant epitopes (B7/N, A2/S, and A24/S) CD8 T cells specific for the immunodominant B7/N epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/NCD8 T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N-specific CD8 T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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http://dx.doi.org/10.1016/j.immuni.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049468PMC
May 2021

Carbamazepine Induces Focused T Cell Responses in Resolved Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Cases But Does Not Perturb the Immunopeptidome for T Cell Recognition.

Front Immunol 2021 12;12:653710. Epub 2021 Apr 12.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Antiseizure medications (ASMs) are frequently implicated in T cell-mediated drug hypersensitivity reactions and cause skin tropic pathologies that range in severity from mild rashes to life-threatening systemic syndromes. During the acute stages of the more severe manifestations of these reactions, drug responsive proinflammatory CD8 T cells display classical features of Th1 cytokine production ( IFNγ) and cytolysis ( granzyme B, perforin). These T cells may be found locally at the site of pathology ( blister cells/fluid), as well as systemically ( blood, organs). What is less understood are the long-lived immunological effects of the memory T cell pool following T cell-mediated drug hypersensitivity reactions. In this study, we examine the ASM carbamazepine (CBZ) and the CBZ-reactive memory T cell pool in patients who have a history of either Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN) from 3-to-20 years following their initial adverse reaction. We show that drug restimulation of CBZ-reactive CD8 T cells results in a proinflammatory profile and produces a mainly focused, yet private, T cell receptor (TCR) usage amongst human leukocyte antigen (HLA)-B*15:02-positive SJS or TEN patients. Additionally, we show that expression of these CBZ-reactive TCRs in a reporter cell line, lacking endogenous αβTCR, recapitulates the features of TCR activation reported for ASM-treated T cell lines/clones, providing a useful tool for further functional validations. Finally, we conduct a comprehensive evaluation of the HLA-B*15:02 immunopeptidome following ASM (or a metabolite) treatment of a HLA-B*15:02-positive B-lymphoblastoid cell line (C1R.B*15:02) and minor perturbation of the peptide repertoire. Collectively, this study shows that the CBZ-reactive T cells characterized require both the drug and HLA-B*15:02 for activation and that reactivation of memory T cells from blood results in a focused TCR profile in patients with resolved disease.
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http://dx.doi.org/10.3389/fimmu.2021.653710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8071863PMC
April 2021

The shared susceptibility epitope of HLA-DR4 binds citrullinated self-antigens and the TCR.

Sci Immunol 2021 Apr;6(58)

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia.

Individuals expressing HLA-DR4 bearing the shared susceptibility epitope (SE) have an increased risk of developing rheumatoid arthritis (RA). Posttranslational modification of self-proteins via citrullination leads to the formation of neoantigens that can be presented by HLA-DR4 SE allomorphs. However, in T cell-mediated autoimmunity, the interplay between the HLA molecule, posttranslationally modified epitope(s), and the responding T cell repertoire remains unclear. In HLA-DR4 transgenic mice, we show that immunization with a Fibβ-74cit peptide led to a population of HLA-DR4 tetramer T cells that exhibited biased T cell receptor (TCR) β chain usage, which was attributable to selective clonal expansion from the preimmune repertoire. Crystal structures of pre- and postimmune TCRs showed that the SE of HLA-DR4 represented a main TCR contact zone. Immunization with a double citrullinated epitope (Fibβ-72,74cit) altered the responding HLA-DR4 tetramer T cell repertoire, which was due to the P2-citrulline residue interacting with the TCR itself. We show that the SE of HLA-DR4 has dual functionality, namely, presentation and a direct TCR recognition determinant. Analogous biased TCR β chain usage toward the Fibβ-74cit peptide was observed in healthy HLA-DR4 individuals and patients with HLA-DR4 RA, thereby suggesting a link to human RA.
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http://dx.doi.org/10.1126/sciimmunol.abe0896DOI Listing
April 2021

Katharina Gaus 1972-2021.

Nat Immunol 2021 05;22(5):535-536

Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

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http://dx.doi.org/10.1038/s41590-021-00915-3DOI Listing
May 2021

Structural plasticity of KIR2DL2 and KIR2DL3 enables altered docking geometries atop HLA-C.

Nat Commun 2021 04 12;12(1):2173. Epub 2021 Apr 12.

