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Aims/hypothesis: The aim of this work was to explore molecular amino acids (AAs) and related structures of HLA-DQA1-DQB1 that underlie its contribution to the progression from stages 1 or 2 to stage 3 type 1 diabetes., Methods: Using high-resolution DQA1 and DQB1 genotypes from 1216 participants in the Diabetes Prevention Trial-Type 1 and the Diabetes Prevention Trial, we applied hierarchically organised haplotype association analysis (HOH) to decipher which AAs contributed to the associations of DQ with disease and their structural properties. HOH relied on the Cox regression to quantify the association of DQ with time-to-onset of type 1 diabetes., Results: By numerating all possible DQ heterodimers of α- and β-chains, we showed that the heterodimerisation increases genetic diversity at the cellular level from 43 empirically observed haplotypes to 186 possible heterodimers. Heterodimerisation turned several neutral haplotypes (DQ2.2, DQ2.3 and DQ4.4) to risk haplotypes (DQ2.2/2.3-DQ4.4 and DQ4.4-DQ2.2). HOH uncovered eight AAs on the α-chain (-16α, -13α, -6α, α22, α23, α44, α72, α157) and six AAs on the β-chain (-18β, β9, β13, β26, β57, β135) that contributed to the association of DQ with progression of type 1 diabetes. The specific AAs concerned the signal peptide (minus sign, possible linkage to expression levels), pockets 1, 4 and 9 in the antigen-binding groove of the α1β1 domain, and the putative homodimerisation of the αβ heterodimers., Conclusions/interpretation: These results unveil the contribution made by DQ to type 1 diabetes progression at individual residues and related protein structures, shedding light on its immunological mechanisms and providing new leads for developing treatment strategies., Data Availability: Clinical trial data and biospecimen samples are available through the National Institute of Diabetes and Digestive and Kidney Diseases Central Repository portal ( https://repository.niddk.nih.gov/studies )., (© 2024. The Author(s).)

Objective: To explore if oral insulin could delay onset of stage 3 type 1 diabetes (T1D) among patients with stage 1/2 who carry HLA DR4-DQ8 and/or have elevated levels of IA-2 autoantibodies (IA-2As)., Research and Methods: Next-generation targeted sequencing technology was used to genotype eight HLA class II genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, and DPB1) in 546 participants in the TrialNet oral insulin preventative trial (TN07). Baseline levels of autoantibodies against insulin (IAA), GAD65 (GADA), and IA-2A were determined prior to treatment assignment. Available clinical and demographic covariables from TN07 were used in this post hoc analysis with the Cox regression model to quantify the preventive efficacy of oral insulin., Results: Oral insulin reduced the frequency of T1D onset among participants with elevated IA-2A levels (HR 0.62; P = 0.012) but had no preventive effect among those with low IA-2A levels (HR 1.03; P = 0.91). High IA-2A levels were positively associated with the HLA DR4-DQ8 haplotype (OR 1.63; P = 6.37 × 10-6) and negatively associated with the HLA DR7-containing DRB1*07:01-DRB4*01:01-DQA1*02:01-DQB1*02:02 extended haplotype (OR 0.49; P = 0.037). Among DR4-DQ8 carriers, oral insulin delayed the progression toward stage 3 T1D onset (HR 0.59; P = 0.027), especially if participants also had high IA-2A level (HR 0.50; P = 0.028)., Conclusions: These results suggest the presence of a T1D endotype characterized by HLA DR4-DQ8 and/or elevated IA-2A levels; for those patients with stage 1/2 disease with such an endotype, oral insulin delays the clinical T1D onset., (© 2024 by the American Diabetes Association.)

This study examined how compensatory and enabling domains of an Age-Friendly City (AFC) moderate the relationship between suspected mental health problems and depressive and anxiety symptoms among older adults. Four thousand six hundred and twenty-five Hong Kong Chinese aged ≥60 years completed a telephone survey between April and July 2022, including PHQ-2 and GAD-2. AFC indices sourced from prior territory-wide study. Linear mixed models showed that enabling AFC domains, namely, social participation, respect and social inclusion, and civic participation and employment, alleviated the effects of suspected mental health problems on respondents' depressive and anxiety symptoms (b = -0.40 to -0.56). Three-way interaction models revealed that the protective effects of all compensatory and enabling AFCC domains (b = -1.23 to -6.18), except civic participation and employment, were stronger in old-old (70-79 years) and oldest-old (≥80 years) than young-old (60-69 years). AFCC-based interventions should focus on compensatory and enabling domains to support older adults' mental health., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objective: To explore associations of HLA class II genes (HLAII) with the progression of islet autoimmunity from asymptomatic to symptomatic type 1 diabetes (T1D)., Research Design and Methods: Next-generation targeted sequencing was used to genotype eight HLAII genes (DQA1, DQB1, DRB1, DRB3, DRB4, DRB5, DPA1, DPB1) in 1,216 participants from the Diabetes Prevention Trial-1 and Randomized Diabetes Prevention Trial with Oral Insulin sponsored by TrialNet. By the linkage disequilibrium, DQA1 and DQB1 are haplotyped to form DQ haplotypes; DP and DR haplotypes are similarly constructed. Together with available clinical covariables, we applied the Cox regression model to assess HLAII immunogenic associations with the disease progression., Results: First, the current investigation updated the previously reported genetic associations of DQA1*03:01-DQB1*03:02 (hazard ratio [HR] = 1.25, P = 3.50*10-3) and DQA1*03:03-DQB1*03:01 (HR = 0.56, P = 1.16*10-3), and also uncovered a risk association with DQA1*05:01-DQB1*02:01 (HR = 1.19, P = 0.041). Second, after adjusting for DQ, DPA1*02:01-DPB1*11:01 and DPA1*01:03-DPB1*03:01 were found to have opposite associations with progression (HR = 1.98 and 0.70, P = 0.021 and 6.16*10-3, respectively). Third, DRB1*03:01-DRB3*01:01 and DRB1*03:01-DRB3*02:02, sharing the DRB1*03:01, had opposite associations (HR = 0.73 and 1.44, P = 0.04 and 0.019, respectively), indicating a role of DRB3. Meanwhile, DRB1*12:01-DRB3*02:02 and DRB1*01:03 alone were found to associate with progression (HR = 2.6 and 2.32, P = 0.018 and 0.039, respectively). Fourth, through enumerating all heterodimers, it was found that both DQ and DP could exhibit associations with disease progression., Conclusions: These results suggest that HLAII polymorphisms influence progression from islet autoimmunity to T1D among at-risk subjects with islet autoantibodies., (© 2024 by the American Diabetes Association.)

