Characterization of Expanded Gamma Delta T Cells from Atypical X-SCID Patient Reveals Preserved Function and IL2RG-Mediated Signaling.

Abnormally high γδ T cell numbers among individuals with atypical SCID have been reported but detailed immunophenotyping and functional characterization of these expanded γδ T cells are limited. We have previously reported atypical SCID phenotype caused by hypomorphic IL2RG (NM_000206.3) c.172C > T;p.(Pro58Ser) variant. Here, we have further investigated the index patient's abnormally large γδ T cell population in terms of function and phenotype by studying IL2RG cell surface expression, STAT tyrosine phosphorylation and blast formation in response to interleukin stimulation, immunophenotyping, TCRvγ sequencing, and target cell killing. In contrast to his âºß T cells, the patient's γδ T cells showed normal IL2RG cell surface expression and normal or enhanced IL2RG-mediated signaling. Vδ2 + population was proportionally increased with a preponderance of memory phenotypes and high overall tendency towards perforin expression. The patient's γδ T cells showed enhanced cytotoxicity towards A549 cancer cells. His TCRvγ repertoire was versatile but sequencing of IL2RG revealed a novel c.534C > A; p.(Phe178Leu) somatic missense variant restricted to γδ T cells. Over time this variant became predominant in γδ T cells, though initially present only in part of them. IL2RG-Pro58Ser/Phe178Leu variant showed higher cell surface expression compared to IL2RG-Pro58Ser variant in stable HEK293 cell lines, suggesting that somatic p.(Phe178Leu) variant may at least partially rescue the pathogenic effect of germline p.(Pro58Ser) variant. In conclusion, our report indicates that expansion of γδ T cells associated with atypical SCID needs further studying and cannot exclusively be deemed as a homeostatic response to low numbers of conventional T cells.

Gut dysbiosis promotes islet-autoimmunity by increasing T-cell attraction in islets via CXCL10 chemokine.

CXCL10 is an IFNγ-inducible chemokine implicated in the pathogenesis of type 1 diabetes. T-cells attracted to pancreatic islets produce IFNγ, but it is unclear what attracts the first IFNγ -producing T-cells in islets. Gut dysbiosis following administration of pathobionts induced CXCL10 expression in pancreatic islets of healthy non-diabetes-prone (C57BL/6) mice and depended on TLR4-signaling, and in non-obese diabetic (NOD) mice, gut dysbiosis induced also CXCR3 chemokine receptor in IGRP-reactive islet-specific T-cells in pancreatic lymph node. In amounts typical to low-grade endotoxemia, bacterial lipopolysaccharide induced CXCL10 production in isolated islets of wild type and RAG1 or IFNG-receptor-deficient but not type-I-IFN-receptor-deficient NOD mice, dissociating lipopolysaccharide-induced CXCL10 production from T-cells and IFNγ. Although mostly myeloid-cell dependent, also ß-cells showed activation of innate immune signaling pathways and Cxcl10 expression in response to lipopolysaccharide indicating their independent sensitivity to dysbiosis. Thus, CXCL10 induction in response to low levels of lipopolysaccharide may allow islet-specific T-cells imprinted in pancreatic lymph node to enter in healthy islets independently of IFN-g, and thus link gut dysbiosis to early islet-autoimmunity via dysbiosis-associated low-grade endotoxemia.

Exposure to systemic antibiotics in outpatient care and the risk of multiple sclerosis.

BACKGROUND: Infections, early life exposures and the microbiome have been associated with the aetiology of multiple sclerosis (MS). Data on any possible roles of antibiotics is scarce and conflicting. OBJECTIVE: The objective of this study was to investigate associations between outpatient systemic antibiotic exposure and the risk of MS in a nationwide case-control setting. METHODS: Patients with MS were identified from the nation MS registry and their exposure to antibiotics was compared with that of persons without MS, provided by the national census authority. Antibiotic exposure was investigated using the national prescription database and analyzed by Anatomical Therapeutic Chemical (ATC) category. RESULTS: Among the 1830 patients with MS and 12765 control persons, there were no associations between exposure to antibiotics in childhood (5-9 years) or adolescence (10-19 years) and the subsequent risk of MS. There was also no association between antibiotic exposure 1-6 years before disease onset and the risk of MS, save for exposure to fluoroquinolones in women (odds ratio: 1.28; 95% confidence interval: 1.03, 1.60; p = 0.028) which is probably associated with the increased infection burden in the MS prodrome. CONCLUSION: Use of systemic prescription antibiotics was not associated with subsequent MS risk.