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

The closely related inhibitory killer-cell immunoglobulin-like receptors (KIR), KIR2DL2 and KIR2DL3, regulate the activation of natural killer cells (NK) by interacting with the human leukocyte antigen-C1 (HLA-C1) group of molecules. KIR2DL2, KIR2DL3 and HLA-C1 are highly polymorphic, with this variation being associated with differences in the onset and progression of some human diseases. However, the molecular bases underlying these associations remain unresolved. Here, we determined the crystal structures of KIR2DL2 and KIR2DL3 in complex with HLA-C*07:02 presenting a self-epitope. KIR2DL2 differed from KIR2DL3 in docking modality over HLA-C*07:02 that correlates with variabilty of recognition of HLA-C1 allotypes. Mutagenesis assays indicated differences in the mechanism of HLA-C1 allotype recognition by KIR2DL2 and KIR2DL3. Similarly, HLA-C1 allotypes differed markedly in their capacity to inhibit activation of primary NK cells. These functional differences derive, in part, from KIR2DS2 suggesting KIR2DL2 and KIR2DL3 binding geometries combine with other factors to distinguish HLA-C1 functional recognition.
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http://dx.doi.org/10.1038/s41467-021-22359-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041999PMC
April 2021

The molecular assembly of the marsupial γμ T cell receptor defines a third T cell lineage.

Science 2021 03;371(6536):1383-1388

Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

αβ and γδ T cell receptors (TCRs) are highly diverse antigen receptors that define two evolutionarily conserved T cell lineages. We describe a population of γμTCRs found exclusively in non-eutherian mammals that consist of a two-domain (Vγ-Cγ) γ-chain paired to a three-domain (Vμ-Vμj-Cμ) μ-chain. γμTCRs were characterized by restricted diversity in the Vγ and Vμj domains and a highly diverse unpaired Vμ domain. Crystal structures of two distinct γμTCRs revealed the structural basis of the association of the γμTCR heterodimer. The Vμ domain shared the characteristics of a single-domain antibody within which the hypervariable CDR3μ loop suggests a major antigen recognition determinant. We define here the molecular basis underpinning the assembly of a third TCR lineage, the γμTCR.
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http://dx.doi.org/10.1126/science.abe7070DOI Listing
March 2021

Mucosal-Associated Invariant T Cell Effector Function Is an Intrinsic Cell Property That Can Be Augmented by the Metabolic Cofactor α-Ketoglutarate.

J Immunol 2021 04 17;206(7):1425-1435. Epub 2021 Feb 17.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia;

Mucosal-associated invariant T (MAIT) cells are an innate-like population of unconventional T cells that respond rapidly to microbial metabolite Ags or cytokine stimulation. Because of this reactivity and surface expression of CD45RO, CD45RA, and CD127, they are described as effector memory cells. Yet, there is heterogeneity in MAIT cell effector response. It is unclear what factors control MAIT cell effector capacity, whether it is fixed or can be modified and if this differs based on whether activation is TCR dependent or independent. To address this, we have taken a systematic approach to examine human MAIT cell effector capacity across healthy individuals in response to ligand and cytokine stimulation. We demonstrate the heterogenous nature of MAIT cell effector capacity and that the ability to produce an effector response is not directly attributable to TCR clonotype or coreceptor expression. Global gene transcription analysis revealed that the MAIT cell effector capacity produced in response to TCR stimulation is associated with increased expression of the epigenetic regulator lysine demethylase 6B (KDM6B). Addition of a KDM6B inhibitor did not alter MAIT cell effector function to Ag or cytokine stimulation. However, addition of the KDM6B cofactor α-ketoglutarate greatly enhanced MAIT cell effector capacity to TCR-dependent stimulation in a partially KDM6B-dependent manner. These results demonstrate that the TCR-dependent effector response of MAIT cells is epigenetically regulated and dependent on the availability of metabolic cofactors.
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http://dx.doi.org/10.4049/jimmunol.2001048DOI Listing
April 2021

Anthem: a user customised tool for fast and accurate prediction of binding between peptides and HLA class I molecules.

Brief Bioinform 2021 Sep;22(5)

Monash Biomedicine Discovery Institute and Biochemistry and Molecular Biology, Monash University, Australia.

Neopeptide-based immunotherapy has been recognised as a promising approach for the treatment of cancers. For neopeptides to be recognised by CD8+ T cells and induce an immune response, their binding to human leukocyte antigen class I (HLA-I) molecules is a necessary first step. Most epitope prediction tools thus rely on the prediction of such binding. With the use of mass spectrometry, the scale of naturally presented HLA ligands that could be used to develop such predictors has been expanded. However, there are rarely efforts that focus on the integration of these experimental data with computational algorithms to efficiently develop up-to-date predictors. Here, we present Anthem for accurate HLA-I binding prediction. In particular, we have developed a user-friendly framework to support the development of customisable HLA-I binding prediction models to meet challenges associated with the rapidly increasing availability of large amounts of immunopeptidomic data. Our extensive evaluation, using both independent and experimental datasets shows that Anthem achieves an overall similar or higher area under curve value compared with other contemporary tools. It is anticipated that Anthem will provide a unique opportunity for the non-expert user to analyse and interpret their own in-house or publicly deposited datasets.
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http://dx.doi.org/10.1093/bib/bbaa415DOI Listing
September 2021

The Role of the HLA Class I α2 Helix in Determining Ligand Hierarchy for the Killer Cell Ig-like Receptor 3DL1.

