Defining the immune phenotype of extremely early-onset type 1 diabetes

Type 1 diabetes is a lifelong autoimmune disease that is very rarely diagnosed in the first two years of life. We recently described a novel T1D phenotype, termed extremely early-onset T1D (here referred to as EXE-T1D) characterised by the development of T1D very early in life, which is associated with rapid and complete b-cell destruction. I therefore hypothesise that EXE-T1D may be the consequence of a more aggressive form of autoimmunity than seen in individuals who develop T1D at a later age. In this thesis this hypothesis was tested by comparing the immune phenotype of individuals with EXE-T1D and individuals with childhood or adulthood-onset T1D. Specifically, the frequency and phenotype of islet-specific T cells was assessed using sensitive FluoroSpot assays, and unbiased immune phenotyping was undertaken using five broad flow cytometry panels. Assessment of b-cell-specific T cell responses showed lower autoreactivity in the blood of EXE-T1D, and a decreased proportion of IL-10 responses close to diagnosis, suggestive of reduced immune regulation. Phenotypic characterisation of the circulating immune system identified increased frequencies of activated circulating follicular helper (aTfh) and peripheral helper (aTph) T cells co-expressing PD-1 and ICOS in EXE-T1D. Detailed examination of the phenotypic characteristics of these cells by single-cell transcriptional profiling revealed minor phenotypic differences between clinical cohorts, including a reduction in cell populations expressing heat shock proteins (HSP) and genes associated with an interferon (IFN) response. This study has revealed novel immune phenotypes associated with EXE-T1D which are currently being investigated as biomarkers of rapid b-cell destruction in larger cohorts of individuals with T1D. This work may therefore guide the selection of patients suitable for specific immune-based therapies to halt disease progression

Association of a single nucleotide polymorphism combination pattern of the Klotho gene with non-cardiovascular death in patients with chronic kidney disease

Chronic kidney disease (CKD) is associated with an elevated risk of all-cause mortality, with cardiovascular death being extensively investigated. However, non-cardiovascular mortality represents the biggest percentage, showing an evident increase in recent years. Klotho is a gene highly expressed in the kidney, with a clear influence on lifespan. Low levels of Klotho have been linked to CKD progression and adverse outcomes. Single nucleotide polymorphisms (SNPs) of the Klotho gene have been associated with several diseases, but studies investigating the association of Klotho SNPs with noncardiovascular death in CKD populations are lacking. The main aim of this study was to assess whether 11 Klotho SNPs were associated with non-cardiovascular death in a subpopulation of the National Observatory of Atherosclerosis in Nephrology (NEFRONA) study (n ¼ 2185 CKD patients). After 48 months of follow-up, 62 cardiovascular deaths and 108 non-cardiovascular deaths were recorded. We identified a high non-cardiovascular death risk combination of SNPs corresponding to individuals carrying the most frequent allele (G) at rs562020, the rare allele (C) at rs2283368 and homozygotes for the rare allele (G) at rs2320762 (rs562020 GG/AG þ rs2283368 CC/CT þ rs2320762 GG). Among the patients with the three SNPs genotyped (n ¼ 1016), 75 (7.4%) showed this combination. Furthermore, 95 (9.3%) patients showed a low-risk combination carrying all the opposite genotypes (rs562020 AA þ rs2283368 TT þ rs2320762 GT/TT). All the other combinations [n ¼ 846 (83.3%)] were considered as normal risk. Using competing risk regression analysis, we confirmed that the proposed combinations are independently associated with a higher fhazard ratio [HR] 3.28 [confidence interval (CI) 1.51-7.12]g and lower [HR 6 × 10- (95% CI 3.3 × 10--1.1 × 10-)] risk of suffering a non-cardiovascular death in the CKD population of the NEFRONA cohort compared with patients with the normal-risk combination. Determination of three SNPs of the Klotho gene could help in the prediction of non-cardiovascular death in CKD

Association of candidate gene polymorphisms with chronic kidney disease : Results of a case-control analysis in the NEFRONA cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2,445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionization-time of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Evaluating T cell responses prior to the onset of type 1 diabetes