Plasmacytoid dendritic cells regulate host immune response to Citrobacter rodentium induced colitis in colon-draining lymph nodes.

Dendritic cells (DCs) are first in line to sense invading microbes and to deliver signals to other immune cells. Plasmacytoid DCs (pDC) produce high amounts of type I interferons (IFNs) but also regulate immune responses. Using the Clec4C (BDCA2)-diphtheria toxin receptor mouse model allowing conditional pDC depletion, we identified an essential role for pDCs in regulating intestinal inflammation locally in the gut. In pDC-depleted mice, Citrobacter rodentium infection led to enhanced activation of conventional DCs and induction of IFN-γ-producing Th1-cells in colon-draining lymph nodes, while induction of Foxp3+ /CD25+ Treg and IL-17-producing Th17 cells was impaired. Concomitantly, F4/80+ macrophages accumulated into the colon lamina propria in excess, and levels of Il-1ß and Tnf transcripts increased and Foxp3+ Treg were fewer. Our results indicate that pDCs control inflammation in the gut during C. rodentium infection and that they have an important immune regulatory role in colon-draining lymph nodes.

Data-Independent Acquisition Mass Spectrometry in Metaproteomics of Gut Microbiota-Implementation and Computational Analysis.

Metagenomic approaches focus on taxonomy or gene annotation but lack power in defining functionality of gut microbiota. Therefore, metaproteomics approaches have been introduced to overcome this limitation. However, the common metaproteomics approach uses data-dependent acquisition mass spectrometry, which is known to have limited reproducibility when analyzing samples with complex microbial composition. In this work, we provide a proof of concept for data-independent acquisition (DIA) metaproteomics. To this end, we analyze metaproteomes using DIA mass spectrometry and introduce an open-source data analysis software package, diatools, which enables accurate and consistent quantification of DIA metaproteomics data. We demonstrate the feasibility of our approach in gut microbiota metaproteomics using laboratory-assembled microbial mixtures as well as human fecal samples.

Novel Hemizygous IL2RG p.(Pro58Ser) Mutation Impairs IL-2 Receptor Complex Expression on Lymphocytes Causing X-Linked Combined Immunodeficiency.

Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient's clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny.

Akkermansia muciniphila induces gut microbiota remodelling and controls islet autoimmunity in NOD mice.

OBJECTIVE: Intestinal microbiota is implicated in the pathogenesis of autoimmune type 1 diabetes in humans and in non-obese diabetic (NOD) mice, but evidence on its causality and on the role of individual microbiota members is limited. We investigated if different diabetes incidence in two NOD colonies was due to microbiota differences and aimed to identify individual microbiota members with potential significance. DESIGN: We profiled intestinal microbiota between two NOD mouse colonies showing high or low diabetes incidence by 16S ribosomal RNA gene sequencing and colonised the high-incidence colony with the microbiota of the low-incidence colony. Based on unaltered incidence, we identified a few taxa which were not effectively transferred and thereafter, transferred experimentally one of these to test its potential significance. RESULTS: Although the high-incidence colony adopted most microbial taxa present in the low-incidence colony, diabetes incidence remained unaltered. Among the few taxa which were not transferred, Akkermansia muciniphila was identified. As A. muciniphila abundancy is inversely correlated to the risk of developing type 1 diabetes-related autoantibodies, we transferred A. muciniphila experimentally to the high-incidence colony. A. muciniphila transfer promoted mucus production and increased expression of antimicrobial peptide Reg3γ, outcompeted Ruminococcus torques from the microbiota, lowered serum endotoxin levels and islet toll-like receptor expression, promoted regulatory immunity and delayed diabetes development. CONCLUSION: Transfer of the whole microbiota may not reduce diabetes incidence despite a major change in gut microbiota, but single symbionts such as A. muciniphila with beneficial metabolic and immune signalling effects may reduce diabetes incidence when administered as a probiotic.

Activation of Plasmacytoid Dendritic Cells in Colon-Draining Lymph Nodes during Citrobacter rodentium Infection Involves Pathogen-Sensing and Inflammatory Pathways Distinct from Conventional Dendritic Cells.

