Human Leukocyte Antigen (HLA)-G is selectively expressed by fetal trophoblast cells that invade maternal tissue and encounter maternal natural killer (NK) cells early in pregnancy. In NK cells, the endosomal receptor KIR2DL4 responds to soluble HLA-G by inducing a broad transcriptional program to support placental development. Structural features of KIR2DL4 that control ligand binding and endocytosis are unclear. Random mutagenesis revealed that three cysteines in the first immunoglobulin domain of KIR2DL4 regulate endocytosis and uptake of HLA-G. We found that the atypical Cys10-Cys28 disulfide bond observed in the KIR2DL4 crystal structure is an allosteric disulfide with potential to switch to a conventional Cys28-Cys74 bond. KIR2DL4 in human cells exists in both disulfide-bonded states, as shown by mass spectrometry analysis. The Cys10-Cys28 bond in a Cys74Ser KIR2DL4 mutant was reduced by protein disulfide isomerase (PDI) in vitro. Inhibition of PDI prevented HLA-G uptake by KIR2DL4 in both transfected 293T cells and primary NK cells. Mutants in the Cys10-Cys28 configuration endocytosed spontaneously but did not bind HLA-G. Conversely, KIR2DL4 with a Cys28-Cys74 bond was at the plasma membrane and responded to soluble HLA-G by endocytosis and transcription of the interferon stimulated gene IFI44L, like wild-type cells. A purified Cys10Leu KIR2DL4 mutant, which exhibited a reduced tendency to oligomerize, bound HLA-G in a peptide dependent manner with an affinity in the low micromolar range. Disulfide switching from the Cys10-Cys28 to the Cys28-Cys74 form correlated with a distant allosteric change, as predicted by AlphaFold, that could reorient a D0 domain loop to facilitate HLA-G binding. Thus, conversion from an inactive state to an HLA-G binding form regulates KIR2DL4 cellular localization and function to promote fetal development.

Understanding host factors driving asymptomatic versus severe disease outcomes is of key importance if we are to control emerging and re-emerging viral infections. HLA-B*15:01 has been associated with asymptomatic SARS-CoV-2 infection in nonhospitalized individuals of European ancestry, with protective immunity attributed to preexisting cross-reactive CD8+ T-cells directed against HLA-B*15:01-restricted Spike-derived S919-927 peptide (B15/S919+CD8+ T-cells). However, fundamental questions remained on the abundance and clonotypic nature of CD8+ T-cell responses in HLA-B*15:01-positive patients who succumbed to life-threatening COVID-19. Here, we analyzed B15/S919+CD8+ T-cell responses in COVID-19 patients from independent HLA-typed COVID-19 patient cohorts across three continents, Australia, Asia and Europe. We assessed B15/S919+CD8+ T-cells in COVID-19 patients across disease outcomes ranging from asymptomatic to hospitalized critical illness. We found that severe/critical COVID-19 patients mounted B15/S919+CD8+ T-cell responses lacking a highly expanded key public B15/S919+CD8+ T-cell receptor (TCR; TRAV9-2/TRBV7-2) which recurred across multiple individuals in COVID-19 patients with a mild disease. Instead, B15/S919+CD8+ T-cell responses in life-threatening disease had a prevalence of an alternate TCR clonotypic motif (TRAV38-2/DV8/TRBV20-1), potentially contributing, at least in part, to why B15/S919+CD8+ T-cells in severe COVID-19 patients were less protective. Interestingly, the frequency, memory phenotype, and activation profiles of circulating B15/S919+CD8+ T-cells did not differ across disease severity. Moreover, B15/S919+CD8+ T-cells were better maintained into convalescence compared to other SARS-CoV-2-specificities. Our study thus provides evidence on the differential nature of the TCR clonal repertoire in 22.37% of HLA-B*15:01-positive COVID-19 patients who developed severe or critical disease in our cohorts, comparing to HLA-B*15:01-expressing individuals with mild COVID-19.

