The central repeat domain 1 of Kaposi's sarcoma-associated herpesvirus (KSHV) latency associated-nuclear antigen 1 (LANA1) prevents cis MHC class I peptide presentation.

KSHV LANA1, a latent protein expressed during chronic infection to maintain a viral genome, inhibits major histocompatibility complex class I (MHC I) peptide presentation in cis as a means of immune evasion. Through deletional cloning, we localized this function to the LANA1 central repeat 1 (CR1) subregion. Other CR subregions retard LANA1 translation and proteasomal processing but do not markedly inhibit LANA1 peptide processing by MHC I. Inhibition of proteasomal processing ablates LANA1 peptide presentation. Direct expression of LANA1 within the endoplasmic reticulum (ER) overcomes CR1 inhibition suggesting that CR1 acts prior to translocation of cytoplasmic peptides into the ER. By physically separating CR1 from other subdomains, we show that LANA1 evades MHC I peptide processing by a mechanism distinct from other herpesviruses including Epstein-Barr virus (EBV). Although LANA1 and EBV EBNA1 are functionally similar, they appear to use different mechanisms to evade host cytotoxic T lymphocyte surveillance.

Natural killer cells from children with type 1 diabetes have defects in NKG2D-dependent function and signaling.

OBJECTIVE: Natural killer (NK) cells from NOD mice have numeric and functional abnormalities, and restoration of NK cell function prevents autoimmune diabetes in NOD mice. However, little is known about the number and function of NK cells in humans affected by type 1 diabetes. Therefore, we evaluated the phenotype and function of NK cells in a large cohort of type 1 diabetic children. RESEARCH DESIGN AND METHODS: Peripheral blood mononuclear blood cells were obtained from subjects whose duration of disease was between 6 months and 2 years. NK cells were characterized by flow cytometry, enzyme-linked immunosorbent spot assays, and cytotoxicity assays. Signaling through the activating NK cell receptor, NKG2D, was assessed by immunoblotting and reverse-phase phosphoprotein lysate microarray. RESULTS: NK cells from type 1 diabetic subjects were present at reduced cell numbers compared with age-matched, nondiabetic control subjects and had diminished responses to the cytokines interleukin (IL)-2 and IL-15. Analysis before and after IL-2 stimulation revealed that unlike NK cells from nondiabetic control subjects, NK cells from type 1 diabetic subjects failed to downregulate the NKG2D ligands, major histocompatibility complex class I-related chains A and B, upon activation. Moreover, type 1 diabetic NK cells also exhibited decreased NKG2D-dependent cytotoxicity and interferon-γ secretion. Finally, type 1 diabetic NK cells showed clear defects in NKG2D-mediated activation of the phosphoinositide 3-kinase-AKT pathway. CONCLUSIONS: These results are the first to demonstrate that type 1 diabetic subjects have aberrant signaling through the NKG2D receptor and suggest that NK cell dysfunction contributes to the autoimmune pathogenesis of type 1 diabetes.

Cutting edge: Increased IL-17-secreting T cells in children with new-onset type 1 diabetes.

CD4(+)FOXP3(+) regulatory T cells are essential for immune tolerance, and murine studies suggest that their dysfunction can lead to type 1 diabetes (T1D). Human studies assessing regulatory T cell dysfunction in T1D have relied on analysis of FOXP3-expressing cells. Recently, distinct subsets of CD4(+)FOXP3(+) T cells with differing function were identified. Notably, CD45RA(-)CD25(int)FOXP3(low) T cells lack suppressive function and secrete the proinflammatory cytokine IL-17. Therefore, we evaluated whether the relative fractions of CD4(+)FOXP3(+) subsets are altered in new-onset T1D subjects. We report that children with new-onset T1D have an increased proportion of CD45RA(-)CD25(int)FOXP3(low) cells that are not suppressive and secrete significantly more IL-17 than other FOXP3(+) subsets. Moreover, these T1D subjects had a higher proportion of both CD4(+) and CD8(+) T cells that secrete IL-17. The bias toward IL-17-secreting T cells in T1D suggests a role for this proinflammatory cytokine in the pathogenesis of disease.

Critical role for IFN-gamma in natural killer cell-mediated protection from diabetes.