Dendritic cells (DCs) bear the main responsibility for initiation of adaptive immune responses necessary for antimicrobial immunity. In the small intestine, afferent lymphatics convey Ags and microbial signals to mesenteric lymph nodes (LNs) to induce adaptive immune responses against microbes and food Ags derived from the small intestine. Whether the large intestine is covered by the same lymphatic system or represents its own lymphoid compartment has not been studied until very recently. We identified three small mesenteric LNs, distinct from small intestinal LNs, which drain lymph specifically from the colon, and studied DC responses to the attaching and effacing pathogen Citrobacter rodentium in these. Transcriptional profiling of conventional (CD11c(high)CD103(high)) DC and plasmacytoid (plasmacytoid DC Ag-1(high)B220(+)CD11c(int)) DC (pDC) populations during steady-state conditions revealed activity of distinct sets of genes in these two DC subsets, both in small intestinal and colon-draining LNs. C. rodentium activated DC especially in colon-draining LNs, and gene expression changed in pDC more profoundly than in conventional DC. Among the genes most upregulated in pDC were C-type lectin receptor CLEC4E, IL-1Rs (IL-1R1 and -2), proinflammatory cytokines (IL-1a and IL-6), and TLR6. Our results indicate that colon immune surveillance is distinct from that of the small intestine in terms of draining LNs, and identify pDC as active sentinels of colonic inflammation and/or microbial dysbiosis.

Autoimmunity, hypogammaglobulinemia, lymphoproliferation, and mycobacterial disease in patients with activating mutations in STAT3.

The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.

Keratin 8 modulates ß-cell stress responses and normoglycaemia.

Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on ß-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8(+/+)) and K8 knockout (K8(-/-)) mice. Islet ß-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8(+/+) and K8(-/-) mice. K8 and K18 were the predominant keratins in islet ß-cells and K8(-/-) mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8(-/-) ß-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8(-/-) mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8(-/-) mice. ß-cell keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8(+/+) mice and in diabetic NOD mice, suggesting a role for keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for keratins in ß-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.

Fermentable fibres condition colon microbiota and promote diabetogenesis in NOD mice.

AIMS/HYPOTHESIS: Gut microbiota (GM) and diet both appear to be important in the pathogenesis of type 1 diabetes. Fermentable fibres (FFs), of which there is an ample supply in natural, diabetes-promoting diets, are used by GM as a source of energy. Our aim was to determine whether FFs modify GM and diabetes incidence in the NOD mouse. METHODS: Female NOD mice were weaned to a semisynthetic diet and the effects of FF supplementation on diabetes incidence and insulitis were evaluated. Real-time quantitative PCR was employed to determine the effects imposed to gene transcripts in the colon and lymph nodes. Changes to GM were analysed by next-generation sequencing. RESULTS: NOD mice fed semisynthetic diets free from FFs were largely protected from diabetes while semisynthetic diets supplemented with the FFs pectin and xylan (PX) resulted in higher diabetes incidence. Semisynthetic diet free from FFs altered GM composition significantly; addition of PX changed the composition of the GM towards that found in natural-diet-fed mice and increased production of FF-derived short-chain fatty acid metabolites in the colon. The highly diabetogenic natural diet was associated with expression of proinflammatory and stress-related genes in the colon, while the semisynthetic diet free from FFs promoted Il4, Il22, Tgfß and Foxp3 transcripts in the colon and/or pancreatic lymph node. PX in the same diet counteracted these effects and promoted stress-related IL-18 activation in gut epithelial cells. 16S RNA sequencing revealed each diet to give rise to its particular GM composition, with different Firmicutes to Bacteroidetes ratios, and enrichment of mucin-degrading Ruminococcaceae following diabetes-protective FF-free diet. CONCLUSIONS/INTERPRETATION: FFs condition microbiota, affect colon homeostasis and are important components of natural, diabetes-promoting diets in NOD mice.

[New dimensions for dendritic cells]. / Dendriittisolujen uudet ulottuvuudet.

Dendritic cells have turned out to be important component in the regulation of immune responses. In addition to various external structures they recognize the body's own intracellular structures and utilize them to construct information about threats affecting the well-being of organs and tissues. Dendritic cells are able to direct immune responses in a manner that among other things opens new dimensions to the prevention and management of autoimmune diseases, allergies, cancer and atherosclerosis. Vaccines directed to dendritic cells and modification of dendritic cells in vitro are becoming a part of the novel, targeted immunotherapy.

Persistent T cell-mediated immune responses against Omicron variants after the third COVID-19 mRNA vaccine dose.