Context: Validated assays to measure autoantigen-specific T-cell frequency and phenotypes are needed for assessing the risk of developing diabetes, monitoring disease progression, evaluating responses to treatment, and personalizing antigen-based therapies., Objective: Toward this end, we performed a technical validation of a tetramer assay for HLA-DRA-DRB1*04:01, a class II allele that is strongly associated with susceptibility to type 1 diabetes (T1D)., Methods: HLA-DRA-DRB1*04:01-restricted T cells specific for immunodominant epitopes from islet cell antigens GAD65, IGRP, preproinsulin, and ZnT8, and a reference influenza epitope, were enumerated and phenotyped in a single staining tube with a tetramer assay. Single and multicenter testing was performed, using a clone-spiked specimen and replicate samples from T1D patients, with a target coefficient of variation (CV) less than 30%. The same assay was applied to an exploratory cross-sectional sample set with 24 T1D patients to evaluate the utility of the assay., Results: Influenza-specific T-cell measurements had mean CVs of 6% for the clone-spiked specimen and 11% for T1D samples in single-center testing, and 20% and 31%, respectively, for multicenter testing. Islet-specific T-cell measurements in these same samples had mean CVs of 14% and 23% for single-center and 23% and 41% for multicenter testing. The cross-sectional study identified relationships between T-cell frequencies and phenotype and disease duration, sex, and autoantibodies. A large fraction of the islet-specific T cells exhibited a naive phenotype., Conclusion: Our results demonstrate that the assay is reproducible and useful to characterize islet-specific T cells and identify correlations between T-cell measures and clinical traits., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Hybrid training mode comprising in-person and teleconferencing sessions is effective and sustainable, yet no standardized principles guide its development for older people. This study aimed to develop a set of principles for hybrid-mode psychoeducation for older people from the experiences of middle-aged and older people in two folds: (1) examining the effects of hybrid-mode community psychoeducation and (2) identifying features that could enhance participants' experience., Methods: We delivered 12-hour Older Person Mental Health First Aid and 3-hour late-life depression training to adults aged 50 and older in in-person and hybrid modes. Hybrid group participants received technology-related support, including in-advance training and on-site support. All participants completed assessments on depression literacy, depression stigma, meaning in life, social support, depressive symptoms, and anxiety pre-and post-intervention and evaluated the program in open-ended questions., Results: A total of 471 in-person and 346 hybrid group participants completed the psychoeducation and post-assessment (80.4 % female, mean age = 64.73 years, SD = 7.29). Linear mixed models revealed improvements in depression literacy, depression stigma, meaning in life, social support, and anxiety (B = -1.43 to 0.13, all p  < .001), with no significant difference between in-person and hybrid groups. Thematic analysis of open-ended questions identified three themes: (1) informational content with case studies, (2) hardcopy course handouts, and (3) interactive learning environment., Discussion/conclusion: Hybrid-mode and in-person psychoeducation had comparable benefits on middle-aged and older people. The TORCH principles, an acronym for Technology provision, On-site technical support, Rehearsal, Connection with group members, and Hardcopy notes, was derived from practice wisdom and qualitative findings to support older people in online learning., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier B.V.)

Background and Objectives: Health and mental health interventions, such as psychotherapy and exercise programs, delivered via information and communication technology (ICT) may improve service access. However, adjustment among older people and in synchronous group interventions is more challenging. Technology affordance concerns the possibilities engendered by technology for various users and purposes and can help understand challenges in ICT-delivered groups and identify possible solutions., Research Design and Methods: Adopting a multiple triangulation approach, we observed ICT-delivered groups of acceptance and commitment therapy and exercise for older people with depressive symptoms, conducted focus groups with older people who had received group psychotherapy with or without an exercise component, and obtained clinical notes from interventionists. We conducted a thematic analysis of the observation notes, focus group transcriptions, and clinical notes., Results: Four focus groups were conducted with 22 participants (mean age = 72.6 years, standard deviation = 7.2, 86% female). We identified 3 challenges: (1) seeing-be seen dilemma, (2) speaking-hearing dilemma, and (3) blurred therapy-home boundary, and 2 solutions: (1) maneuvering layouts and collaborative tools, and (2) cross-platform mediated strategies. Participants struggled to observe the interventionist while simultaneously demonstrating their posture in front of a camera. Remaining silent and moderated turn-taking allowed for clearer hearing but limited interactions. Interruptions from the background environment and intersections of family living spaces disrupted audio-visual communication and jeopardized the sense of security. As a solution, interventionists maneuvered layouts and collaborative tools on teleconferencing applications to achieve intervention goals and provided support through different media., Discussion and Implications: The identified challenges and potential solutions can be understood from interactivity, portability, temporality, persistence, and multimediality. Technology affordance can guide ICT-delivered group design by matching the affordance of various technologies and communication media with the characteristics of the intervention and users to enhance efficacy and avoid an unnecessary digital divide., Competing Interests: None., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Gerontological Society of America.)

Background: Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions., Objective: This study aimed to determine whether inherent qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT)., Methods: We first performed ex vivo T-cell profiling on peanut-reactive CD154 + CD137 + T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4 + T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT., Results: Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6 - CRT H 2 + and CCR6 + CRT H 2 - ). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of T H 2A pTeff cells. PnOIT induced elimination of T H 2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of T H 2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation., Conclusion: Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies., (Copyright © 2023 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Introduction: The nonstructural protein 12 (NSP12) of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) has a high sequence identity with common cold coronaviruses (CCC)., Methods: Here, we comprehensively assessed the breadth and specificity of the NSP12-specific T-cell response after in vitro T-cell expansion with 185 overlapping 15-mer peptides covering the entire SARS-CoV-2 NSP12 at single-peptide resolution in a cohort of 27 coronavirus disease 2019 (COVID-19) patients. Samples of nine uninfected seronegative individuals, as well as five pre-pandemic controls, were also examined to assess potential cross-reactivity with CCCs., Results: Surprisingly, there was a comparable breadth of individual NSP12 peptide-specific CD4 + T-cell responses between COVID-19 patients (mean: 12.82 responses; range: 0-25) and seronegative controls including pre-pandemic samples (mean: 12.71 responses; range: 0-21). However, the NSP12-specific T-cell responses detected in acute COVID-19 patients were on average of a higher magnitude. The most frequently detected CD4 + T-cell peptide specificities in COVID-19 patients were aa236-250 (37%) and aa246-260 (44%), whereas the peptide specificities aa686-700 (50%) and aa741-755 (36%), were the most frequently detected in seronegative controls. In CCC-specific peptide-expanded T-cell cultures of seronegative individuals, the corresponding SARS-CoV-2 NSP12 peptide specificities also elicited responses in vitro . However, the NSP12 peptide-specific CD4 + T-cell response repertoire only partially overlapped in patients analyzed longitudinally before and after a SARS-CoV-2 infection., Discussion: The results of the current study indicate the presence of pre-primed, cross-reactive CCC-specific T-cell responses targeting conserved regions of SARS-CoV-2, but they also underline the complexity of the analysis and the limited understanding of the role of the SARS-CoV-2 specific T-cell response and cross-reactivity with the CCCs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Westphal, Mader, Karsten, Cords, Knapp, Schulte, Hermanussen, Peine, Ditt, Grifoni, Addo, Huber, Sette, Lütgehetmann, Pischke, Kwok, Sidney and Schulze zur Wiesch.)