J Immunol 2021 02 13;206(4):849-860. Epub 2021 Jan 13.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria 3000, Australia;

HLA class I molecules that represent ligands for the inhibitory killer cell Ig-like receptor (KIR) 3DL1 found on NK cells are categorically defined as those HLA-A and HLA-B allotypes containing the Bw4 motif, yet KIR3DL1 demonstrates hierarchical recognition of these HLA-Bw4 ligands. To better understand the molecular basis underpinning differential KIR3DL1 recognition, the HLA-A family of allotypes were investigated. Transfected human 721.221 cells expressing HLA-A*32:01 strongly inhibited primary human KIR3DL1 NK cells, whereas HLA-A*24:02 and HLA-A*23:01 displayed intermediate potency and HLA-A*25:01 failed to inhibit activation of KIR3DL1 NK cells. Structural studies demonstrated that recognition of HLA-A*24:02 by KIR3DL1 used identical contacts as the potent HLA-B*57:01 ligand. Namely, the D1-D2 domains of KIR3DL1 were placed over the α1 helix and α2 helix of the HLA-A*24:02 binding cleft, respectively, whereas the D0 domain contacted the side of the HLA-A*24:02 molecule. Nevertheless, functional analyses showed KIR3DL1 recognition of HLA-A*24:02 was more sensitive to substitutions within the α2 helix of HLA-A*24:02, including residues Ile and Lys Furthermore, the presence of Thr in the α2 helix of HLA-A*25:01 abrogated KIR3DL1 NK inhibition. Together, these data demonstrate a role for the HLA class I α2 helix in determining the hierarchy of KIR3DL1 ligands. Thus, recognition of HLA class I is dependent on a complex interplay between the peptide repertoire, polymorphisms within and proximal to the Bw4 motif, and the α2 helix. Collectively, the data furthers our understanding of KIR3DL1 ligands and will inform genetic association and immunogenetics studies examining the role of KIR3DL1 in disease settings.
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http://dx.doi.org/10.4049/jimmunol.2001109DOI Listing
February 2021

Human skin is colonized by T cells that recognize CD1a independently of lipid.

J Clin Invest 2021 01;131(1)

Division of Rheumatology, Inflammation and Immunity, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

CD1a-autoreactive T cells contribute to skin disease, but the identity of immunodominant self-lipid antigens and their mode of recognition are not yet solved. In most models, MHC and CD1 proteins serve as display platforms for smaller antigens. Here, we showed that CD1a tetramers without added antigen stained large T cell pools in every subject tested, accounting for approximately 1% of skin T cells. The mechanism of tetramer binding to T cells did not require any defined antigen. Binding occurred with approximately 100 lipid ligands carried by CD1a proteins, but could be tuned upward or downward with certain natural self-lipids. TCR recognition mapped to the outer A' roof of CD1a at sites remote from the antigen exit portal, explaining how TCRs can bind CD1a rather than carried lipids. Thus, a major antigenic target of CD1a T cell autoreactivity in vivo is CD1a itself. Based on their high frequency and prevalence among donors, we conclude that CD1a-specific, lipid-independent T cells are a normal component of the human skin T cell repertoire. Bypassing the need to select antigens and effector molecules, CD1a tetramers represent a simple method to track such CD1a-specific T cells from tissues and in any clinical disease.
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http://dx.doi.org/10.1172/JCI140706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773353PMC
January 2021

The cryo-EM structure of the endocytic receptor DEC-205.

J Biol Chem 2021 Jan-Jun;296:100127. Epub 2020 Dec 3.

Infection and Immunity Program, Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia; Australian Research Council Centre of Excellence for Advanced Molecular Imaging, Monash University, Clayton, Victoria, Australia. Electronic address:

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine-guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.
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http://dx.doi.org/10.1074/jbc.RA120.016451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948739PMC
August 2021

Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility.

