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Background Mucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and therefore virus shedding. Parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior mucosal infection compared with injections of a parenteral vaccine in a mucosally naive subject. We investigated whether this was also the case for the BNT162b2 coronavirus disease 2019 (COVID-19) messenger RNA vaccine. Methods Twenty recovered COVID-19 subjects (RCSs) and 23 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–naive subjects were vaccinated with, respectively, 1 and 2 doses of the BNT162b2 COVID-19 vaccine. Nasal epithelial lining fluid (NELF) and plasma were collected before and after vaccination and assessed for immunoglobulin G (IgG) and IgA antibody levels to Spike and for their ability to neutralize binding of Spike to angiotensin-converting enzyme-2 receptor. Blood was analyzed 1 week after vaccination for the number of Spike-specific antibody-secreting cells (ASCs) with a mucosal tropism. Results All RCSs had both nasal and blood SARS-CoV-2–specific antibodies at least 90 days after initial diagnosis. In RCSs, a single dose of vaccine amplified preexisting Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including Delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2–naive subjects after 2 vaccine doses compared with RCSs after 1 dose. Conclusions Mucosal immune response to the SARS-CoV-2 Spike protein is higher in RCSs after a single vaccine dose compared with SARS-CoV-2–naive subjects after 2 doses.

Background: Facioscapulohumeral muscular dystrophy (FSHD) is one of the most common myopathies in adults, displaying a progressive, frequently asymmetric involvement of a typical muscles’ pattern. FSHD is associated with epigenetic derepression of the polymorphic D4Z4 repeat on chromosome 4q, leading to DUX4 retrogene toxic expression in skeletal muscles. Identifying biomarkers that correlate with disease severity would facilitate clinical management and assess potential FSHD therapeutics’ efficacy. Objectives: This study purpose was to analyze serum cytokines to identify potential biomarkers in a large cohort of adult patients with FSHD. Methods: We retrospectively measured the levels of 20 pro-inflammatory and regulatory cytokines in sera from 100 genetically confirmed adult FSHD1 patients. Associations between cytokine concentrations and various clinical scores were investigated. We then measured serum and muscle interleukin 6 (IL-6) levels in a validated FSHD-like mouse model, ranging in severity and DUX4 expression. Results: IL-6 was identified as the only cytokine with a concentration correlating with several clinical severity and functional scores, including Clinical Severity Score, Manual Muscle Testing sum score, Brooke and Vignos scores. Further, FSHD patients displayed overall IL-6 levels more than twice high as control, and patients with milder phenotypes exhibited lower IL-6 serum concentration than those with severe muscular weakness. Lastly, an FSHD-like mouse model analysis confirmed that IL-6 levels positively correlate with disease severity and DUX4 expression. Conclusions: Serum IL-6, therefore, shows promise as a serum biomarker of FSHD severity in a large cohort of FSHD1 adult patients.

Neuroinflammation has been proposed to impact symptomatology in patients with schizophrenia spectrum disorders. While previous studies have shown equivocal effects of treatments with add-on anti-inflammatory drugs such as Aspirin, N-acetylcysteine and Celecoxib, none have used a subset of prospectively recruited patients exhibiting an inflammatory profile. The aim of the study is to evaluate the efficacy and safety as well as the cost-effectiveness of a treatment with 400 mg Celecoxib added to an ongoing antipsychotic treatment in patients with schizophrenia spectrum disorders exhibiting an inflammatory profile. The “Add-on Celecoxib treatment in patients with schizophrenia spectrum disorders and inflammatory cytokine profile trial (TargetFlame)” is a multicentre randomized, placebo-controlled phase III investigator-initiated clinical trial with the following two arms: patients exhibiting an inflammatory profile receiving either add-on Celecoxib 400 mg/day or add-on placebo. A total of 199 patients will be assessed for eligibility by measuring blood levels of three pro-inflammatory cytokines, and 109 patients with an inflammatory profile, i.e. inflamed, will be randomized, treated for 8 weeks and followed-up for additional four months. The primary endpoint will be changes in symptom severity as assessed by total Positive and Negative Syndrome Scale (PANSS) score changes from baseline to week 8. Secondary endpoints include various other measures of psychopathology and safety. Additional health economic analyses will be performed. TargetFlame is the first study aimed at evaluating the efficacy, safety and cost-effectiveness of the antiphlogistic agent Celecoxib in a subset of patients with schizophrenia spectrum disorders exhibiting an inflammatory profile. With TargetFlame, we intended to investigate a novel precision medicine approach towards anti-inflammatory antipsychotic treatment augmentation using drug repurposing. Clinical trial registration:http://www.drks.de/DRKS00029044 and https://trialsearch.who.int/Trial2.aspx?TrialID=DRKS00029044