Awareness of COVID-19 infection risk and oscillation patterns ('waves') may affect older people's mental health. Empirical data from populations experiencing multiple waves of community outbreaks can inform guidance for maintaining mental health. This study aims to investigate the effects of COVID-19 infection risk and oscillations on depression among community-dwelling older people in Hong Kong., A rolling cross-sectional telephone survey method was used. Screening for depression risk was conducted among 8,163 older people (age ≥ 60) using the Patient Health Questionnaire-2 (PHQ-2) from February to August 2020. The relationships between PHQ-2, COVID-19 infection risk proxies - change in newly infected cases and effective reproductive number (R t ), and oscillations - stage of a 'wave' reported in the media, were analysed using correlation and regression., 8.4% of survey respondents screened positive for depression risk. Being female ( β = .08), having a pre-existing mental health issue ( β = .21), change in newly infected cases ( β = .05), and screening during the latency period before the media called out new waves ( β = .03), contributed to higher depression risk ( R 2 = .06, all p <.01)., While depression risk does not appear alarming in this sample, our results highlight that older people are sensitive to reporting of infection, particularly among those with existing mental health needs. Future public health communication should balance awareness of infection risks with mental health protection.

Introduction: Previous studies verify the formation of enzymatically post-translationally modified (PTM) self-peptides and their preferred recognition by T cells in subjects with type 1 diabetes (T1D). However, questions remain about the relative prevalence of T cells that recognize PTM self-peptides derived from different antigens, their functional phenotypes, and whether their presence correlates with a specific disease endotype., Methods: To address this question, we identified a cohort of subjects with T1D who had diverse levels of residual beta cell function. Using previously developed HLA class II tetramer reagents, we enumerated T cells that recognize PTM GAD epitopes in the context of DRB1*04:01 or PTM IA2 epitopes in the context of DQB1*03:02 (DQ8)., Results: Consistent with prior studies, we observed higher overall frequencies and a greater proportion of memory T cells in subjects with T1D than in HLA matched controls. There were significantly higher numbers of GAD specific T cells than IA2 specific T cells in subjects with T1D. T cells specific for both groups of epitopes could be expanded from the peripheral blood of subjects with established T1D and at-risk subjects. Expanded neo-epitope specific T cells primarily produced interferon gamma in both groups, but a greater proportion of T cells were interferon gamma positive in subjects with T1D, including some poly-functional cells that also produced IL-4. Based on direct surface phenotyping, neo-epitope specific T cells exhibited diverse combinations of chemokine receptors. However, the largest proportion had markers associated with a Th1-like phenotype. Notably, DQ8 restricted responses to PTM IA2 were over-represented in subjects with lower residual beta cell function. Neo-epitope specific T cells were present in at-risk subjects, and those with multiple autoantibodies have higher interferon gamma to IL-4 ratios than those with single autoantibodies, suggesting a shift in polarization during progression., Discussion: These results reinforce the relevance of PTM neo-epitopes in human disease and suggest that distinct responses to neo-antigens promote a more rapid decline in beta cell function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Nguyen, Arribas-Layton, Chow, Speake, Kwok, Hessner, Greenbaum and James.)

This study aimed to examine depressive symptoms of community-dwelling older people amidst COVID-19 and explore how naturally occurring coping strategies were associated with depression. A mixed-method cross-sectional telephone survey was conducted with 375 older people aged 60 years and above between March and May 2020 in Hong Kong. Trained social workers interviewed participants and assessed depressive symptoms with the Patient Health Questionnaire-9 (PHQ-9). Attribute coding and thematic analysis were adopted for qualitative data analyses. Generalized linear models (GLM) were used to examine the effects of demographics, self-reported risk factors and coping strategies on PHQ-9 scores. Participants' average PHQ-9 score was 1.9 ( SD  = 2.9), suggesting a low risk for depression in general. Over half of the participants reported adaptive coping strategies, including learning new things, staying physically, mentally, and socially active, and having a positive mind-set. GLM results indicated that living with family members (other than spouse) and/or others, maladaptive coping, and self-reported risk factors were significantly associated with higher PHQ-9 scores, while adaptive coping was significantly associated with lower PHQ-9 scores. Our study contributed to the growing literature on older people's resilience and adaptive coping during the pandemic, and the results may have implications for mental health promotion and community care.

HLA-DR3 (DR3) is one of the dominant HLA-DR alleles associated with systemic lupus erythematosus (SLE) susceptibility. Our previous studies showed multiple intramolecular DR3 restricted T cell epitopes in the Smith D (SmD) protein, from which we generated a non-homologous, bacterial epitope mimics library. From this library we identified ABC 247-261 Mimic as one new DR3 restricted bacterial T cell epitope from the ABC transporter ATP-binding protein in Clostridium tetani . It activated and induced autoreactive SmD 66-80 -specific T cells and induced autoantibodies to lupus-related autoantigens in vivo . Compared to healthy donors, SLE patients have a greater percentage of cross-reactive T cells to ABC 247-261 Mimic and SmD 66-80 . In addition, we analyzed the ability of single DR3 restricted Tetanus toxoid (TT) T cell epitopes to induce autoimmune T cells. We found that the immunodominant TT epitope TT 826-845 stimulated SmD 66-80 reactive T cells but failed to induce persistent anti-SmD autoantibodies compared to the ABC 247-261 Mimic. Thus, exposure to the ABC 247-261 Mimic epitope may contribute to autoimmunity in susceptible DR3 individuals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhao, Anderson, Kannapell, Kwok, Gaskin and Fu.)

Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic β-cells. One of the earliest aspects of this process is the development of autoantibodies and T cells directed at an epitope in the B-chain of insulin (insB:9-23). Analysis of microbial protein sequences with homology to the insB:9-23 sequence revealed 17 peptides showing >50% identity to insB:9-23. Of these 17 peptides, the hprt4-18 peptide, found in the normal human gut commensal Parabacteroides distasonis , activated both human T cell clones from T1D patients and T cell hybridomas from nonobese diabetic (NOD) mice specific to insB:9-23. Immunization of NOD mice with P. distasonis insB:9-23 peptide mimic or insB:9-23 peptide verified immune cross-reactivity. Colonization of female NOD mice with P. distasonis accelerated the development of T1D, increasing macrophages, dendritic cells, and destructive CD8 + T cells, while decreasing FoxP3 + regulatory T cells. Western blot analysis identified P. distasonis -reacting antibodies in sera of NOD mice colonized with P. distasonis and human T1D patients. Furthermore, adoptive transfer of splenocytes from P. distasonis -treated mice to NOD/SCID mice enhanced disease phenotype in the recipients. Finally, analysis of human children gut microbiome data from a longitudinal DIABIMMUNE study revealed that seroconversion rates (i.e., the proportion of individuals developing two or more autoantibodies) were consistently higher in children whose microbiome harbored sequences capable of producing the hprt4-18 peptide compared to individuals who did not harbor it. Taken together, these data demonstrate the potential role of a gut microbiota-derived insB:9-23-mimic peptide as a molecular trigger of T1D pathogenesis.