Cross-reactive αβ T cell receptors (TCRs) recognizing multiple peptide variants can provide effective control of rapidly evolving viruses yet remain understudied. By screening 12 naturally occurring influenza-derived HLA-B*35:01–restricted nucleoprotein (NP) 418–426 epitopes (B*35:01-NP 418 ) that emerged since 1918 within influenza A viruses, including 2024 A/H5N1 viruses, we identified functional broadly cross-reactive T cells universally recognizing NP 418 variants. Binding studies demonstrated that TCR cross-reactivity was concomitant with diminished antigen sensitivity. Primary human B*35:01/NP 418 + CD8 + T cell lines displayed reduced cross-reactivity in the absence of CD8 coreceptor binding, validating the low avidity of cross-reactive B*35:01-NP 418 + CD8 + T cell responses. Six TCR–HLA-B*35:01/NP 418 crystal structures showed how cross-reactive TCRs recognized multiple B*35:01/NP 418 epitope variants. Specific TCR interactions were formed with invariant and conserved peptide-HLA features, thus remaining distal from highly varied positions of the NP 418 epitope. Our study defines molecular mechanisms associated with extensive TCR cross-reactivity toward naturally occurring viral variants highly relevant to universal protective immunity against influenza.

Most COVID-19 vaccines elicit immunity against the SARS-CoV-2 Spike protein. However, Spike protein mutations in emerging strains and immune evasion by the SARS-CoV-2 virus demonstrates the need to develop more broadly targeting vaccines. To facilitate this, we use mass spectrometry to identify immunopeptides derived from seven relatively conserved structural and non-structural SARS-CoV-2 proteins (N, E, Nsp1/4/5/8/9). We use two different B-lymphoblastoid cell lines to map Human Leukocyte Antigen (HLA) class I and class II immunopeptidomes covering some of the prevalent HLA types across the global human population. We employ DNA plasmid transfection and direct antigen delivery approaches to sample different antigens and find 248 unique HLA class I and HLA class II bound peptides with 71 derived from N, 12 from E, 28 from Nsp1, 19 from Nsp4, 73 from Nsp8 and 45 peptides derived from Nsp9. Over half of the viral peptides are unpublished. T cell reactivity tested against 56 of the detected peptides shows CD8+ and CD4+ T cell responses against several peptides from the N, E, and Nsp9 proteins. Results from this study will aid the development of next-generation COVID vaccines targeting epitopes from across a number of SARS-CoV-2 proteins.

T cell activity is governed by T cell receptor (TCR) signaling and constrained by immune checkpoint molecules, including programmed cell death protein 1 (PD-1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), and lymphocyte activation gene 3 (LAG-3). The basis for how LAG-3 binds to human leukocyte antigen class II molecules (HLA-II) remains unknown. Here, we present the 3.4-angstrom crystal structure of a LAG-3-peptide-HLA-II complex and probe the energetics of the complex interface. Coincident with the HLA-II binding site of the ancestrally related, monomeric CD4 receptor, the LAG-3 homodimer laterally engages two HLA-II molecules via distal D1 domain surfaces, imposing a 38 degrees angular offset. The LAG-3-HLA-II interface is discontinuous and lacks involvement of the D1 extra loop, a binding site for anti-LAG-3 therapeutic monoclonal antibodies. Upon HLA-II binding, intrinsically mobile loops of the LAG-3 molecule become ordered, with contact residues highly conserved across HLA-DR, DQ, and DP allomorphs. Our data provide a structural foundation for development of immunomodulatory approaches targeting LAG-3.