Introduction: The prime-boost COVID-19 mRNA vaccination strategy has proven to be effective against severe COVID-19 disease and death. However, concerns have been raised due to decreasing neutralizing antibody levels after COVID-19 vaccination and due to the emergence of new immuno-evasive SARS-CoV-2 variants that may require additional booster vaccinations. Methods: In this study, we analyzed the humoral and cell-mediated immune responses against the Omicron BA.1 and BA.2 subvariants in Finnish healthcare workers (HCWs) vaccinated with three doses of COVID-19 mRNA vaccines. We used enzyme immunoassay and microneutralization test to analyze the levels of SARS-CoV-2 specific IgG antibodies in the sera of the vaccinees and the in vitro neutralization capacity of the sera. Activation induced marker assay together with flow cytometry and extracellular cytokine analysis was used to determine responses in SARS-CoV-2 spike protein stimulated PBMCs. Results: Here we show that within the HCWs, the third mRNA vaccine dose recalls both humoral and T cell-mediated immune responses and induces high levels of neutralizing antibodies against Omicron BA.1 and BA.2 variants. Three weeks after the third vaccine dose, SARS-CoV-2 wild type spike protein-specific CD4+ and CD8+ T cells are observed in 82% and 71% of HCWs, respectively, and the T cells cross-recognize both Omicron BA.1 and BA.2 spike peptides. Although the levels of neutralizing antibodies against Omicron BA.1 and BA.2 decline 2.5 to 3.8-fold three months after the third dose, memory CD4+ T cell responses are maintained for at least eight months post the second dose and three months post the third vaccine dose. Discussion: We show that after the administration of the third mRNA vaccine dose the levels of both humoral and cell-mediated immune responses are effectively activated, and the levels of the spike-specific antibodies are further elevated compared to the levels after the second vaccine dose. Even though at three months after the third vaccine dose antibody levels in sera decrease at a similar rate as after the second vaccine dose, the levels of spike-specific CD4+ and CD8+ T cells remain relatively stable. Additionally, the T cells retain efficiency in cross-recognizing spike protein peptide pools derived from Omicron BA.1 and BA.2 subvariants. Altogether our results suggest durable cellmediated immunity and protection against SARS-CoV-2.

Ly6C supports preferential homing of central memory CD8+ T cells into lymph nodes.

Ly6C is a murine cell-surface antigen expressed by plasma cells, subsets of myeloid cells and many T cells, including memory T cells. We previously documented that Ly6C crosslinking induces LFA-1 clustering on naïve CD8(+) T cells. Here, we show that in vitro and in vivo differentiation of naïve CD8(+) T cells into central (Tcm) but not effector (Tem) memory T cells enhances Ly6C expression, and its crosslinking induces strong LFA-1 clustering on Tcm. Blocking Ly6C function inhibits in vivo Tcm homing to LNs as efficiently as blocking L-selectin but it does not potentiate the inhibition provided by blocking either L-selectin or LFA-1 function. Thus, Ly6C, L-selectin and LFA-1 all appear to be part of a common homing pathway. In vitro, Ly6C crosslinking enhances Tcm adherence to ICAM-1 in the presence of CCL21. In summary, Tcm homing involves Ly6C, in addition to L-selectin and LFA-1, and appears to potentiate firm adhesion of Tcm to ICAM-1 in synergy with a chemokine. We propose that Ly6C augments Tcm compartmentalization into LNs during their homing.

Infection with the enteric pathogen C. rodentium promotes islet-specific autoimmunity by activating a lymphatic route from the gut to pancreatic lymph node.

In nonobese diabetic (NOD) mice, C. rodentium promotes priming of islet-specific T-cells in pancreatic lymph nodes (PaLN), which is a critical step in initiation and perpetuation of islet-autoimmunity. To investigate mechanisms by which C. rodentium promotes T-cell priming in PaLN, we used fluorescent imaging of lymphatic vasculature emanating from colon, followed dendritic cell (DC) migration from colon using photoconvertible-reporter mice, and evaluated the translocation of bacteria to lymph nodes with GFP-C. rodentium and in situ hybridization of bacterial DNA. Fluorescent dextran injected in the colon wall accumulated under subcapsular sinus of PaLN indicating the existence of a lymphatic route from colon to PaLN. Infection with C. rodentium induced DC migration from colon to PaLN and bacterial DNA was detected in medullary sinus and inner cortex of PaLN. Following infection with GFP-C. rodentium, fluorescence appeared in macrophages and gut-derived (CD103+) and resident (CD103-/XCR1+) DC, indicating transportation of bacteria from colon to PaLN both by DC and by lymph itself. This induced proinflammatory cytokine transcripts, activation of DC and islet-specific T-cells in PaLN of NOD mice. Our findings demonstrate the existence of a direct, enteric pathogen-activated route for lymph, cells, and bacteria from colon, which promotes activation of islet-specific T-cells in PaLN.