Background: PR3-ANCA vasculitis has a genetic association with HLA-DPB1. We explored immunologic and clinical features related to the interaction of HLA-DPB1*04:01 with a strongly binding PR3 peptide epitope (PR3 225-239 )., Methods: Patients with ANCA vasculitis with active disease and disease in remission were followed longitudinally. Peripheral blood mononuclear cells from patients and healthy controls with HLA-DPB1*04:01 were tested for HLA-DPB1*04:01 expression and interaction with a PR3 peptide identified via in silico and in vitro assays. Tetramers (HLA/peptide multimers) identified autoreactive T cells in vitro. RESULTS: The HLA-DPB1*04:01 genotype was associated with risk of relapse in PR3-ANCA (HR for relapse 2.06; 95% CI, 1.01 to 4.20) but not in myeloperoxidase (MPO)-ANCA or the combined cohort. In silico predictions of HLA and PR3 peptide interactions demonstrated strong affinity between ATRLFPDFFTRVALY (PR3 225-239 ) and HLA-DPB1*04:01 that was confirmed by in vitro competitive binding studies. The interaction was tested in ex vivo flow cytometry studies of labeled peptide and HLA-DPB1*04:01-expressing cells. We demonstrated PR3 225-239 specific autoreactive T cells using synthetic HLA multimers (tetramers). Patients in long-term remission off therapy had autoantigenic peptide and HLA interaction comparable to that of healthy volunteers., Conclusions: The risk allele HLA-DPB1*04:01 has been associated with PR3-ANCA, but its immunopathologic role was unclear. These studies demonstrate that HLA-DPB1*04:01 and PR3 225-239 initiate an immune response. Autoreactive T cells specifically recognized PR3 225-239 presented by HLA-DPB1*04:01. Although larger studies should validate these findings, the pathobiology may explain the observed increased risk of relapse in our cohort. Moreover, lack of HLA and autoantigen interaction observed during long-term remission signals immunologic nonresponsiveness., (Copyright © 2022 by the American Society of Nephrology.)

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4 + T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4 + T-cell response in both patients, with higher frequencies of virus-specific CD4 + T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4 + T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4 + T-cells with CD45RA - CXCR5 + PD-1 + circulating T follicular helper cell (cT FH ) phenotype. Furthermore, we observed a delayed contraction of CD127 - virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4 + T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell-T-cell interaction, a robust virus-specific CD4 + T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection.

Atherosclerosis is accompanied by a CD4 T cell response to apolipoprotein B (APOB). Major Histocompatibility Complex (MHC)-II tetramers can be used to isolate antigen-specific CD4 T cells by flow sorting. Here, we produce, validate and use an MHC-II tetramer, DRB1*07:01 APOB-p18, to sort APOB-p18-specific cells from peripheral blood mononuclear cell samples from 8 DRB1*07:01+ women with and without subclinical cardiovascular disease (sCVD). Single cell RNA sequencing showed that transcriptomes of tetramer+ cells were between regulatory and memory T cells in healthy women and moved closer to memory T cells in women with sCVD. TCR sequencing of tetramer+ cells showed clonal expansion and V and J segment usage similar to those found in regulatory T cells. These findings suggest that APOB-specific regulatory T cells may switch to a more memory-like phenotype in women with atherosclerosis. Mouse studies showed that such switched cells promote atherosclerosis., Competing Interests: DISCLOSURES There are no conflicts of interest.

Background: Late-life depression is common, modifiable, yet under-treated. Service silos and human resources shortage contribute to insufficient prevention and intervention. We describe an implementation research protocol of collaborative stepped care and peer support model that integrates community mental health and aged care services to address service fragmentation, using productive ageing and recovery principles to involve older people as peer supporters to address human resource issue., Methods/design: This is a non-randomised controlled trial examining the effectiveness and cost-effectiveness of the "Jockey Club Holistic Support Project for Elderly Mental Wellness" (JC JoyAge) model versus care as usual (CAU) in community aged care and community mental health service units in 12 months. Older people aged 60 years and over with mild to moderate depressive symptoms or risk factors for developing depression will be included. JoyAge service users will receive group-based activities and psychoeducation, low-intensity psychotherapy, or high-intensity psychotherapy according to the stepped care protocol in addition to usual community mental health or aged care, with support from an older peer supporter. The primary clinical outcome, depressive symptoms, and secondary outcomes, self-harm risk, anxiety symptoms, and loneliness, will be measured with the Patient Health Questionnaire-9 (PHQ-9), Self-Harm Inventory, Generalized Anxiety Disorder 7-item scale (GAD-7), and UCLA Loneliness 3-item scale (UCLA-3) respectively. Cost-effectiveness analysis will assess health-related quality of life using the EQ-5D-5L and service utilisation using the Client Service Receipt Inventory (CSRI). We use multilevel linear mixed models to compare outcomes change between groups and calculate the incremental cost-effectiveness ratio in terms of quality-adjusted life years., Discussion: This study will provide evidence about outcomes for older persons with mental health needs receiving collaborative stepped care service without silos and with trained young-old volunteers to support engagement, treatment, and transitions. Cost-effectiveness findings from this study will inform resource allocation in this under-treated population., Trial Registration: ClinicalTrials.gov NCT03593889. Registered on 20 July 2018., (© 2022. The Author(s).)

Background: Atopic dermatitis (AD) is one of the most common inflammatory skin diseases worldwide and Staphylococcus aureus colonization and secondary infections occur in the majority of AD patients. Allergic sensitizations against microbial antigens have been discussed as possible trigger factors of AD. Recently, we reported IgE sensitization against fibronectin-binding protein 1 (FBP1), an essential virulence component in S. aureus, in a subgroup of patients suffering from AD. To expand these findings by investigating delayed-type immune reactions, the objective of this study was to detect and phenotypically characterize FBP1-specific T cells as possible trigger factors in AD., Methods: Immunodominant T-cell epitopes were mapped by proliferation testing of patient-derived FBP1-specific T-cell lines after stimulation with single 15mer peptides, which were derived from different functional domains of the FBP1 sequence. Major histocompatibility complex class II tetramers carrying immunodominant epitopes successfully stained T helper cells in 8 out of 8 HLA-matched, IgE-sensitized AD patients., Results: Cytokine profiling of multimer-sorted cells revealed that predominantly the type 2 cytokines IL-13 and IL-4 were secreted by these cells. In contrast, IL-17, the marker cytokine for response to extracellular pathogens, was scarcely detectable., Conclusions: We demonstrate that FBP1 contains immunodominant peptides that induce a specific pro-inflammatory T helper cell response with increased Th2 levels that can drive an allergic inflammation in sensitized AD patients., (© 2021 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Tumor-specific T cells likely underpin effective immune checkpoint-blockade therapies. Yet, most studies focus on Treg cells and CD8 + tumor-infiltrating lymphocytes (TILs). Here, we study CD4 + TILs in human lung and colorectal cancers and observe that non-Treg CD4 + TILs average more than 70% of total CD4 + TILs in both cancer types. Leveraging high dimensional analyses including mass cytometry, we reveal that CD4 + TILs are phenotypically heterogeneous, within each tumor and across patients. Consistently, we find different subsets of CD4 + TILs showing characteristics of effectors, tissue resident memory (Trm) or exhausted cells (expressing PD-1, CTLA-4 and CD39). In both cancer types, the frequencies of CD39 - non-Treg CD4 + TILs strongly correlate with frequencies of CD39 - CD8 + TILs, which we and others have previously shown to be enriched for cells specific for cancer-unrelated antigens (bystanders). Ex-vivo , we demonstrate that CD39 - CD4 + TILs can be specific for cancer-unrelated antigens, such as HCMV epitopes. Overall, our findings highlight that CD4 + TILs can also recognize cancer-unrelated antigens and suggest measuring CD39 expression as a straightforward way to quantify or isolate bystander CD4 + T cells., Competing Interests: E.W.N is a co-founder, advisor and shareholder of ImmunoScape Pte. Ltd. E.W.N. is an advisor for Neogene Therapeutics and Nanostring Technologies. All other authors declare no potential conflicts of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Objectives: Whether and how symptom clusters are associated with care utilization remains understudied. This study aims to investigate the economic impact of symptom clusters., Methods: We conducted cross-sectional analyses of data collected from 3255 older adults aged 60 years and over in Hong Kong using the Patient Health Questionnaire-9 and the Client Service Receipt Inventory to measure depressive symptoms and service utilization to calculate 1-year care expenditure. Based on Research Domain Criteria framework, we categorized depressive symptoms into four clusters: Negative Valance Systems and Externalizing (NVSE; anhedonia and depression), Negative Valance Systems and Internalizing (guilt and self-harm), Arousal and Regulatory Systems (sleep, fatigue, and appetite), and Cognitive and Sensorimotor Systems (CSS; concentration and psychomotor). Two-part models were used with four symptom clusters to estimate economic impacts on care utilization., Results: Core affective symptoms had the largest economic impact on non-psychiatric care expenditure; a one-point increase in NVSE was associated with USD$ 571 additional non-psychiatric care expenditure. The economic impacts of CSS on non-psychiatric care expenditure was attenuated when the severity level of NVSE was higher., Conclusions: Our findings highlight the importance of understanding economic impacts on care utilization based on symptom profiles with a particular emphasis on symptom combinations. Policymakers should optimize care allocation based on older adults' depressive symptom profiles rather than simply considering their depression sum-score or the severity defined by cut-off points., (© 2021 John Wiley & Sons Ltd.)