Long COVID occurs in a small but important minority of patients following COVID-19, reducing quality of life and contributing to healthcare burden. Although research into underlying mechanisms is evolving, immunity is understudied. SARS- CoV-2- specific T cell responses are of key importance for viral clearance and COVID-19 recovery. However, in long COVID, the establishment and persistence of SARS- CoV-2- specific T cells are far from clear, especially beyond 12 mo postinfection and postvaccination. We defined ex vivo antigen- specific B cell and T cell responses and their T cell receptors (TCR) repertoires across 2 y postinfection in people with long COVID. Using 13 SARS-CoV-2 peptide-HLA tetramers, spanning 11 HLA allotypes, as well as spike and nucleocapsid probes, we tracked SARS- CoV-2- specific CD8+ and CD4+ T cells and B- cells in individuals from their first SARS-CoV-2 infection through primary vaccination over 24 mo. The frequencies of ORF1a- and nucleocapsid- specific T cells and B cells remained stable over 24 mo. Spike- specific CD8+ and CD4+ T cells and B cells were boosted by SARS-CoV-2 vaccination, indicating immunization, in fully recovered and people with long COVID, altered the immunodominance hierarchy of SARS-CoV-2 T cell epitopes. Meanwhile, influenza- specific CD8+ T cells were stable across 24 mo, suggesting no bystander- activation. Compared to total T cell populations, SARS- CoV-2- specific T cells were enriched for central memory phenotype, although the proportion of central memory T cells decreased following acute illness. Importantly, TCR repertoire composition was maintained throughout long COVID, including post- vaccination, to 2 y postinfection. Overall, we defined ex vivo SARS- CoV-2- specific B cells and T cells to understand primary and recall responses, providing key insights into antigen- specific responses in people with long COVID.

Influenza B viruses (IBVs) cause substantive morbidity and mortality, and yet immunity towards IBVs remains understudied. CD8+ T-cells provide broadly cross-reactive immunity and alleviate disease severity by recognizing conserved epitopes. Despite the IBV burden, only 18 IBV-specific T-cell epitopes restricted by 5 HLAs have been identified currently. A broader array of conserved IBV T-cell epitopes is needed to develop effective cross-reactive T-cell based IBV vaccines. Here we identify 9 highly conserved IBV CD8+ T-cell epitopes restricted to HLA-B*07:02, HLA-B*08:01 and HLA-B*35:01. Memory IBV-specific tetramer+CD8+ T-cells are present within blood and tissues. Frequencies of IBV-specific CD8+ T-cells decline with age, but maintain a central memory phenotype. HLA-B*07:02 and HLA-B*08:01-restricted NP30-38 epitope-specific T-cells have distinct T-cell receptor repertoires. We provide structural basis for the IBV HLA-B*07:02-restricted NS1196-206 (11-mer) and HLA-B*07:02-restricted NP30-38 epitope presentation. Our study increases the number of IBV CD8+ T-cell epitopes, and defines IBV-specific CD8+ T-cells at cellular and molecular levels, across tissues and age.

Algae encode up to five different types of cryptochrome photoreceptors. So far, relatively little is known about the biological functions of the DASH (Drosophila, Arabidopsis, Synechocystis and Homo)-type cryptochromes. The green alga Chlamydomonas reinhardtii encodes two of them. CRY-DASH1 also called DCRY1 has its maximal absorption peak in the UV-A range. It is localized in the chloroplast and plays an important role in balancing the photosynthetic machinery. Here, we performed a comparative analysis of chloroplast proteins from wild type and a knockout mutant of CRY-DASH1 named cry-dash1mut, using label-free quantitative proteomics as well as immunoblotting. Our results show upregulation of enzymes involved in specific pathways in the mutant including key enzymes of chlorophyll and carotenoid biosynthesis consistent with increased levels of photosynthetic pigments in cry-dash1mut. There is also an increase in certain redox as well as photosystem I and II proteins, including D1. Strikingly, CRY-DASH1 is coregulated in a D1 deletion mutant, where its amount is increased. In contrast, key proteins of the central carbon metabolism, including glycolysis/gluconeogenesis, dark fermentation and the oxidative pentose phosphate pathway are downregulated in cry-dash1mut. Similarly, enzymes of histidine biosynthesis are downregulated in cry-dash1mut leading to a reduction in the amount of free histidine. Yet, transcripts encoding for several of these proteins are at a similar level in the wild type and cry-dash1mut or even opposite. We show that CRY-DASH1 can bind to RNA, taking the psbA RNA encoding D1 as target. These data suggest that CRY-DASH1 regulates plastidial metabolic pathways at the posttranscriptional level.

