Cutting Edge: a novel, human-specific interacting protein couples FOXP3 to a chromatin-remodeling complex that contains KAP1/TRIM28.

Regulatory T cells (Tregs) play a pivotal role in the maintenance of immunological self-tolerance. Deficiency or dysfunction of Tregs leads to severe autoimmune diseases. Although the forkhead/winged-helix family member FOXP3 is critical for Treg differentiation and function, the molecular basis for FOXP3 function remains unclear. In this study, we identified and characterized a human-specific FOXP3-interacting protein, referred to as FIK (FOXP3-interacting KRAB domain-containing protein). FIK is highly expressed in Tregs and acts as a bridging molecule to link FOXP3 with the chromatin-remodeling scaffold protein KAP1 (TIF-1ß/TRIM28). Disruption of the FOXP3-FIK-KAP1 complex in Tregs restored expression of FOXP3-target genes and abrogated the suppressor activity of the Tregs. These data demonstrate a critical role for FIK in regulating FOXP3 activity and Treg function.

Circulating cytokines are associated with human islet graft function in type 1 diabetes.

Islet cell transplantation has considerable potential as a cure for type 1 diabetes, but recurrent autoimmunity and allograft rejection in which both cytokines play an important role are major obstacles. Using a new approach considering confounders by regression analysis, we investigated circulating cytokines and their association with graft function in type 1 diabetes patients who underwent either simultaneous islet kidney (SIK) or islet after kidney (IAK) transplantation. After transplantation, interleukin (IL)-10 was lower in SIK recipients with subsequent loss of graft function in comparison to recipients maintaining graft function. Before transplantation, high IL-13 and IL-18 concentrations were prospectively associated for subsequent loss of graft function in IAK recipients, whereas in SIK recipients, high macrophage migration inhibitory factor (MIF) concentrations were associated with subsequent loss of graft function. Circulating cytokines are associated with islet graft function in patients with long-standing type 1 diabetes when considering confounders.

Association of T-cell reactivity with beta-cell function in recent onset type 1 diabetes patients.

OBJECTIVE: The aim of the current study was to investigate whether autoantigen directed T-cell reactivity relates to beta-cell function during the first 78 weeks after diagnosis of type 1 diabetes. RESEARCH DESIGN AND METHODS: 50 adults and 49 children (mean age 27.3 and 10.9 years respectively) with recent onset type 1 diabetes who participated in a placebo-controlled trial of immune intervention with DiaPep277 were analyzed. Secretion of interferon (IFN)-gamma, interleukin (IL)-5, IL-13 and IL-10 by single peripheral mononuclear cells (PBMC) upon stimulation with islet antigens GAD65, heat shock protein 60 (Hsp60) protein-tyrosine-phosphatase-like-antigen (pIA2) or tetanus toxoid (TT) was determined applying ELISPOT; beta-cell function was evaluated by glucagon stimulated C-peptide. Multivariate regression analysis was applied. RESULTS: In general, number of islet antigen-reactive cells decreased over 78 weeks in both adults and children, whereas reactivity to TT was not reduced. In addition, there was an association between the quality of immune cell responses and beta-cell function. Overall, increased responses by IFN-gamma secreting cells were associated with lower beta-cell function whereas IL-5, IL-13 and IL-10 cytokine responses were positively associated with beta-cell function in adults and children. Essentially, the same results were obtained with three different models of regression analysis. CONCLUSIONS: The number of detectable islet-reactive immune cells decreases within 1-2 years after diagnosis of type 1 diabetes. Cytokine production by antigen-specific PBMC reactivity is related to beta-cell function as measured by stimulated C-peptide. Cellular immunity appears to regress soon after disease diagnosis and begin of insulin therapy.

Effect of serum content and diluent selection on assay sensitivity and signal intensity in multiplex bead-based immunoassays.