We aimed to search for common features in the autoreactive T cell receptor (TCR) repertoire in patients with rheumatoid arthritis (RA), focusing on the newly identified candidate antigen citrullinated Tenascin C (cit-TNC). Mononuclear cells from peripheral blood or synovial fluid of eight RA-patients positive for the RA-associated HLA-DRB1*04:01 allele were in-vitro cultured with recently identified citrullinated peptides from Tenascin C. Antigen-specific T cells were isolated using peptide-HLA tetramer staining and subsequently single-cell sequenced for paired alpha/beta TCR analyses by bioinformatic tools. TCRs were re-expressed for further studies of antigen-specificity and T cell responses. Autoreactive T cell lines could be grown out from both peripheral blood and synovial fluid. We demonstrate the feasibility of retrieving true autoreactive TCR sequences by validating antigen-specificity in T cell lines with re-expressed TCRs. One of the Tenascin C peptides, cit-TNC22, gave the most robust T cell responses including biased TCR gene usage patterns. The shared TCR-beta chain signature among the cit-TNC22-specific TCRs was evident in blood and synovial fluid of different patients. The identification of common elements in the autoreactive TCR repertoire gives promise to the possibility of both immune monitoring of the autoimmune components in RA and of future antigen- or TCR-targeted specific intervention in subsets of patients., (© 2021. The Author(s).)

Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct ex vivo staining of PBMC with tetramer reagents was used to define immunodominant and subdominant T cell epitopes and estimate the frequencies of these T cells in SARS-CoV-2 exposed and naïve individuals. Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies., Competing Interests: The authors have declared that no competing interests exist.

Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single-cell RNA sequencing (scRNA-Seq) to identify T cell receptors (TCRs) in IAR T cells, we have identified a class of TCRs that share TCRα chains between individuals ("public" chains). We isolated IAR T cells from blood of healthy, new-onset T1D and established T1D donors using multiplexed CD154 enrichment and identified paired TCRαβ sequences from 2767 individual cells. More than a quarter of cells shared TCR junctions between 2 or more cells ("expanded"), and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCRα junctions were most prominent in new-onset T1D. Public TCR sequences were more germline like than expanded unique, or "private," TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCRα junctions were often paired with mismatched TCRβ junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity toward distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCRα junctions and germline-constrained antigen recognition properties. Since these "innate-like" TCRs differ from previously described immunodominant TCRβ chains in autoimmunity, they have implications for fundamental studies of disease mechanisms. Self-reactive restricted TCRα chains and their associated epitopes should be considered in fundamental and translational investigations of TCRs in T1D.

Antibody production following vaccination can provide protective immunity to subsequent infection by pathogens such as influenza viruses. However, circumstances where antibody formation is impaired after vaccination, such as in older people, require us to better understand the cellular and molecular mechanisms that underpin successful vaccination in order to improve vaccine design for at-risk groups. Here, by studying the breadth of anti-haemagglutinin (HA) IgG, serum cytokines, and B and T cell responses by flow cytometry before and after influenza vaccination, we show that formation of circulating T follicular helper (cTfh) cells was associated with high-titre antibody responses. Using Major Histocompatability Complex (MHC) class II tetramers, we demonstrate that HA-specific cTfh cells can derive from pre-existing memory CD4 + T cells and have a diverse T cell receptor (TCR) repertoire. In older people, the differentiation of HA-specific cells into cTfh cells was impaired. This age-dependent defect in cTfh cell formation was not due to a contraction of the TCR repertoire, but rather was linked with an increased inflammatory gene signature in cTfh cells. Together, this suggests that strategies that temporarily dampen inflammation at the time of vaccination may be a viable strategy to boost optimal antibody generation upon immunisation of older people., Competing Interests: DH, CW, SI, JL, JD, JW, EJ, JL, WK, MZ, AL, EC, ML No competing interests declared, (© 2021, Hill et al.)

The aim of this study was to define the breadth and specificity of dominant SARS-CoV-2-specific T cell epitopes using a comprehensive set of 135 overlapping 15-mer peptides covering the SARS-CoV-2 envelope (E), membrane (M) and nucleoprotein (N) in a cohort of 34 individuals with acute (n = 10) and resolved (n = 24) COVID-19. Following short-term virus-specific in vitro cultivation, the single peptide-specific CD4+ T cell response of each patient was screened using enzyme linked immuno spot assay (ELISpot) and confirmed by single-peptide intracellular cytokine staining (ICS) for interferon-γ (IFN-γ) production. 97% (n = 33) of patients elicited one or more N, M or E-specific CD4+ T cell responses and each patient targeted on average 21.7 (range 0-79) peptide specificities. Overall, we identified 10 N, M or E-specific peptides that showed a response frequency of more than 36% and five of them showed high binding affinity to multiple HLA class II binders in subsequent in vitro HLA binding assays. Three peptides elicited CD4+ T cell responses in more than 55% of all patients, namely Mem&#95;P30 (aa146-160), Mem&#95;P36 (aa176-190), both located within the M protein, and Ncl&#95;P18 (aa86-100) located within the N protein. These peptides were further defined in terms of length and HLA restriction. Based on this epitope and restriction data we developed a novel DRB*11 tetramer (Mem&#95;aa145-164) and examined the ex vivo phenotype of SARS-CoV-2-specific CD4+ T cells in one patient. This detailed characterization of single T cell peptide responses demonstrates that SARS-CoV-2 infection universally primes a broad T cell response directed against multiple specificities located within the N, M and E structural protein., Competing Interests: The authors have declared that no competing interests exist.