Autoimmune diabetes in nonobese diabetic (NOD) mice can be prevented by a single injection of complete Freund's adjuvant (CFA), but the mechanisms mediating protection remains unclear. We previously showed that NOD mice immunized with CFA have a markedly reduced incidence of diabetes that is associated with a significant decrease in the number of beta-cell-specific, autoreactive cytotoxic T lymphocytes and, furthermore, that the effect of CFA is mediated by natural killer (NK) cells. In this study, we report one mechanism by which NK cells regulate the onset of diabetes. Administration of CFA produced a rapid increase in NK cell frequency and function, including cytotoxicity and IFN-gamma secretion. By co-transferring NK cells from IFN-gamma-deficient (or wild-type) NOD mice and spleen cells from diabetic NOD mice to NOD/SCID recipients, we show that IFN-gamma secretion by NK cells significantly influences the effect of CFA protection. In contrast, NK cytotoxicity does not appear to participate in CFA-mediated protection from diabetes. Our findings demonstrate that NK cells mediate the protective effects of CFA through secretion of IFN-gamma.

Recognition of HLA class I-restricted beta-cell epitopes in type 1 diabetes.

Type 1 diabetes results from the autoimmune destruction of insulin-producing pancreatic beta-cells by cytotoxic T-lymphocytes (CTLs). In humans, few beta-cell epitopes have been reported, thereby limiting the study of beta-cell-specific CTLs in type 1 diabetes. To identify additional epitopes, HLA class I peptide affinity algorithms were used to identify a panel of peptides derived from the beta-cell proteins islet amyloid polypeptide (IAPP), islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), insulin, insulinoma-associated antigen 2 (IA-2), and phogrin that were predicted to bind HLA-A*0201. Peripheral blood mononuclear cells from 24 HLA-A*0201 recent-onset type 1 diabetic patients and 11 nondiabetic control subjects were evaluated for gamma-interferon secretion in response to peptide stimulation in enzyme-linked immunospot assays. We identified peptides IAPP9-17, IGRP215-223, IGRP152-160, islet IA-2(172-180), and IA-2(482-490) as novel HLA-A*0201-restricted T-cell epitopes in type 1 diabetic patients. Interestingly, we observed a strong inverse correlation between the binding affinity of beta-cell peptides to HLA-A*0201 and CTL responses against those peptides in recent-onset type 1 diabetic patients. In addition, we found that self-reactive CTLs with specificity for an insulin peptide are frequently present in healthy individuals. These data suggest that many beta-cell epitopes are recognized by CTLs in recent-onset type 1 diabetic patients. These epitopes may be important in the pathogenesis of type 1 diabetes.

The complement C5b-9 complexes induced injury of glomerular mesangial cells in rats with Thy-1 nephritis by increasing nitric oxide synthesis.

Thy-1 nephritis (Thy-1 N), namely, anti-Thy-1 or anti-thymocyte serum (ATS) induced nephritis (ATSN), is a typical model of human mesangioproliferative glomerulonephritis. The pathologic changes of glomerular mesangial cells (GMCs) in Thy-1 N are complement-dependent, especially C5b-9 complexes, but the role of C5b-9 in the mechanism of Thy-1 N has not been defined. Because previous studies have demonstrated that sublytic C5b-9 can increase production of several inflammatory mediators from resident glomerular cells, we utilized the isolated human membrane-bound C5b-9 complexes to stimulate the cultured rat GMCs and examined whether the GMCs can also induce the synthesis of nitric oxide (NO) in vitro. Simultaneously, the effects of antiserum against rat C5b-9 and NG-monomethyl-L-arginine (L-NMMA, NO inhibitor), including interfering with the formation of C5b-9, reducing NO production and GMCs injury were observed. The results showed that sublytic C5b-9 can increase synthesis of inducible NO from the stimulated GMCs, and that the anti-C5b-9 antiserum can obviously inhibit the pathologic changes in Thy-1 N, while L-NMMA can decrease the GMCs damage although the effect is not so significant as that of the anti-C5b-9 antiserum. These findings indicate that the synthesis of NO by GMCs can be promoted by sublytic C5b-9, and that lesions of GMCs in rats with Thy-1 N are prevented by either inhibiting C5b-9 formation or NO elevation in advance. The pathologic changes of GMCs in Thy-1 N are indeed complement C5b-9-dependent, and the glomerular injury can be mediated in part through elevation of NO from the GMCs after the sublytic C5b-9 stimulation.