AIMS: In the current study we aimed to evaluat T cell phenotypes and metabolic profiles in high‐risk individuals who progressed to type 1 diabetes compared to those remaining disease free. METHODS: A Fluorspot assay was used to examine T cell responses to a panel of islet autoantigen peptides in samples obtained 6‐ and 30‐months preceding disease onset and at the same timepoints in non‐progressors. RESULTS: We noted a significant increase in the magnitude of the proinflammatory interferon‐γ response to proinsulin and insulin peptides in individuals who progressed to type 1 diabetes. In contrast, in the non‐progressors, we observed an increase in the regulatory IL‐10 response to proinsulin peptides. Furthermore, the T cell responses to the islet peptide panel predisposed towards a proinflammatory interferon‐γ bias in the progressors. CONCLUSIONS: Collectively, these data suggest that a proinflammatory T cell response is prevalent in high‐risk individuals who progress to type 1 diabetes and can be detected up to 6 months prior to onset of disease. This observation, albeit in a small cohort, can potentially be harnessed in disease staging, particularly in identifying autoantibody‐positive individuals transitioning from stage 2 (dysglycemia present and pre‐symptomatic) to stage 3 (dysglycemia present and symptomatic). The detection of these different T cell phenotypes in progressors and non‐progressors suggests the presence of disease endotypes

Evaluating clinical effectiveness and impact of anti-pneumococcal vaccination in adults after universal childhood PCV13 implementation in Catalonia, 2017–2018

Background: At present, because of indirect effects derived from routine childhood immunisation, clinical benefits vaccinating adults with the 23-valent pneumococcal polysaccharide vaccine (PPsV23) and/or the 13-valent pneumococcal conjugate vaccine (PCV13) are uncertain. This study investigated clinical effectiveness for both PPsV23/PCV13 in preventing pneumonia among Catalonian adults during an earlier 2-year period post-PCV13 free (publicly funded) approval for infants. Methods: We conducted a Population-based cohort study involving 2,059,645 adults ≥ 50 years in Catalonia, Spain, who were followed between 01/01/2017–31/12/2018. Primary outcomes were hospitalisation from pneumococcal pneumonia (PP) or all-cause pneumonia (ACP) and main explanatory variable was PCV13/PPsV23 vaccination status. Cox regression models were used to estimate vaccination effectiveness adjusted by age/sex and underlying-risk conditions. Results: Cohort members were followed for 3,958,528 person-years (32,328 PCV13-vaccinated, 1,532,186 PPsV23-vaccinated), observing 3592 PP (131 in PCV13-vaccinated vs 2476 in PPsV23-vaccinated) and 24,136 ACP (876 in PCV13-vaccinated vs 17,550 in PPsV23-vaccinated). Incidence rates (per 100,000 person-years) were 90.7 for PP (394.2 in PCV13-vaccinated vs 161.6 in PPsV23-vaccinated) and 609.7 for ACP (2636.3 in PCV13-vaccinated vs 1145.4 in PPsV23-vaccinated). The PCV13 was associated with an increased risk of PP (hazard ratio [HR]: 1.24; 95% CI: 1.00–1.52; p = 0.046) and ACP (HR: 1.38; 95% CI: 1.28–1.49; p < 0.001) whereas the PPsV23 did not alter the risk of PP (HR: 1.07; 95% CI: 0.98–1.18; p = 0.153) and slightly increased the risk of ACP (HR: 1.14; 95% CI: 1.10–1.18; p < 0.001). In supplementary analyses focused on at-risk individuals (i.e., elderly persons, immunocompromissing and other chronic illnesses) protective effects of vaccination did not emerge either. Conclusions: Data does not support clinical benefits from pneumococcal vaccination (nor PCV13 neither PPsV23) against pneumonia among Catalonian middle-aged and older adults in the current era of universal PCV13 childhood immunisation in our setting. New extended valency PCVs are greatly needed

Mapping T Cell Responses to Native and Neo-Islet Antigen Epitopes in at Risk and Type 1 Diabetes Subjects