Omicron s escape to vaccine-induced systemic antibody responses has been shown in several studies in Omicron-infected patients and vaccine controls.In the present study we compared mucosal antibody response to Omicron to mucosal antibody response to ancestral strain and Delta variant. This was done on nasal epithelial lining fluid (NELF) prospectively collected in 84 otherwise healthy healthcare workers who had never exhibited PCR-documented COVID-19 and had received three doses of the Pfizer-BioNTech COVID-19 mRNA vaccine. NELF was collected prior to Omicron detection in the geographical area of inclusion.We show that NELF antibodies from vaccinated individuals were less efficient at inhibiting the binding of the Omicron Spike protein to ACE-2 compared to those of Delta or the ancestral strain.These findings may explain the increased risk of onward transmission of Omicron, consistent with its successful global displacement of Delta in countries with a high vaccination coverage.

Background Fragile X syndrome (FXS) is the most frequent cause of inherited intellectual disability and the most commonly identified monogenic cause of autism. Recent studies have shown that long-term pathological consequences of FXS are not solely confined to the central nervous system (CNS) but rather extend to other physiological dysfunctions in peripheral organs. To gain insights into possible immune dysfunctions in FXS, we profiled a large panel of immune-related biomarkers in the serum of FXS patients and healthy controls. Methods We have used a sensitive and robust Electro Chemi Luminescence (ECL)-based immunoassay to measure the levels of 52 cytokines in the serum of n = 25 FXS patients and n = 29 healthy controls. We then used univariate statistics and multivariate analysis, as well as an advanced unsupervised clustering method, to identify combinations of immune-related biomarkers that could discriminate FXS patients from healthy individuals. Results While the majority of the tested cytokines were present at similar levels in FXS patients and healthy individuals, nine chemokines, CCL2, CCL3, CCL4, CCL11, CCL13, CCL17, CCL22, CCL26 and CXCL10, were present at much lower levels in FXS patients. Using robust regression, we show that six of these biomarkers (CCL2, CCL3, CCL11, CCL22, CCL26 and CXCL10) were negatively associated with FXS diagnosis. Finally, applying the K-sparse unsupervised clustering method to the biomarker dataset allowed for the identification of two subsets of individuals, which essentially matched the FXS and healthy control categories. Conclusions Our data show that FXS patients exhibit reduced serum levels of several chemokines and may therefore exhibit impaired immune responses. The present study also highlights the power of unsupervised clustering methods to identify combinations of biomarkers for diagnosis and prognosis in medicine.

Antigens derived from engulfed apoptotic bodies that are presented by dendritic cells can amplify Ag-specific T-cells. Accelerated co-cultured DC (acDC) strategy keeps lymphocytes in contact with differentiating DCs. Therefore, Ag-specific T-cell activation can occur during DC maturation. Our aim was to prepare DCs by acDC method and check the subsequent engulfment of the apoptotic body by acDC. We have proposed that this method could be feasible if we transfect the apoptotic bodies with the antigen. DCs were prepared using acDC method and their maturation markers were confirmed by flow cytometry. Ultraviolet was used for inducing apoptosis in the PBMCs and induction of apoptosis checked by propidium iodide and 7-aminoactinomycin D staining. Flow cytometry and immunohistochemistry were used for checking the uptake of apoptotic bodies by the DCs. The alloreactivity against apoptotic bodies was examined by enzyme-linked immunospot (ELISPOT) assay. Results showed that 40.4% of DCs could efficiently engulf the apoptotic bodies. The results indicated that acDC method is capable to isolate a high yield of DCs, and these cells could properly engulf the apoptotic bodies, more works should be performed to use this method for Ag discovery through delivering the Ag by apoptotic bodies into the DCs.