Progression of spontaneous autoimmune diabetes is associated with a switch in the killing mechanism used by autoreactive CTL.

Autoimmune (type 1) diabetes mellitus results from the destruction of insulin-producing pancreatic beta-cells by T lymphocytes. Beta-cell death that is induced by autoreactive CTL in diabetes involves both Fas/Fas ligand (FasL)- and perforin/granzyme-mediated pathways, although their relative contributions during the progression of the disease remain unknown. We demonstrate here that despite the preferential use of the Fas/FasL pathway for cytolysis of beta-cell targets, transgenic beta-cell-specific CTL were able to kill targets via the perforin pathway when triggered by a higher affinity stimulus. In addition, we show that the killing mechanism used by islet-associated CD8(+) T cells from non-obese diabetic mice changed as the mice aged and correspondingly, with the stage of diabetes. These results provide direct evidence for age-related changes in the cytotoxic pathways used by diabetogenic T cells during the progression of autoimmune diabetes.

Regulation of autoimmune diabetes by complete Freund's adjuvant is mediated by NK cells.

Autoimmune (type 1) diabetes results from a loss of beta cells that is mediated by self-reactive T cells. Previous studies have shown that a single injection of CFA prevents diabetes in nonobese diabetic (NOD) mice, but the mechanism(s) of protection remain unknown. We show here that NOD mice immunized with CFA have a markedly reduced incidence of diabetes and that this reduced incidence is associated with a decrease in the number of beta cell-specific, autoreactive CTL. In addition, the adoptive transfer of diabetes into syngeneic NOD/SCID recipients was prevented by CFA immunization, and the protective effects of CFA were lost when cells expressing the NK cell marker, asialo GM1, were removed from both donor cells and recipient mice. Returning a population of CD3-DX5+ cells to the adoptive transfer restored the protective effects of CFA. Therefore, NK cells mediate the protective effects of CFA possibly through the down-regulation of autoreactive CTL and stimulation of NK cells represents a novel approach to the prevention of autoimmune diabetes.

Identification of a beta-cell-specific HLA class I restricted epitope in type 1 diabetes.

Type 1 diabetes is an autoimmune disease in which pancreatic beta-cells are destroyed by cytotoxic T-cells that recognize peptide epitopes presented by HLA class I molecules. The identification of human beta-cell epitopes may significantly improve the prospects for immunodiagnosis and immunotherapy in type 1 diabetes. Using algorithms to predict nonameric beta-cell peptides that would bind to the common HLA allele, HLA-A*0201, we identified a potential epitope from the leader sequence of islet amyloid polypeptide (human islet amyloid polypeptide [IAPP] precursor protein [preproIAPP] 5-13: KLQVFLIVL). Peripheral blood mononuclear cells (PBMCs) were isolated from 18 HLA-A*0201 patients with type 1 diabetes (9 with recent-onset [<180 days; range, 1-120 days] and 9 with long-standing diabetes [>180 days; range, 183-3,273 days]) and 9 healthy, nondiabetic control subjects. PBMCs were screened for peptide recognition using interferon-gamma enzyme-linked immunospot (ELISpot) assays. Of the nine patients with recent-onset type 1 diabetes, six had ELISpot responses to preproIAPP 5-13 that were >3 SDs above the mean of the nondiabetic control subjects (P = 0.002). In contrast, no patients with type 1 diabetes for >180 days had a response above this threshold. In summary, preproIAPP 5-13 is a novel HLA class I epitope recognized by a significant proportion of cytotoxic T-cells from HLA-A*0201 patients with recent-onset type 1 diabetes and may prove to be a useful tool for the prediction and/or prevention of this disease.

Effects of "moxibustion serum" on proliferation and phenotypes of tumor infiltrating lymphocytes.

Tumor infiltrating lymphocytes (TIL) were cultured with "moxibustion serum" (MS), and the results were examined by flow cytometry. The results indicated that MS could enhance the proliferation of TIL, accelerate it to reach the exponential growth phase, and assist recombinant interleukin 2 (rIL-2) to enhance successively the percentage of CD3+ positive cells, maintain the number of CD4+ positive T cells, promote greatly the percentage of CD8+ positive T cells among TILs, and reverse the CD4+/CD8+ ratio. Such cooperative effects rely on relative specificity of acupoints. It is suggested that MS is beneficial to the growth of TIL both in the aspects of proliferation and phenotypes.