AIMS: Recent studies highlight the potentially important role of neoepitopes in breaking immune tolerance in type 1 diabetes. T cell reactivity to these neoepitopes has been reported, but how this response compares quantitatively and phenotypically with previous reports on native epitopes is not known. Thus, an understanding of the relationship between native and neoepitopes and their role as tolerance breakers or disease drivers in type 1 diabetes is required. We set out to compare T cell reactivity and phenotype against a panel of neo- and native islet autoantigenic epitopes to examine how this relates to stages of type 1 diabetes development. METHODS: Fifty-four subjects comprising patients with T1D, and autoantibody-positive unaffected family members were tested against a panel of neo- and native epitopes by ELISPOT (IFN-γ, IL-10, and IL-17). A further subset of two patients was analyzed by Single Cell Immune Profiling (RNAseq and TCR α/β) after stimulation with pools of native and neoepitope peptides. RESULTS: T cell responses to native and neoepitopes were present in patients with type 1 diabetes and at-risk subjects, and overall, there were no significant differences in the frequency, magnitude, or phenotype between the two sets of peptide stimuli. Single cell RNAseq on responder T cells revealed a similar profile in T1D patients stimulated with either neo- or native epitopes. A pro-inflammatory gene expression profile (TNF-α, IFN-γ) was dominant in both native and neoepitope stimulated T cells. TCRs with identical clonotypes were found in T cell responding to both native and neoepitopes. CONCLUSION/INTERPRETATION: These data suggest that in peripheral blood, T cell responses to both native and neoepitopes are similar in terms of frequency and phenotype in patients with type 1 diabetes and high-risk unaffected family members. Furthermore, using a combination of transcriptomic and clonotypic analyses, albeit using a limited panel of peptides, we show that neoepitopes are comparable to native epitopes currently in use for immune-monitoring studies

Humoral and cellular immunogenicity to a second dose of COVID-19 vaccine BNT162b2 in people receiving methotrexate or targeted immunosuppression : a longitudinal cohort study

Background: COVID-19 vaccines have robust immunogenicity in the general population. However, data for individuals with immune-mediated inflammatory diseases who are taking immunosuppressants remains scarce. Our previously published cohort study showed that methotrexate, but not targeted biologics, impaired functional humoral immunity to a single dose of COVID-19 vaccine BNT162b2 (Pfizer-BioNTech), whereas cellular responses were similar. Here, we aimed to assess immune responses following the second dose. Methods: In this longitudinal cohort study, we recruited individuals with psoriasis who were receiving methotrexate or targeted biological monotherapy (ie, tumour necrosis factor [TNF] inhibitors, interleukin [IL]-17 inhibitors, or IL-23 inhibitors) from a specialist psoriasis centre serving London and South-East England. The healthy control cohort were volunteers without psoriasis, not receiving immunosuppression. Immunogenicity was evaluated immediately before, on day 28 after the first BNT162b2 vaccination and on day 14 after the second dose (administered according to an extended interval regimen). Here, we report immune responses following the second dose. The primary outcomes were humoral immunity to the SARS-CoV-2 spike glycoprotein, defined as titres of total spike-specific IgG and of neutralising antibody to wild-type, alpha (B.1.1.7), and delta (B.1.617.2) SARS-CoV-2 variants, and cellular immunity defined as spike-specific T-cell responses (including numbers of cells producing interferon-γ, IL-2, IL-21). Findings: Between Jan 14 and April 4, 2021, 121 individuals were recruited, and data were available for 82 participants after the second vaccination. The study population included patients with psoriasis receiving methotrexate (n=14), TNF inhibitors (n=19), IL-17 inhibitors (n=14), IL-23 inhibitors (n=20), and 15 healthy controls, who had received both vaccine doses. The median age of the study population was 44 years (IQR 33–52), with 43 (52%) males and 71 (87%) participants of White ethnicity. All participants had detectable spike-specific antibodies following the second dose, and all groups (methotrexate, targeted biologics, and healthy controls) demonstrated similar neutralising antibody titres against wild-type, alpha, and delta variants. By contrast, a lower proportion of participants on methotrexate (eight [62%] of 13, 95% CI 32–86) and targeted biologics (37 [74%] of 50, 60–85; p=0·38) had detectable T-cell responses following the second vaccine dose, compared with controls (14 [100%] of 14, 77–100; p=0·022). There was no difference in the magnitude of T-cell responses between patients receiving methotrexate (median cytokine-secreting cells per 106 cells 160 [IQR 10–625]), targeted biologics (169 [25–503], p=0·56), and controls (185 [133–328], p=0·41). Interpretation: Functional humoral immunity (ie, neutralising antibody responses) at 14 days following a second dose of BNT162b2 was not impaired by methotrexate or targeted biologics. A proportion of patients on immunosuppression did not have detectable T-cell responses following the second dose. The longevity of vaccine-elicited antibody responses is unknown in this population. Funding: NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London; The Psoriasis Association.</p