Indigenous peoples globally are at increased risk of COVID‐19‐associated morbidity and mortality. However, data that describe immune responses to SARS‐CoV‐2 infection in Indigenous populations are lacking. We evaluated immune responses in Australian First Nations peoples hospitalized with COVID‐19. Our work comprehensively mapped out inflammatory, humoral and adaptive immune responses following SARS‐CoV‐2 infection. Patients were recruited early following the lifting of strict public health measures in the Northern Territory, Australia, between November 2021 and May 2022. Australian First Nations peoples recovering from COVID‐19 showed increased levels of MCP‐1 and IL‐8 cytokines, IgG‐antibodies against Delta‐RBD and memory SARS‐CoV‐2‐specific T cell responses prior to hospital discharge in comparison with hospital admission, with resolution of hyperactivated HLA‐DR + CD38 + T cells. SARS‐CoV‐2 infection elicited coordinated ASC, Tfh and CD8 + T cell responses in concert with CD4 + T cell responses. Delta and Omicron RBD‐IgG, as well as Ancestral N‐IgG antibodies, strongly correlated with Ancestral RBD‐IgG antibodies and Spike‐specific memory B cells. We provide evidence of broad and robust immune responses following SARS‐CoV‐2 infection in Indigenous peoples, resembling those of non‐Indigenous COVID‐19 hospitalized patients.

Although the protective role of neutralizing antibodies against COVID-19 is well established, questions remain about the relative importance of cellular immunity. Using 6 pMHC multimers in a cohort with early and frequent sampling, we define the phenotype and kinetics of recalled and primary T cell responses following Delta or Omicron breakthrough infection in previously vaccinated individuals. Recall of spike-specific CD4+ T cells was rapid, with cellular proliferation and extensive activation evident as early as 1 day post symptom onset. Similarly, spike-specific CD8+ T cells were rapidly activated but showed variable degrees of expansion. The frequency of activated SARS-CoV-2-specific CD8+ T cells at baseline and peak inversely correlated with peak SARS-CoV-2 RNA levels in nasal swabs and accelerated viral clearance. Our study demonstrates that a rapid and extensive recall of memory T cell populations occurs early after breakthrough infection and suggests that CD8+ T cells contribute to the control of viral replication in breakthrough SARS-CoV-2 infections.

Immunocompromised hematology patients are vulnerable to severe COVID-19 and respond poorly to vacci-nation. Relative deficits in immunity are, however, unclear, especially after 3 vaccine doses. We evaluated immune responses in hematology patients across three COVID-19 vaccination doses. Seropositivity was low after a first dose of BNT162b2 and ChAdOx1 (-26%), increased to 59%-75% after a second dose, and increased to 85% after a third dose. While prototypical antibody-secreting cells (ASCs) and T follicular helper (Tfh) cell responses were elicited in healthy participants, hematology patients showed prolonged ASCs and skewed Tfh2/17 responses. Importantly, vaccine-induced expansions of spike-specific and pep-tide-HLA tetramer-specific CD4+/CD8+ T cells, together with their T cell receptor (TCR) repertoires, were robust in hematology patients, irrespective of B cell numbers, and comparable to healthy participants. Vacci-nated patients with breakthrough infections developed higher antibody responses, while T cell responses were comparable to healthy groups. COVID-19 vaccination induces robust T cell immunity in hematology patients of varying diseases and treatments irrespective of B cell numbers and antibody response.