INTRODUCTION: In the Luminex multiplexed immunoassay system, complex samples such as human serum are diluted to minimize disturbing matrix effects with a specific diluent. This diluent has to imitate the sample matrix to allow interpolation and has to provide optimal cytokine-antibody binding for all cytokines. Because diluents influence multiplex immunoassay results, this paper explores several methods to determine the quality of a chosen diluent. MATERIALS AND METHODS: Two commercially available diluents, DY997 and RD6 from R&D Systems, were compared in a 19-plex immunoassay setup from Luminex. RESULTS: Using diluent DY997, multiplex signal intensity was reduced by 55% when spiked samples (chemokines and cytokines at 100 pg/mL) contained 50% v/v human serum, compared to samples containing 25% v/v. When using diluent RD6, signal intensity was reduced by 20% when samples contained 50% v/v human serum, compared to 25% v/v human serum. Diluent DY997 showed decreasing multiplex assay sensitivity with increasing protein concentrations, but not as low as in the presence of 50% v/v human serum. CONCLUSIONS: In a 19-plex setup, this paper describes signal intensity, assay sensitivity and background signal levels in relation to the total volume-fraction of serum and protein concentration. For the determination of cytokines in serum samples with the multiplexed system Luminex the diluent RD6 seems more appropriate than the diluent DY997.

KAP1 Regulates Regulatory T Cell Function and Proliferation in Both Foxp3-Dependent and -Independent Manners.

Regulatory T cells (Tregs) are indispensable for the establishment of tolerance of self-antigens in animals. The transcriptional regulator Foxp3 is critical for Treg development and function, controlling the expression of genes important for Tregs through interactions with binding partners. We previously reported KAP1 as a binding partner of FOXP3 in human Tregs, but the mechanisms by which KAP1 affects Treg function were unclear. In this study, we analyzed mice with Treg-specific deletion of KAP1 and found that they develop spontaneous autoimmune disease. KAP1-deficient Tregs failed to induce Foxp3-regulated Treg signature genes. In addition, KAP1-deficient Tregs were less proliferative due to the decreased expression of Slc1a5, whose expression was KAP1 dependent but Foxp3 independent. This reduced expression of Slc1a5 resulted in reduced mTORC1 activation. Thus, our data suggest that KAP1 regulates Treg function in a Foxp3-dependent manner and also controls Treg proliferation in a Foxp3-independent manner.

Association of IL-1ra and adiponectin with C-peptide and remission in patients with type 1 diabetes.

OBJECTIVE: We investigated the association of anti-inflammatory cytokine interleukin (IL)-1 receptor antagonist (IL-1ra), adiponectin, proinflammatory cytokines IL-1 beta, IL-6, and CCL2, and tumor necrosis factor-alpha with beta-cell function, metabolic status, and clinical remission in patients with recent-onset type 1 diabetes. RESEARCH DESIGN AND METHODS: Serum was obtained from 256 newly diagnosed patients (122 males and 134 females, median age 9.6 years). Stimulated C-peptide, blood glucose, and A1C were determined in addition to circulating concentration of cytokines at 1, 6, and 12 months after diagnosis. Analyses were adjusted for sex, age, and BMI percentile. RESULTS: Anti-inflammatory IL-1ra was positively associated with C-peptide after 6 (P = 0.0009) and 12 (P = 0.009) months. The beneficial association of IL-1ra on beta-cell function was complemented by the negative association of IL-1 beta with C-peptide after 1 month (P = 0.009). In contrast, anti-inflammatory adiponectin was elevated in patients with poor metabolic control after 6 and 12 months (P < 0.05) and positively correlated with A1C after 1 month (P = 0.0004). Proinflammatory IL-6 was elevated in patients with good metabolic control after 1 month (P = 0.009) and showed a positive association with blood glucose disposal after 12 months (P = 0.047). CONCLUSIONS: IL-1ra is associated with preserved beta-cell capacity in type 1 diabetes. This novel finding indicates that administration of IL-1ra, successfully improving beta-cell function in type 2 diabetes, may also be a new therapeutic approach in type 1 diabetes. The relation of adiponectin and IL-6 with remission and metabolic status transfers observations from in vitro and animal models into the human situation in vivo.