The effect of methotrexate and targeted immunosuppression on humoral and cellular immune responses to the COVID-19 vaccine BNT162b2 : a cohort study

Background: Patients on therapeutic immunosuppressants for immune-mediated inflammatory diseases were excluded from COVID-19 vaccine trials. We therefore aimed to evaluate humoral and cellular immune responses to COVID-19 vaccine BNT162b2 (Pfizer-BioNTech) in patients taking methotrexate and commonly used targeted biological therapies, compared with healthy controls. Given the roll-out of extended interval vaccination programmes to maximise population coverage, we present findings after the first dose. Methods: In this cohort study, we recruited consecutive patients with a dermatologist-confirmed diagnosis of psoriasis who were receiving methotrexate or targeted biological monotherapy (tumour necrosis factor [TNF] inhibitors, interleukin [IL]-17 inhibitors, or IL-23 inhibitors) from a specialist psoriasis centre serving London and South East England. Consecutive volunteers without psoriasis and not receiving systemic immunosuppression who presented for vaccination at Guy's and St Thomas' NHS Foundation Trust (London, UK) were included as the healthy control cohort. All participants had to be eligible to receive the BNT162b2 vaccine. Immunogenicity was evaluated immediately before and on day 28 (±2 days) after vaccination. The primary outcomes were humoral immunity to the SARS-CoV-2 spike glycoprotein, defined as neutralising antibody responses to wild-type SARS-CoV-2, and spike-specific T-cell responses (including interferon-γ, IL-2, and IL-21) 28 days after vaccination. Findings: Between Jan 14 and April 4, 2021, 84 patients with psoriasis (17 on methotrexate, 27 on TNF inhibitors, 15 on IL-17 inhibitors, and 25 on IL-23 inhibitors) and 17 healthy controls were included. The study population had a median age of 43 years (IQR 31–52), with 56 (55%) males, 45 (45%) females, and 85 (84%) participants of White ethnicity. Seroconversion rates were lower in patients receiving immunosuppressants (60 [78%; 95% CI 67–87] of 77) than in controls (17 [100%; 80–100] of 17), with the lowest rate in those receiving methotrexate (seven [47%; 21–73] of 15). Neutralising activity against wild-type SARS-CoV-2 was significantly lower in patients receiving methotrexate (median 50% inhibitory dilution 129 [IQR 40–236]) than in controls (317 [213–487], p=0·0032), but was preserved in those receiving targeted biologics (269 [141–418]). Neutralising titres against the B.1.1.7 variant were similarly low in all participants. Cellular immune responses were induced in all groups, and were not attenuated in patients receiving methotrexate or targeted biologics compared with controls. Interpretation: Functional humoral immunity to a single dose of BNT162b2 is impaired by methotrexate but not by targeted biologics, whereas cellular responses are preserved. Seroconversion alone might not adequately reflect vaccine immunogenicity in individuals with immune-mediated inflammatory diseases receiving therapeutic immunosuppression. Real-world pharmacovigilance studies will determine how these findings reflect clinical effectiveness. Funding: UK National Institute for Health Research.</p

Human inherited PD-L1 deficiency is clinically and immunologically less severe than PD-1 deficiency.

International audienceWe previously reported two siblings with inherited PD-1 deficiency who died from autoimmune pneumonitis at 3 and 11 years of age after developing other autoimmune manifestations, including type 1 diabetes (T1D). We report here two siblings, aged 10 and 11 years, with neonatal-onset T1D (diagnosed at the ages of 1 day and 7 wk), who are homozygous for a splice-site variant of CD274 (encoding PD-L1). This variant results in the exclusive expression of an alternative, loss-of-function PD-L1 protein isoform in overexpression experiments and in the patients' primary leukocytes. Surprisingly, cytometric immunophenotyping and single-cell RNA sequencing analysis on blood leukocytes showed largely normal development and transcriptional profiles across lymphoid and myeloid subsets in the PD-L1-deficient siblings, contrasting with the extensive dysregulation of both lymphoid and myeloid leukocyte compartments in PD-1 deficiency. Our findings suggest that PD-1 and PD-L1 are essential for preventing early-onset T1D but that, unlike PD-1 deficiency, PD-L1 deficiency does not lead to fatal autoimmunity with extensive leukocytic dysregulation