Background: The immunogenicity of a two-dose mRNA COVID-19 vaccine regimen is low in kidney transplant (KT) recipients. Here, we provide a thorough assessment of the immunogenicity of a three-dose COVID-19 vaccine regimen in this population. Methods: We performed a prospective longitudinal study in sixty-one KT recipients given three doses of the BNT162b2 COVID-19 vaccine. We performed semi-structured pharmacovigilance interviews and monitored donor-specific antibodies and kidney function. We compared levels of anti-spike IgG, pseudo-neutralization activity against vaccine homologous and heterologous variants, frequency of spike-specific interferon (IFN)-γ-secreting cells, and antigen-induced cytokine production 28 days after the second and third doses. Findings: Reactions to vaccine were mild. One patient developed donor-specific anti-HLA antibodies after the second dose which could be explained by non-adherence to immunosuppressive therapy. Spike-specific IgG seroconversion raised from 44·3% (n=27) after the second dose to 62·3% (n=38) after the third dose (p

Most researchers working in the field of immunopsychiatry would agree with the statement that "severe psychiatric disorders are associated with inflammation and more broadly with changes in immune variables". However, as many other fields in biology and medicine, immunopsychiatry suffers from a replication crisis characterized by lack of reproducibility. In this paper, we will comment on four types of immune variables which have been studied in psychiatric disorders: Acute Phase Proteins (AAPs), cytokines, lipid mediators of inflammation and immune cell parameters, and discuss the rationale for looking at them in blood. We will briefly describe the analytical methods that are currently used to measure the levels of these biomarkers and comment on overlooked analytical and statistical methodological issues that may explain some of the conflicting data reported in the literature. Lastly, we will briefly summarize what cross-sectional, longitudinal and mendelian randomization studies have brought to our understanding of schizophrenia (SZ).

International audience; Bipolar disorder (BD) diagnosis currently relies on assessment of clinical symptoms, mainly retrospective and subject to memory bias. BD is often misdiagnosed as Major Depressive Disorder (MDD) resulting in ineffective treatment and worsened clinical outcome. The primary purpose of this study was to identify blood biomarkers that discriminate MDD from BD patients when in a depressed state. We have used clinical data and serum samples from two independent naturalistic cohorts of patients with a Major Depressive Episode (MDE) who fulfilled the criteria of either BD or MDD at inclusion. The discovery and replication cohorts consisted of 462 and 133 patients respectively. Patients were clinically assessed using standard diagnostic interviews, and clinical variables including current treatments were recorded. Blood was collected and serum assessed for levels of 31 cytokines using a sensitive multiplex assay. A penalized logistic regression model combined with nonparametric bootstrap was subsequently used to identify cytokines associated with BD. Interleukin (IL)-6, IL-10, IL-15, IL-27 and C-X-C ligand chemokine (CXCL)-10 were positively associated with BD in the discovery cohort. Of the five cytokines identified as discriminant features in the discovery cohort, IL-10, IL-15 and IL-27 were also positively associated with BD in the replication cohort therefore providing an external validation to our finding. Should our results be validated in a prospective cohort, they could provide new insights into the pathophysiological mechanisms of mood disorders.

In contrast to other fields of medicine, and despite extensive research to identify neuroimaging, electroencephalography, or blood-based biomarkers, neither trait nor state biomarkers are currently available in psychiatry. Would such biomarkers be available, they could help clinicians to answer some of the simplest questions to which they are faced in their daily practice. Previous studies have shown that most severe mental disorders are associated with altered blood levels of cytokines, a group of small soluble proteins that are mainly produced by immune cells. Because cytokine levels can be measured using sensitive and cost-effective analytical methods, many authors have investigated whether they could be useful biomarkers in psychiatry. Despite hundreds of papers in this field, no single cytokine, or combination of cytokines, has emerged as a biomarker that would be robust enough to be used in clinical settings. Here, we will argue that this failure may have been due at least in part to overlooked methodological issues not only for preparation, storage, and analysis of the biological samples but also for statistical analysis of the datasets. We will also propose some guidelines for future studies and advocate for the adoption of common Standard Operating Procedures.