CD4 + T cell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and life-threatening invasive pneumococcal diseases (IPDs), including pneumonia, sepsis, and meningitis [1][2][3].With the emergence of new pneumococcal strains, there is an urgent need for vaccines that elicit broader population coverage against conserved pneumococcal antigens, irrespective of capsular serotype [2].Pneumolysin (Ply) is a key pneumococcal virulence factor belonging to a family of cholesterol-dependent cytolysins (CDCs) that disrupts host cell defence mechanisms and immune cell function.This cytotoxin is expressed by virtually all pneumococcal strains and pneumococcal carriage and infection induce natural immunity to Ply [3][4].Consequently, a detoxified form of this protein has been tested as a potential serotype-independent vaccine candidate to protect against IPD [5][6][7].In this study, we identified a highly immunogenic human CD4 + T cell epitope in Ply, presented by globally abundant HLA allotypes.We reveal that within the Ply epitope-specific T cell receptor (TCR) repertoire of healthy HLA-typed adults, there are a diverse array of public (shared between individuals) and private (individual-specific) TCRs.Moreover, the immunogenicity of the Ply epitope was underpinned by conserved residues in the core region of the peptide which enables cross-recognition of other bacterial pathogens expressing CDCs.Our structural data further showed that the HLA-Ply antigenic complex was engaged similarly by one representative public TCR and two distinct private TCRs.Taken together, these findings provide detailed molecular insights into CD4 + T cell-mediated recognition of a trans-species bacterial epitope, which may lead to the design of T cell-based interventions against infections involving S. pneumoniae as well as other CDC-producing bacterial pathogens.

Most human killer cell immunoglobulin-like receptors (KIR) are expressed by natural killer (NK) cells and recognize HLA class I molecules as ligands. KIR3DL3 is a conserved but polymorphic inhibitory KIR recognizing a B7 family ligand, HHLA2, and is implicated for immune checkpoint targeting. The expression profile and biological function of KIR3DL3 have been somewhat elusive, so we searched extensively for KIR3DL3 transcripts, revealing highly enriched expression in γδ and CD8+ T cells rather than NK cells. These KIR3DL3-expressing cells are rare in the blood and thymus but more common in the lungs and digestive tract. High-resolution flow cytometry and single-cell transcriptomics showed that peripheral blood KIR3DL3+ T cells have an activated transitional memory phenotype and are hypofunctional. The T cell receptor (TCR) usage is biased toward genes from early rearranged TCR-α variable segments or Vδ1 chains. In addition, we show that TCR-mediated stimulation can be inhibited through KIR3DL3 ligation. Whereas we detected no impact of KIR3DL3 polymorphism on ligand binding, variants in the proximal promoter and at residue 86 can reduce expression. Together, we demonstrate that KIR3DL3 is up-regulated alongside unconventional T cell stimulation and that individuals may vary in their ability to express KIR3DL3. These results have implications for the personalized targeting of KIR3DL3/HHLA2 checkpoint inhibition.

CD4+ T cell-mediated immunity against Streptococcus pneumoniae (pneumococcus) can protect against recurrent bacterial colonization and invasive pneumococcal diseases (IPDs). Although such immune re-sponses are common, the pertinent antigens have remained elusive. We identified an immunodominant CD4+ T cell epitope derived from pneumolysin (Ply), a member of the bacterial cholesterol-dependent cyto-lysins (CDCs). This epitope was broadly immunogenic as a consequence of presentation by the pervasive hu-man leukocyte antigen (HLA) allotypes DPB1*02 and DPB1*04 and recognition via architecturally diverse T cell receptors (TCRs). Moreover, the immunogenicity of Ply427-444 was underpinned by core residues in the conserved undecapeptide region (ECTGLAWEWWR), enabling cross-recognition of heterologous bacte-rial pathogens expressing CDCs. Molecular studies further showed that HLA-DP4-Ply427-441 was engaged similarly by private and public TCRs. Collectively, these findings reveal the mechanistic determinants of near-global immune focusing on a trans-phyla bacterial epitope, which could inform ancillary strategies to combat various life-threatening infectious diseases, including IPDs.