For an emerging disease like COVID-19, systems immunology tools may quickly identify and quantitatively characterize cells associated with disease progression or clinical response. With repeated sampling, immune monitoring creates a real-time portrait of the cells reacting to a novel virus before disease-specific knowledge and tools are established. However, single cell analysis tools can struggle to reveal rare cells that are under 0.1% of the population. Here, the machine learning workflow Tracking Responders EXpanding (T-REX) was created to identify changes in both rare and common cells across human immune monitoring settings. T-REX identified cells with highly similar phenotypes that localized to hotspots of significant change during rhinovirus and SARS-CoV-2 infections. Specialized MHCII tetramer reagents that mark rhinovirus-specific CD4+ cells were left out during analysis and then used to test whether T-REX identified biologically significant cells. T-REX identified rhinovirus-specific CD4+ T cells based on phenotypically homogeneous cells expanding by ≥95% following infection. T-REX successfully identified hotspots of virus-specific T cells by comparing infection (day 7) to either pre-infection (day 0) or post-infection (day 28) samples. Plotting the direction and degree of change for each individual donor provided a useful summary view and revealed patterns of immune system behavior across immune monitoring settings. For example, the magnitude and direction of change in some COVID-19 patients was comparable to blast crisis acute myeloid leukemia patients undergoing a complete response to chemotherapy. Other COVID-19 patients instead displayed an immune trajectory like that seen in rhinovirus infection or checkpoint inhibitor therapy for melanoma. The T-REX algorithm thus rapidly identifies and characterizes mechanistically significant cells and places emerging diseases into a systems immunology context for comparison to well-studied immune changes., Competing Interests: SB, WK, RT, JW none, AP became an employee of Cytek Biosciences, Inc after performing this research at University of Virginia. LM None, JL became a paid consultant of Cytek Biosciences, Inc after performing this research at University of Virginia. JI was a co-founder and a board member of Cytobank Inc and received unrelated research support from Incyte Corp, Janssen, and Pharmacyclics., (© 2021, Barone et al.)

Recent advances in single-cell sequencing technologies enable the generation of large-scale data sets of paired TCR sequences from patients with autoimmune disease. Methods to validate and characterize patient-derived TCR data are needed, as well as relevant model systems that can support the development of antigen-specific tolerance inducing drugs. We have generated a pipeline to allow streamlined generation of 'artificial' T cells in a robust and reasonably high throughput manner for in vitro and in vivo studies of antigen-specific and patient-derived immune responses. Hereby chimeric (mouse-human) TCR alpha and beta constructs are re-expressed in three different formats for further studies: ( i ) transiently in HEK cells for peptide-HLA tetramer validation experiments, ( ii ) stably in the TCR-negative 58 ​T cell line for functional readouts such as IL-2 production and NFAT-signaling, and lastly ( iii ) in human HLA-transgenic mice for studies of autoimmune disease and therapeutic interventions. As a proof of concept, we have used human HLA-DRB1∗04:01 restricted TCR sequences specific for a type I diabetes-associated GAD peptide, and an influenza-derived HA peptide. We show that the same chimeric TCR constructs can be used in each of the described assays facilitating sequential validation and prioritization steps leading to humanized animal models., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: COVID-19-related information on social media is overabundant and sometimes questionable, resulting in an "infodemic" during the pandemic. While previous studies suggest social media usage increases the risk of developing anxiety symptoms, how induced anxiety affects attitudes and behaviors is less discussed, let alone during a global pandemic. Little is known about the relationship between older adults using social media during a pandemic and their anxiety, their attitudes toward social trust in information, and behaviors to avoid contracting COVID-19., Objective: The goal of this study was to investigate the associations between using social media for COVID-19-related information and anxiety symptoms as well as the mediation effect of anxiety symptoms on social trust in information and COVID-safe behaviors among older adults., Methods: A cross-sectional telephone survey was conducted in Hong Kong between May and August 2020. A rapid warm-call protocol was developed to train social workers and volunteers from participant nongovernmental organizations to conduct the telephone surveys. Questions related to COVID-safe behaviors, social trust in information, social media use, anxiety and depressive symptoms, and sociodemographic information were asked. The number of confirmed COVID-19 cases at the community level was used to account for the risk of contracting COVID-19. Ordinary least squares regressions examined the associations between social media use and anxiety symptoms, and how they were associated with social trust in information and COVID-safe behaviors. Structural equation modeling further mapped out these relationships to identify the mediation effects of anxiety symptoms., Results: This study collected information regarding 3421 adults aged 60 years and older. Use of social media for COVID-19-related information was associated with more anxiety symptoms and lower social trust in information but had no significant relationship with COVID-safe behaviors. Anxiety symptoms predicted lower social trust in information and higher COVID-safe behaviors. Lower social trust in information was predicted by using social media for COVID-19 information, mediated by anxiety symptoms, while no mediation effect was found for COVID-safe behaviors., Conclusions: Older adults who rely on social media for COVID-19-related information exhibited more anxiety symptoms, while showing mixed effects on attitudes and behaviors. Social trust in information may be challenged by unverified and contradictory information online. The negligible impact on COVID-safe behaviors suggested that social media may have caused more confusion than consolidating a consistent effort against the pandemic. Media literacy education is recommended to promote critical evaluation of COVID-19-related information and responsible sharing among older adults., (©Frankie Ho Chun Wong, Tianyin Liu, Dara Kiu Yi Leung, Anna Y Zhang, Walker Siu Hong Au, Wai Wai Kwok, Angie K Y Shum, Gloria Hoi Yan Wong, Terry Yat-Sang Lum. Originally published in the Journal of Medical Internet Research (http://www.jmir.org), 11.02.2021.)

Aims: Late-life depression has substantial impacts on individuals, families and society. Knowledge gaps remain in estimating the economic impacts associated with late-life depression by symptom severity, which has implications for resource prioritisation and research design (such as in modelling). This study examined the incremental health and social care expenditure of depressive symptoms by severity., Methods: We analysed data collected from 2707 older adults aged 60 years and over in Hong Kong. The Patient Health Questionnaire-9 (PHQ-9) and the Client Service Receipt Inventory were used, respectively, to measure depressive symptoms and service utilisation as a basis for calculating care expenditure. Two-part models were used to estimate the incremental expenditure associated with symptom severity over 1 year., Results: The average PHQ-9 score was 6.3 (standard deviation, s.d. = 4.0). The percentages of respondents with mild, moderate and moderately severe symptoms and non-depressed were 51.8%, 13.5%, 3.7% and 31.0%, respectively. Overall, the moderately severe group generated the largest average incremental expenditure (US$5886; 95% CI 1126-10 647 or a 272% increase), followed by the mild group (US$3849; 95% CI 2520-5177 or a 176% increase) and the moderate group (US$1843; 95% CI 854-2831, or 85% increase). Non-psychiatric healthcare was the main cost component in a mild symptom group, after controlling for other chronic conditions and covariates. The average incremental association between PHQ-9 score and overall care expenditure peaked at PHQ-9 score of 4 (US$691; 95% CI 444-939), then gradually fell to negative between scores of 12 (US$ - 35; 95% CI - 530 to 460) and 19 (US$ -171; 95% CI - 417 to 76) and soared to positive and rebounded at the score of 23 (US$601; 95% CI -1652 to 2854)., Conclusions: The association between depressive symptoms and care expenditure is stronger among older adults with mild and moderately severe symptoms. Older adults with the same symptom severity have different care utilisation and expenditure patterns. Non-psychiatric healthcare is the major cost element. These findings inform ways to optimise policy efforts to improve the financial sustainability of health and long-term care systems, including the involvement of primary care physicians and other geriatric healthcare providers in preventing and treating depression among older adults and related budgeting and accounting issues across services.