Background: The immunogenicity of a two-dose mRNA COVID-19 vaccine regimen is low in kidney transplant (KT) recipients. We report the first complete assessment of safety and cross-variant immunogenicity of a three-dose vaccine regimen in KT recipients. Methods: We performed a prospective longitudinal study in sixty-one KT recipients given three doses of the BNT162b2 COVID-19 vaccine. We performed semi-structured pharmacovigilance interviews and monitored donor-specific antibodies and kidney function. We compared geometric mean titers (GMT) of anti-spike IgG, pseudo-neutralization activity against vaccine homologous and heterologous variants, frequency of spike-specific interferon (IFN)-γ-secreting cells, and antigen-induced cytokine production 28 days after the second and third doses. The immunoassays were also performed in non-transplanted individuals 28 days after the second dose to obtain reference values. Findings: Reactions to vaccine were mild. One patient developed donor-specific anti-HLA antibodies after the second dose which could be explained by non-adherence to immunosuppressive therapy. Spike-specific IgG seroconversion raised from 44·3% (n=27) after the second dose to 62·3% (n=38) after the third dose (p

Background Patients with a First-Episode of Psychosis (FEP) exhibit low-grade inflammation as demonstrated by elevated levels of C reactive protein (CRP) and pro-inflammatory cytokines. Aims The primary goal of this study was to investigate the association between pro-inflammatory biomarkers and clinical outcomes in unmedicated FEP patients. Method We used clinical data and biological samples from 289 FEP patients participating to the Optimization of Treatment and Management of Schizophrenia in Europe (OPTIMISE) clinical trial. Patients were assessed at baseline and 4-5 weeks after treatment with amisulpride. Baseline serum levels of interleukin (IL)-6, IL-8, Tumor Necrosis Factor (TNF)-α and CRP were measured. We first used multivariable regression to investigate the association between each of the four tested biomarkers and the following clinical outcomes: Positive And Negative Syndrome Scale (PANSS), Calgary Depression Score for Schizophrenia (CDSS), remission according to Andreasen’s criteria and Serious Adverse Events (SAEs). As a complementary approach, we used an unsupervised clustering method to stratify patients into an “inflamed” or a “non-inflamed” biotype based on baseline levels of IL-6, IL-8 and TNF-α. We then used linear and logistic regressions to investigate the association between the patient biotype and clinical outcomes. Results After adjusting for covariates and confounders, we did not find any association between IL-6, IL-8, TNF-α, CRP or the patient biotype and clinical outcomes. Implications Our results do not support the existence of an association between baseline levels of CRP and proinflammatory cytokines and early response to amisulpride in unmedicated FEP patients.

BackgroundMucosal antibodies can prevent virus entry and replication in mucosal epithelial cells and hence virus shedding. Preclinical and clinical studies have shown that a parenteral booster injection of a vaccine against a mucosal pathogen promotes stronger mucosal immune responses following prior infection compared to two injections of a parenteral vaccine. We investigated whether this was also the case for a COVID-19 mRNA vaccine.MethodsTwenty-three COVID-19 convalescent patients and 20 SARS-CoV-2-naive subjects were vaccinated with respectively one and two doses of the Pfizer-BioNTech COVID-19 RNA vaccine. Nasal Epithelial Lining Fluid (NELF) and plasma were collected before and after vaccination and assessed for Immunoglobulin (Ig)G and IgA to Spike and for their ability to inhibit the binding of Spike to its ACE-2 receptor. Blood was analyzed one week after vaccination for the number of Spike-specific Antibody Secreting Cells (ASCs) with a mucosal tropism.ResultsIn COVID-19 convalescent patients, a single dose of vaccine amplified pre-existing Spike-specific IgG and IgA antibody responses in both NELF and blood against both vaccine homologous and variant strains, including delta. These responses were associated with Spike-specific IgG and IgA ASCs with a mucosal tropism in blood. Nasal IgA and IgG antibody responses were lower in magnitude in SARS-CoV-2-naive subjects after two vaccine dosesConclusionThis study showed that a parenteral booster injection of a COVID-19 RNA vaccine promoted stronger mucosal immune responses in COVID-19 convalescent patients compared to SARS-CoV-2 naive subjects who had received a first vaccine dose.