Long-Lasting T Cell Responses in BNT162b2 COVID-19 mRNA Vaccinees and COVID-19 Convalescent Patients.

The emergence of novel variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made it more difficult to prevent the virus from spreading despite available vaccines. Reports of breakthrough infections and decreased capacity of antibodies to neutralize variants raise the question whether current vaccines can still protect against COVID-19 disease. We studied the dynamics and persistence of T cell responses using activation induced marker (AIM) assay and Th1 type cytokine production in peripheral blood mononuclear cells obtained from BNT162b2 COVID-19 mRNA vaccinated health care workers and COVID-19 patients. We demonstrate that equally high T cell responses following vaccination and infection persist at least for 6 months against Alpha, Beta, Gamma, and Delta variants despite the decline in antibody levels.

[Immunological regulation of acute inflammatory reaction]. / Akuutin tulehdusreaktion immunologinen säätely.

Inflammation is an acute manifestation of the activation of the immune defense. The immunological regulation of an inflammatory reaction is disturbed in many diseases, and inflammation is also involved in numerous conditions that are often considered non-inflammatory such as atherosclerosis, ischemic tissue injury, obesity and Alzheimer's disease. Especially recent progress in the research on the innate immune system has improved our understanding on the manifold cause-effect relationships in the regulation of the immune reactions and opened up new possibilities to manage inflammation.

Pertussis seroprevalence among adults of reproductive age (20-39 years) in fourteen European countries.

The reported incidence of pertussis in European countries varies considerably. We aimed to study specific Bordetella pertussis seroprevalence in Europe by measuring serum IgG antibody levels to pertussis toxin (anti-PT IgG). Fourteen national laboratories participated in this study including Belgium, Denmark, Finland, Greece, Hungary, Italy, Lithuania, Malta, Norway, Poland, Portugal, Romania, Spain, and Sweden. Each country collected approximately 250 samples (N = 7903) from the age groups 20-29 years (N = 3976) and 30-39 years (N = 3927) during 2010-2013. Samples were anonymous residual sera from diagnostic laboratories and were analyzed at the national laboratories by a Swedish reference method, a commercial ELISA kit, or were sent to Sweden for analysis. The median anti-PT IgG concentrations ranged from 4 to 13.6 IU/mL. The proportion of samples with anti-PT IgG ≥100 IU/mL, indicating a recent infection ranged from 0.2% (Hungary) to 5.7% (Portugal). The highest proportion of sera with anti-PT IgG levels between 50 and <100 IU/mL, indicating an infection within the last few years, was found in Portugal (12.3%) and Italy (13.9%). This study shows that the circulation of B. pertussis is quite extensive in adults, aged 20-39 years, despite well-established vaccination programs in Europe.

Characterization of CD4+ T cells specific for glutamic acid decarboxylase (GAD65) and proinsulin in a patient with stiff-person syndrome but without type 1 diabetes.

BACKGROUND: Glutamic acid decarboxylase (GAD) is a rate-limiting enzyme in the synthesis of gamma-amino butyric acid (GABA) and an important autoantigen both in patients with type 1 diabetes (T1D) and stiff-person syndrome (SPS). Autoantibodies (GADA) to the 65-kDa isoform of GAD are a characteristic feature in both diseases. Approximately 30% of patients with SPS develop diabetes, yet, it is unclear to which extent co-existing autoimmunity to GAD65 and other islet autoantigens determines the risk of developing T1D. METHODS: In this study, we monitored CD4+ T-cell responses to GAD65 and proinsulin in a patient with SPS who remained normoglycaemic during the 46-month follow-up. RESULTS: Fluctuating but persistent T-cell reactivity to GAD65 was identified, as well as T-cell reactivity to proinsulin at one time point. The majority of the T-cell clones isolated from the patient with SPS produced high levels of Th2 cytokines (IL-13, IL-5 and IL-4). We also examined levels of GADA, insulin and IA-2 autoantibodies, and epitope specificity of GADA. In both serum and cerebrospinal fluid (CSF), GADA levels were high, and GADA persisted throughout the follow-up. Despite T-cell reactivity to both GAD65 and proinsulin, autoantibodies to other islet autoantigens did not develop. CONCLUSIONS: Further follow-up will determine whether the beta-cell autoimmunity observed in this patient will eventually lead to T1D.