Celiac disease (CeD) is a chronic inflammatory autoimmune-like condition characterised by disease-relevant HLA-DQ2.5/8molecules that present gluten epitopes derived from wheat, rye and barley to reactive T cells [1,2].We investigated CD4+ T cell responses towards immunodominant gluten epitopes; DQ2.5-glia-α1a (PFPQPELPY) and DQ2.5-glia-ω1 (PFPQPEQPF) by examining patient T cell repertoire [3,4].It was found to be composed of highly specific and cross-reactive T cell clones (TCCs) [4].Next, we determined the ternary complex structures of cross-reactive T cell receptor (TCR) bound to HLA-DQ2.5-glia-α1a and HLA-DQ2.5-glia-ω1 and epitope-specific TCR bound to HLA-DQ2.5-glia-ω1 specific TCR [3,5].The interactions at the interface of TCR: peptide-HLA-DQ2.5 required for specificity provide an insight into how the TCRs distinguish between these highly similar peptides [5].Comparison of the TCR footprint contacts of the ternary complexes for both peptides revealed similar canonical TCR docking with slight shift in docking angle [3,5, 6].The hypervariable CDR3 loops were shown to be responsible for differential TCR recognition capacities of the cross-reactive versus discriminatory TCRs [5,7].We measured SPR binding affinities of the TCR-pHLA interaction and found that the cross-reactive TCR is selectively promiscuous in binding as this TCR only bound these epitopes with similar affinity [5,7].This study highlights that cross-reactive T cells may also contribute to CeD pathogenesis [5,7].Furthermore, these cross-reactive and discriminatory T cells may prove to be important therapeutic targets in treatment of CeD [5,7, 8].

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

Summary SARS-CoV-2 mRNA vaccines induce robust anti-spike (S) antibody and CD4+ T cell responses. It is not yet clear whether vaccine-induced follicular helper CD4+ T (TFH) cell responses contribute to this outstanding immunogenicity. Using fine needle aspiration of draining axillary lymph nodes from individuals who received the BNT162b2 mRNA vaccine, we show that frequency of TFH correlates with that of S-binding germinal center B cells. Mining of the responding TFH T cell receptor repertoire revealed a strikingly immunodominant HLADPB1* 04-restricted response to S167-180 in individuals with this allele, which is among the most common HLA alleles in humans. Paired blood and lymph node specimens show that while circulating S-specific TFH cells peak one week after the second immunization, S-specific TFH persist at nearly constant frequencies for at least six months. Collectively, our results underscore the key role that robust TFH cell responses play in establishing long-term immunity by this efficacious human vaccine.

Lymphocyte activation gene 3 (LAG3) is an important checkpoint inhibitor molecule of immunotherapeutic interest. New crystal structures of LAG3 provide important insight into its molecular architecture, laying the groundwork for future basic and applied investigations.

As the establishment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cell memory in children remains largely unexplored, we recruited convalescent COVID-19 children and adults to define their circulating memory SARS-CoV-2-specific CD4+ and CD8+ T cells prior to vaccination. We analyzed epitope-specific T cells directly ex vivo using seven HLA class I and class II tetramers presenting SARS-CoV-2 epitopes, together with Spike-specific B cells. Unvaccinated children who seroconverted had comparable Spike-specific but lower ORF1a- and N-specific memory T cell responses compared with adults. This agreed with our TCR sequencing data showing reduced clonal expansion in children. A strong stem cell memory phenotype and common T cell receptor motifs were detected within tetramer-specific T cells in seroconverted children. Conversely, children who did not seroconvert had tetramer-specific T cells of predominantly naive phenotypes and diverse TCRαβ repertoires. Our study demonstrates the generation of SARS-CoV-2-specific T cell memory with common TCRαβ motifs in unvaccinated seroconverted children after their first virus encounter.