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Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

OBJECTIVE Insulin in pancreatic β-cells is a target of autoimmunity in type 1 diabetes. In the NOD mouse model of type 1 diabetes, oral or nasal administration of insulin induces immune tolerance to insulin and protects against autoimmune diabetes. Evidence for tolerance to mucosally administered insulin or other autoantigens is poorly documented in humans. Adults with recent-onset type 1 diabetes in whom the disease process is subacute afford an opportunity to determine whether mucosal insulin induces tolerance to insulin subsequently injected for treatment. RESEARCH DESIGN AND METHODS We randomized 52 adults with recent-onset, noninsulin-requiring type 1 diabetes to nasal insulin or placebo for 12 months. Fasting blood glucose and serum C-peptide, glucagon-stimulated serum C-peptide, and serum antibodies to islet antigens were monitored three times monthly for 24 months. An enhanced ELISpot assay was used to measure the T-cell response to human proinsulin. RESULTS β-Cell function declined by 35% overall, and 23 of 52 participants (44%) progressed to insulin treatment. Metabolic parameters remained similar between nasal insulin and placebo groups, but the insulin antibody response to injected insulin was significantly blunted in a sustained manner in those who had received nasal insulin. In a small cohort, the interferon-γ response of blood T-cells to proinsulin was suppressed after nasal insulin. CONCLUSIONS Although nasal insulin did not retard loss of residual β-cell function in adults with established type 1 diabetes, evidence that it induced immune tolerance to insulin provides a rationale for its application to prevent diabetes in at-risk individuals.

Type 2 diabetes (T2D) is a chronic metabolic disorder in which beta-cells are destroyed. The islet amyloid polypeptide (IAPP) produced by beta-cells has been reported to influence beta-cell destruction.To evaluate if IAPP can act as an autoantigen and therefore, to see if CD8+ T-cells specific for this protein might be present in T2D patients.Peripheral blood mononuclear cells (PBMC) were obtained from human leukocyte antigen (HLA)-A2+ T2D patients and non-diabetic healthy subjects. Cells were then screened for peptide recognition using ELISPOT assay for the presence of IFN-γ producing CD8+ T-cells against two HLA Class I-restricted epitopes derived from IAPP (IAPP5-13 and IAPP9-17) and common viral antigenic minimal epitopes Flu MP 58-66, CMV495-503, EBV280-288 and HIV77-85 as controls.A total of 36.4% of patients and 56.2% of healthy subjects showed a response against IAPP5-13 peptide. No significant difference in response against this peptide was noted between the patients and the healthy donors. With respect to peptide IAPP9-17, although healthy subjects showed a higher mean number of spot forming cells than the patients, the difference was not significant; 36.4% of patients and 37.5% of controls responded to this peptide. The response of healthy subjects to the common viral peptides was stronger than that of the patients, though the result was not significant.It is unlikely that IAPP would be a target for CD8+ T-cells in diabetic patients; however, the trend observed toward a lower response of T2D patients against IAPP and common viral peptides may imply a decreased immune response in these patients.

Anti-Hu antibody–associated paraneoplastic neurological syndromes (Hu-PNSs) are severe and often precede the detection of a malignancy, usually small-cell lung cancer. In Hu-PNS, it is hypothesized that neuronal cells are destroyed by T cells targeted against HuD, a protein expressed by small-cell lung cancer cells and neurons. There is only limited evidence for the existence of HuD-specific T cells. To detect these T cells in the blood of Hu-PNS patients, we employed 3 highly sensitive assays that included T cell stimulation with dendritic cells (DCs) to specifically expand the number of any HuD-specific T cells. A total of 17 Hu-PNS patients were tested with 1 or more of the following 3 assays: (1) tetramer staining after stimulation of T cells with conventionally generated DCs (n = 9), (2) interleukin (IL)-13 enzyme-linked immunosorbent spot (ELISpot; n = 3), IL-4 and IL-5 and interferon (IFN)–γ multiplex cytokine bead array (n = 2) to assay cytokine production by T cells after stimulation with conventionally generated DCs, and (iii) IFN-γ ELISpot and tetramer staining after T cell stimulation with accelerated co-cultured DCs (n = 11). No circulating HuD-specific T cells were found. We suggest that either autoaggressive T cells in Hu-PNS are not targeted against HuD or that their numbers in the blood are too low for detection by highly sensitive techniques.