J Exp Med 2021 02;218(2)

Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.
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http://dx.doi.org/10.1084/jem.20200476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595743PMC
February 2021

Cytomegalovirus replication is associated with enrichment of distinct γδ T cell subsets following lung transplantation: A novel therapeutic approach?

J Heart Lung Transplant 2020 Aug 26. Epub 2020 Aug 26.

Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia; Lung Transplant Service, The Alfred Hospital, Melbourne, Victoria, Australia. Electronic address:

Background: Anti-viral treatments to control cytomegalovirus (CMV) after lung transplantation (LTx) are associated with toxicity and anti-viral resistance. Cellular immunotherapy with virus-specific cytotoxic T cells has yielded promising results but requires donor/recipient matching. γδ T cells are involved in anti-viral immunity and can recognize antigens independently of major histocompatibility complex molecules and may not require the same level of matching. We assessed the phenotype of circulating γδ T cells after LTx to identify the candidate populations for CMV immunotherapy.

Methods: Peripheral blood mononuclear cells were isolated from lung transplant recipients before transplantation and at routine bronchoscopies after LTx. Patients were stratified by risk of CMV disease into moderate risk (recipient CMV seropositive, n = 15) or high risk (HR) (recipient CMV seronegative/donor CMV seropositive, n = 10). CMV replication was classified as polymerase chain reaction positive (>150 copies/ml) in blood and/or bronchoalveolar lavage within the first 18 months. The phenotype of γδ T cells was assessed by multicolor flow cytometry, and T-cell receptor (TCR) sequences were determined by deep sequencing.

Results: In HR lung transplant recipients with CMV replication, we observed striking phenotypic changes in γδ T cells, marked by an increase in the proportion of effector Vδ1+ γδ T cells expressing the activating natural killer cell receptor NKG2C. Moreover, we observed a remarkable increase in TCR diversity.

Conclusions: NKG2C+ Vδ1+ γδ T cells were associated with CMV replication and may indicate their potential to control infection. As such, we propose that they could be a potential target for cellular therapy against CMV.
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http://dx.doi.org/10.1016/j.healun.2020.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448790PMC
August 2020

Endoplasmic reticulum chaperones stabilize ligand-receptive MR1 molecules for efficient presentation of metabolite antigens.

Proc Natl Acad Sci U S A 2020 10 21;117(40):24974-24985. Epub 2020 Sep 21.

Department of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia;

The antigen-presenting molecule MR1 (MHC class I-related protein 1) presents metabolite antigens derived from microbial vitamin B synthesis to activate mucosal-associated invariant T (MAIT) cells. Key aspects of this evolutionarily conserved pathway remain uncharacterized, including where MR1 acquires ligands and what accessory proteins assist ligand binding. We answer these questions by using a fluorophore-labeled stable MR1 antigen analog, a conformation-specific MR1 mAb, proteomic analysis, and a genome-wide CRISPR/Cas9 library screen. We show that the endoplasmic reticulum (ER) contains a pool of two unliganded MR1 conformers stabilized via interactions with chaperones tapasin and tapasin-related protein. This pool is the primary source of MR1 molecules for the presentation of exogenous metabolite antigens to MAIT cells. Deletion of these chaperones reduces the ER-resident MR1 pool and hampers antigen presentation and MAIT cell activation. The MR1 antigen-presentation pathway thus co-opts ER chaperones to fulfill its unique ability to present exogenous metabolite antigens captured within the ER.
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http://dx.doi.org/10.1073/pnas.2011260117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547156PMC
October 2020

Suboptimal SARS-CoV-2-specific CD8 T cell response associated with the prominent HLA-A*02:01 phenotype.

Proc Natl Acad Sci U S A 2020 09 10;117(39):24384-24391. Epub 2020 Sep 10.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia;

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8 T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8 and CD4 T cells in vitro, with CD4 T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8 T cell epitopes, A2/S and A2/Orf1ab Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S CD8 and A2/Orf1ab CD8 populations indicated that A2/S CD8 T cells were detected at comparable frequencies (∼1.3 × 10) in acute and convalescent HLA-A*02:01 patients. These frequencies were higher than those found in uninfected HLA-A*02:01 donors (∼2.5 × 10), but low when compared to frequencies for influenza-specific (A2/M1) and Epstein-Barr virus (EBV)-specific (A2/BMLF) (∼1.38 × 10) populations. Phenotyping A2/S CD8 T cells from COVID-19 convalescents ex vivo showed that A2/S CD8 T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8 T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S CD8 T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8 T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8 T cell immunity in COVID-19.
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http://dx.doi.org/10.1073/pnas.2015486117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533701PMC
September 2020
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