Background & Aims: Gluten challenge is used to diagnose celiac disease (CeD) and for clinical research. Sustained gluten exposure reliably induces histologic changes but is burdensome. We investigated the relative abilities of multiple biomarkers to assess disease activity induced by 2 gluten doses, and aimed to identify biomarkers to supplement or replace histology., Methods: In this randomized, double-blind, 2-dose gluten-challenge trial conducted in 2 US centers (Boston, MA), 14 adults with biopsy-proven CeD were randomized to 3 g or 10 g gluten/d for 14 days. The study was powered to detect changes in villous height to crypt depth, and stopped at planned interim analysis on reaching this end point. Additional end points included gluten-specific cluster of differentiation (CD)4 T-cell analysis with HLA-DQ2-gluten tetramers and enzyme-linked immune absorbent spot, gut-homing CD8 T cells, interleukin-2, symptoms, video capsule endoscopy, intraepithelial leukocytes, and tissue multiplex immunofluorescence., Results: All assessments showed changes with gluten challenge. However, time to maximal change, change magnitude, and gluten dose-response relationship varied. Villous height to crypt depth, video capsule endoscopy enteropathy score, enzyme-linked immune absorbent spot, gut-homing CD8 T cells, intraepithelial leukocyte counts, and HLA-DQ2-restricted gluten-specific CD4 T cells showed significant changes from baseline at 10 g gluten only; symptoms were significant at 3 g. Symptoms and plasma interleukin-2 levels increased significantly or near significantly at both doses. Interleukin-2 appeared to be the earliest, most sensitive marker of acute gluten exposure., Conclusions: Modern biomarkers are sensitive and responsive to gluten exposure, potentially allowing less invasive, lower-dose, shorter-duration gluten ingestion. This work provides a preliminary framework for rational design of gluten challenge for CeD research. ClinicalTrials.gov number, NCT03409796., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Peptide-major histocompatibility complex class II (pMHCII) multimers have emerged as a convenient and powerful tool for characterization of CD4 T cell immune responses in a large variety of human diseases. Peptide-MHCII multimers can rapidly identify peptide antigens recognized by CD4 T cells via high-throughput peptide screening procedures. The specificity and phenotype of antigen-specific CD4 T cells can be effectively visualized by pMHCII multimers from unmanipulated immune cell populations. Functional attributes of antigen-specific CD4 T cells can also be defined with the multimer technology in combination with immune functional assays such as intracellular cytokine staining (ICS).

Objective: Loneliness is a significant and independent risk factor for depression in later life. Particularly in Asian culture, older people may find it less stigmatising to express loneliness than depression. This study aimed to adapt a simple loneliness screen for use in older Chinese, and to ascertain its relevance in detecting depressive symptoms as a community screening tool., Design, Setting and Participants: This cross-sectional study was conducted among 1653 older adults aged 60 years or above living in the community in Hong Kong. This was a convenient sample recruited from four local non-governmental organisations providing community eldercare or mental healthcare services. All data was collected by trained social workers through face-to-face interviews., Measures: Loneliness was measured using an adapted Chinese version of UCLA 3-item Loneliness Scale, depression symptoms were assessed using the Patient Health Questionnaire-9 (PHQ-9), and social support with emotional and instrumental support proxies (number of people who can offer help). Basic demographics including age, gender, education and living arrangement were also recorded., Results: The average loneliness score was 3.9±3.0, and it had a moderate correlation with depressive symptoms (r=0.41, p<0.01). A loneliness score of 3 can distinguish those without depression from those with mild or more significant depressive symptoms, defined as a PHQ-9 score of ≥5 (sensitivity 76%, specificity 62%, area under the curve=0.73±0.01). Loneliness explained 18% unique variance of depressive symptoms, adding to age, living arrangement and emotional support as significant predictors., Conclusion: A 3-item loneliness scale can reasonably identify older Chinese who are experiencing depressive symptoms as a quick community screening tool. Its wider use may facilitate early detection of depression, especially in cultures with strong mental health stigma., Trial Registration Number: ClinicalTrials.gov NCT03593889., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4 + T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4 + T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4 + T cells in MS., Competing Interests: Declaration of Interests R.M. received unrestricted grants (Biogen and Novartis) and compensation for advice/lecturing (Biogen, Novartis, Sanofi Genzyme, Merck, Hoffmann La Roche, Neuway, CellProtect, and Third Rock Ventures). R.M., M.S., and A.L. are listed as inventor on several NIH- and University of Zurich-held patents and are co-founders and co-owners of Cellerys. I.J. received compensation for advice by Sanofi Genzyme, none of which has affected this work. R.M. and M.S. are listed as co-inventors on a patent on “Immunodominant proteins and fragments in autoimmune diseases,” including RAGSRP2., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

HLA-DQA1 and -DQB1 genes have significant and potentially causal associations with autoimmune type 1 diabetes (T1D). To follow up on the earlier analysis on high-risk HLA-DQ2.5 and DQ8.1, the current analysis uncovers seven residues (αa1, α157, α196, β9, β30, β57, and β70) that are resistant to T1D among subjects with DQ4-, 5-, 6-, and 7 -resistant DQ haplotypes. These 7 residues form 13 common motifs: 6 motifs are significantly resistant, 6 motifs have modest or no associations ( P values >0.05), and 1 motif has 7 copies observed among control subjects only. The motifs "DAAFYDG," "DAAYHDG," and "DAAYYDR" have significant resistance to T1D (odds ratios [ORs] 0.03, 0.25, and 0.18; P = 6.11 × 10 -24 , 3.54 × 10 -15 , and 1.03 × 10 -21 , respectively). Remarkably, a change of a single residue from the motif "DAAYHDG" to "DAAYHSG" (D to S at β57) alters the resistance potential, from resistant motif (OR 0.15; P = 3.54 × 10 -15 ) to a neutral motif ( P = 0.183), the change of which was significant (Fisher P value = 0.0065). The extended set of linked residues associated with T1D resistance and unique to each cluster of HLA-DQ haplotypes represents facets of all known features and functions of these molecules: antigenic peptide binding, peptide-MHC class II complex stability, β167-169 RGD loop, T-cell receptor binding, formation of homodimer of α-β heterodimers, and cholesterol binding in the cell membrane rafts. Identification of these residues is a novel understanding of resistant DQ associations with T1D. Our analyses endow potential molecular approaches to identify immunological mechanisms that control disease susceptibility or resistance to provide novel targets for immunotherapeutic strategies., (© 2020 by the American Diabetes Association.)