A phase II clinical trial with glutamic acid decarboxylase (GAD) 65 formulated with aluminium hydroxide (GAD-alum) has shown efficacy in preserving residual insulin secretion in children and adolescents with recent-onset type 1 diabetes (T1D). We have performed a 4-year follow-up study of 59 of the original 70 patients to investigate long-term cellular and humoral immune responses after GAD-alum-treatment. Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with GAD(65). Frequencies of naive, central and effector memory CD4+ and CD8+ T cells were measured, together with cytokine secretion, proliferation, gene expression and serum GAD(65) autoantibody (GADA) levels. We here show that GAD-alum-treated patients display increased memory T-cell frequencies and prompt T-cell activation upon in vitro stimulation with GAD(65), but not with control antigens, compared with placebo subjects. GAD(65)-induced T-cell activation was accompanied by secretion of T helper (Th) 1, Th2 and T regulatory cytokines and by induction of T-cell inhibitory pathways. Moreover, post-treatment serum GADA titres remained persistently increased in the GAD-alum arm, but did not inhibit GAD(65) enzymatic activity. In conclusion, memory T- and B-cell responses persist 4 years after GAD-alum-treatment. In parallel to a GAD(65)-induced T-cell activation, our results show induction of T-cell inhibitory pathways important for regulating the GAD(65) immunity. Funding Agencies|Swedish Research Council|K2008-55x-20652-01-3|Swedish Child Diabetes Foundation (Barndiabetesfonden)||Medical Research Council of Southeast Sweden||Juvenile Diabetes Research Foundation (JDRF)|1-2008-106|Ile-de-France CODDIM||Inserm Avenir Program

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease targeting pancreatic beta-cells. Despite this textbook definition, it is quite striking that neither the diagnosis nor the therapy nor the follow-up of T1D "belong" to immunologists, but rather to endocrinologists whose only option is to limit the consequences of the disease. Immune therapies would seem better suited to correct the causes of T1D, but critical laboratory tools are missing for early diagnosis, prognostic stratification, and therapeutic follow-up. The immune markers routinely available are limited to autoantibodies, which have some intrinsic limitations. Because T cells are central pathogenic actors of T1D, the quest for their measurement appeared to offer a path towards new autoimmune markers. Given the strong association between T1D susceptibility and the HLA class II locus, investigators have long been focused on CD4(+) T cells. However, data gathered in the NOD mouse and the examination of human insulitis point to a critical role of CD8(+) T cells in the pathogenesis of T1D. These observations have revived interest in trying to measure CD8(+) T cell responses in human T1D. Achievement of this goal mainly depends on two factors. First, the relevant epitopes need to be identified. Second, appropriate readouts and measurement techniques need to be selected. This review summarizes recent advances on both of these battlefronts, and discusses the potential clinical applications of T cell assays.

Donor-derived cytokine-induced killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild grafts versus host (GVH) events were observed. To extend this strategy across major HLA barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells' alloreactivity are needed. We hypothesized that alloreactivity and anti-tumor activity of CIK cells segregate within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from seven patients who underwent HCT as treatment of metastatic colorectal cancer. We found that CIK cells maintained their alloreactivity across major HLA barriers when tested as bulk population; after CD56-positive selection, anti-tumor activity was restricted to the CD3+/CD56+ cell fraction and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+/CD56- cell fraction. Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host- or donor-derived PBMC, confirming their low potential for GVH across minor HLA barriers. Moreover, we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor-derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+/CD56- cells might reduce the risk of GVH without affecting the tumor-killing capacity and could help extending CIK infusions across major HLA barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells.

International audience; The identification of parameters maximizing detection sensitivity in ELISpot assays is important to transfer this technology into the clinical setting for identifying rare Ag-specific CD8(+) T cells. We have therefore considered human IFN-gamma CD8(+) T cell responses against viral epitopes to analyze different variables which could be critical during the epitope-specific stimulation period. Two parameters were found to greatly enhance detection sensitivity (i.e., to specifically increase epitope-driven signal while keeping background noise to a minimum): use of human serum-free vs. serum-supplemented culture medium (2.4-fold median increase) and addition of low dose IL-7 (1.5-fold increase). Incorporating both of these parameters into the ELISpot procedure proved capable of greatly amplifying (35.1-fold increase) the low grade CD8(+) T cell responses directed against beta-cell epitopes of type 1 diabetes patients, as compared to a previously optimized procedure using human serum-supplemented medium and low dose IL-2. Implementation of this ELISpot procedure should expedite development of "immune staging" protocols for autoimmune as well as tumor and infectious diseases.

CD8+ T cells play an important role in the initiation of insulitis and in the destructive stage leading to insulin-dependent diabetes mellitus. A string of recent studies has led to the identification of numerous HLA-A2-restricted epitopes derived from pancreatic β cell Ags. It is hoped that assays detecting responses of patient PBMC to such epitopes might be instrumental for early diagnosis of β cell-directed autoimmunity and for monitoring trials of immunointervention. However, it remains unclear whether the results of assays studying PBMC reflect responses of islet-infiltrating lymphocytes, and to what extent they correlate with disease risk and/or activity. We have used female and male humanized NOD mice expressing HLA-A2 in addition to murine MHC class I molecules to study spontaneous responses of islet-infiltrating blood, spleen, and lymph node lymphocytes of various age groups to a panel of 16 epitopes. Twelve of these are restricted by HLA-A2, have previously been shown to be recognized by patient CTL, and have identical sequences in human and murine autoantigens. Using an IFN-γ ELISPOT assay, we find highly similar hierarchies of epitope immunodominance in the different T cell compartments, including peripheral blood and pancreatic islets. Moreover, we demonstrate that most of the epitopes eliciting dominant responses in humans display similar status in the mouse model. These results emphasize the potential of humanized mice as tools for studying spontaneous autoimmune CTL responses, and they provide a strong rationale for the development and use of assays monitoring responses of CD8+ PBMC in human type 1 diabetes.

OBJECTIVE—Islet-reactive CD8+ T-cells play a key role in the pathogenesis of type 1 diabetes in the NOD mouse. The predominant T-cell specificities change over time, but whether similar shifts also occur after clinical diagnosis and insulin treatment in type 1 diabetic patients is unknown.RESEARCH DESIGN AND METHODS—We took advantage of a recently validated islet-specific CD8+ T-cell γ-interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2+ adult type 1 diabetic patients close to diagnosis and at a second time point 7–16 months later.RESULTS—CD8+ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60–67 to 20% (P < 0.02). The previously subdominant IA-2206–214 and IGRP265–273 peptides were newly targeted, thus becoming the immunodominant epitopes.CONCLUSIONS—Shifts both in frequency and in immunodominance of CD8+ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

Despite the understanding that type 1 diabetes pathogenesis is mediated by T-cells, detection of these rare lymphocytes remains largely elusive. Suitable T-cell assays are highly needed, since they could offer preclinical diagnoses and immune surrogate end points for clinical trials. Although CD4+ T-cell assays have met with limited success, CD8+ T-cells are increasingly recognized as key actors in the diabetes of the NOD mouse. CD8+ T-cells are likely to play a role also in humans and may provide new markers of β-cell autoimmunity. Taking advantage of a panel of HLA-A2–restricted β-cell epitopes derived from preproinsulin, GAD, and islet glucose-6-phosphatase catalytic subunit-related protein (IGRP), we have implemented an islet-specific CD8+ T-cell interferon-γ enzyme-linked immunospot (ISL8Spot) assay. The ISL8Spot assay is capable of detecting and quantifying β-cell–reactive CD8+ T-cells directly ex vivo, without any preliminary expansion, using either fresh or frozen samples. Positive ISL8Spot responses separate new-onset diabetic and healthy samples with high accuracy (86% sensitivity, 91% specificity), using as few as five immunodominant epitopes. Moreover, sensitivity reaches 100% when the ISL8Spot assay is complemented by antibody determinations. Combination of CD8+ T-cell measurements with immune intervention strategies may open new avenues toward type 1 diabetes prediction and prevention.