Monitoring the frequency of circulatory CXCR5 + (cCXCR5 + ) CD4 + T cells in periphery blood provides a potential biomarker to draw inferences about T follicular helper (T FH ) activity within germinal center. However, cCXCR5 + T cells are highly heterogeneous in their expression of ICOS, PD1 and CD38 and the relationship between different cCXCR5 subsets as delineated by these markers remains unclear. We applied class II tetramer reagents and mass cytometry to investigate the ontogeny of different subsets of cCXCR5 + T cell following yellow fever immunization. Through unsupervised analyses of mass cytometry data, we show yellow fever virus-specific cCXCR5 T cells elicited by vaccination were initially CD38 + ICOS + PD1 + , but then transitioned to become CD38 + ICOS - PD1 + and CD38 - ICOS - PD1 + before coming to rest as a CD38 - ICOS - PD1 - subset. These results imply that most antigen-specific cCXCR5 + T cells, including the CD38 - ICOS - PD1 - CXCR5 + T cells are derived from the CXCR5 + CD38 + ICOS + PD1 + subset, the subset that most resembles preT FH /T FH in the germinal center.

Background: Allergic asthmatic subjects are uniquely susceptible to acute wheezing episodes provoked by rhinovirus. However, the underlying immune mechanisms and interaction between rhinovirus and allergy remain enigmatic, and current paradigms are controversial., Objective: We sought to perform a comprehensive analysis of type 1 and type 2 innate and adaptive responses in allergic asthmatic subjects infected with rhinovirus., Methods: Circulating virus-specific T H 1 cells and allergen-specific T H 2 cells were precisely monitored before and after rhinovirus challenge in allergic asthmatic subjects (total IgE, 133-4692 IU/mL; n = 28) and healthy nonallergic controls (n = 12) using peptide/MHCII tetramers. T cells were sampled for up to 11 weeks to capture steady-state and postinfection phases. T-cell responses were analyzed in parallel with 18 cytokines in the nose, upper and lower airway symptoms, and lung function. The influence of in vivo IgE blockade was also examined., Results: In uninfected asthmatic subjects, higher numbers of circulating virus-specific PD-1 + T H 1 cells, but not allergen-specific T H 2 cells, were linked to worse lung function. Rhinovirus infection induced an amplified antiviral T H 1 response in asthmatic subjects versus controls, with synchronized allergen-specific T H 2 expansion, and production of type 1 and 2 cytokines in the nose. In contrast, T H 2 responses were absent in infected asthmatic subjects who had normal lung function, and in those receiving anti-IgE. Across all subjects, early induction of a minimal set of nasal cytokines that discriminated high responders (G-CSF, IFN-γ, TNF-α) correlated with both egress of circulating virus-specific T H 1 cells and worse symptoms., Conclusions: Rhinovirus induces robust T H 1 responses in allergic asthmatic subjects that may promote disease, even after the infection resolves., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

HLA-DQA1 and -DQB1 are strongly associated with type 1 diabetes (T1D), and DQ8.1 and DQ2.5 are major risk haplotypes. Next-generation targeted sequencing of HLA-DQA1 and -DQB1 in Swedish newly diagnosed 1- to 18 year-old patients ( n = 962) and control subjects ( n = 636) was used to construct abbreviated DQ haplotypes, converted into amino acid (AA) residues, and assessed for their associations with T1D. A hierarchically organized haplotype (HOH) association analysis allowed 45 unique DQ haplotypes to be categorized into seven clusters. The DQ8/9 cluster included two DQ8.1 risk and the DQ9 resistant haplotypes, and the DQ2 cluster included the DQ2.5 risk and DQ2.2 resistant haplotypes. Within each cluster, HOH found residues α44Q (odds ratio [OR] 3.29, P = 2.38 * 10 -85 ) and β57A (OR 3.44, P = 3.80 * 10 -84 ) to be associated with T1D in the DQ8/9 cluster representing all ten residues (α22, α23, α44, α49, α51, α53, α54, α73, α184, β57) due to complete linkage disequilibrium (LD) of α44 with eight such residues. Within the DQ2 cluster and due to LD, HOH analysis found α44C and β135D to share the risk for T1D (OR 2.10, P = 1.96 * 10 -20 ). The motif "QAD" of α44, β57, and β135 captured the T1D risk association of DQ8.1 (OR 3.44, P = 3.80 * 10 -84 ), and the corresponding motif "CAD" captured the risk association of DQ2.5 (OR 2.10, P = 1.96 * 10 -20 ). Two risk associations were related to GAD65 autoantibody (GADA) and IA-2 autoantibody (IA-2A) but in opposite directions. CAD was positively associated with GADA (OR 1.56, P = 6.35 * 10 -8 ) but negatively with IA-2A (OR 0.59, P = 6.55 * 10 -11 ). QAD was negatively associated with GADA (OR 0.88; P = 3.70 * 10 -3 ) but positively with IA-2A (OR 1.64; P = 2.40 * 10 -14 ), despite a single difference at α44. The residues are found in and around anchor pockets 1 and 9, as potential T-cell receptor contacts, in the areas for CD4 binding and putative homodimer formation. The identification of three HLA-DQ AAs (α44, β57, β135) conferring T1D risk should sharpen functional and translational studies., (© 2020 by the American Diabetes Association.)

T cells isolated from the pancreatic infiltrates of nonobese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry, and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by using a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T-cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel posttranslational modification that may contribute to the loss of peripheral tolerance in T1D., (© 2020 by the American Diabetes Association.)

Background: The direct-instillation nasal allergen challenge (NAC) and the environmental exposure chamber (EEC) are 2 methods of conducting controlled allergen provocations. The clinical and biological comparability of these methods has not been thoroughly investigated., Objective: We sought to compare clinical and immunologic responses to cat allergen in NAC versus EEC., Methods: Twenty-four participants were randomized to receive either NAC followed by a 2-day challenge in an EEC or a 2-day challenge in an EEC followed by NAC. Challenges were separated by 28-day washout periods. We measured total nasal symptom scores, peak nasal inspiratory flow, nasal (0-8 hours) and serum cytokines, serum antibodies, peripheral blood antigen-specific T lymphocytes, and gene expression in nasal scrapings. The primary outcome was the total nasal symptom score area under the curve for the first 3 hours after allergen exposure in NAC or after initiation of exposure in EEC., Results: Both challenges increased IL-5 and IL-13 in nasal fluids and serum and resulted in altered nasal cell expression of gene modules related to mucosal biology and transcriptional regulation. Changes in gene modules, more so than cytokine measurements, showed significant associations with total nasal symptom score and peak nasal inspiratory flow. Overall, EEC exposure generated larger responses and more early terminations compared with NAC. Although the 2 challenges did not correlate in symptom magnitude or temporality, striking correlations were observed in cytokine levels., Conclusions: Although clinical outcomes of NAC and EEC were temporally different and nonequivalent in magnitude, immunologic responses were similar. Selection of a particular allergen challenge method should depend on considerations of study objectives and cost., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. All rights reserved.)