Introduction Plusieurs essais cliniques de vaccination a l’aide d’antigenes des cellules beta tels que l’insuline et la GAD se sont reveles decevants chez les patients diabetiques de type 1. Il est donc important de comprendre les effets immunologiques induits par ces traitements afin d’ameliorer les essais cliniques ulterieurs. Patients et methodes Nous avons participe a l’analyse des reponses des anticorps et des lymphocytes T diriges contre l’insuline et la GAD apres traitement par ces deux antigenes dans le cadre de deux protocoles cliniques: un protocole australien de vaccination par insuline intra-nasale, sans adjuvant, administree chez des patients diabetiques adultes non encore traites par insuline; et une vaccination sous-cutanee avec la GAD en presence de l’adjuvant alun chez des enfants diabetiques de type 1 suedois. Resultats Le traitement par insuline intra-nasale n’a pas retarde la perte de fonction beta-cellulaire residuelle ni le passage a l’insulinotherapie chez les patients traites par rapport au groupe placebo. Toutefois, les analyses immunologiques ont revele une induction efficace de la tolerance immunitaire vis-a-vis de l’insuline, au niveau de reponses des lymphocytes T ainsi que des reponses anticorpales. Le traitement par GAD-alun en sous-cutane chez les enfants diabetiques suedois a ete associe a une meilleur preservation de la fonction beta-cellulaire pour les patients traites precocement. Le traitement a induit aussi une augmentation des titres des anticorps anti-GAD associes a des reponses T productrices des cytokines de type Th1 (IFN-γ, TNF-α), Th2 (IL-5, IL-13) et T regulatrices (IL-10). Conclusion Ces resultats suggerent que la formulation antigenique (sans ou avec adjuvant) et la voie de vaccination induisent des effets immunologiques differents. La presence de biomarqueurs de tolerance immunitaire ne s’associe pas a des benefices cliniques significatifs, suggerant la necessite d’intervenir plus precocement ou d’associer plusieurs antigenes pour obtenir un effet de tolerance plus complet.

Cytokine activated cells (CIK) are ex-vivo expanded and activated cells expressing both T and NK surface markers (NKT type II, NKT-II). These cells have a broad non-MHC restricted antitumor cytotoxicity against autologous and allogeneic hematopoietic malignancies, while few data are available about their effectiveness against solid tumors. Murine studies demonstrated a reduced GVHD capacity of NKT-II cells when transplanted across major MHC barriers, making them a potential tool for adoptive therapy strategies after allogeneic hematopoietic cell transplant (HCT). Alloreactive potential of human NKT-II has been less investigated as well as their expansion from patients receiving an immunosuppressive treatment after HCT. We explored the alloreactive potential of human NKT- II and investigated NKT-II cytotoxicity against colon cancer cells. Additionally we looked at the possibility of expanding NKT-II from patients with solid tumors during immunosuppressive treatment after nonmyeloablative HCT (NM-HCT). NKT-II were obtained from PBMC, cultured for 3–4 weeks with timed addition of IL2, IFN-γ and anti-CD3. NKT-II with a CD3+/CD56+ phenotype were successfully expanded (median 140 folds, range 26–500) from both healthy donors (n=6) and patients (n=5); CD56+/CD3+ cells were either CD8+ (median 60%, range 18–86), CD4+ (7%, 1–27) or CD4/CD8 double negative (14%, 2–73). Expanded NKT-II cells efficiently proliferated in response to allogeneic irradiated PBMC as measured by 3H thymidine incorporation assay (n=2); this alloreactivity was comparable to a classic allogeneic mixed lymphocyte reaction used as control. Expanded NKT-II efficiently lysed 3 different colon cancer cell lines in standard chromium release assay (n=9). Interestingly, when NKT-II cultures were purified by positive selection of CD56+ cells, the CD56+ fraction did not either proliferate or have cytolitic activity against allogeneic PBMC, but fully retained the tumor killing capacity. Conversely, the CD56- fraction maintained the alloreactive potential but did not show any antitumor activity. These data suggest that the observed alloreactivity in the bulk NKT-II population is driven by CD56-/CD3+ cells, while the antitumor activity is due to CD56+/CD3+ T cells. The presence of pure NK cells (CD56+/CD3−) was negligible. When NKT-II cells were expanded after NM-HCT from patients affected by solid cancers (4 colorectal carcinoma, 1 osteosarcoma), they did not show significant differences as compared to NKT-II cells from healthy donors in terms of expansion and tumor killing. NKT-II were expanded from patients’ PBMC collected at a median of 42 days after a NM-HCT, when all patients were receiving cyclosporine as immunosuppression. In 2 cases analyzed, the degree of donor chimerism on expanded cells was comparable to the pre-expansion levels. In conclusion our data suggest that human NKT-II are able to kill colon cancer cells, even when expanded from chimeric patients treated with cyclosporine after allogeneic NM-HCT. The CD56+/CD3+ fraction is responsible for the antitumor activity and does not have a significant alloreactive potential across major MHC barriers. The presence of alloreactive CD56-/CD3+ cells in the bulk NKT-II population after the expansion should be taken into consideration for future application in the allogeneic setting, where a preventive CD56+ cell purification is advisable.