Dimorphic histopathology of long-standing childhood-onset diabetes.

AIMS/HYPOTHESIS: Childhood diabetes is thought to usually result from autoimmune beta cell destruction (type 1A) with eventual total loss of beta cells. Analysis of C-peptide in children characterised at diabetes onset for autoantibodies shows heterogeneous preservation of insulin secretion in long-standing diabetes. The aim of this study was to characterise the pancreases of childhood-onset diabetes in order to define the pathological basis of this heterogeneity. METHODS: We evaluated 20 cadaveric organ donor pancreases of childhood-onset long-term patients for disease heterogeneity and obtained corresponding C-peptide measurements. RESULTS: Pancreases from the majority of cadaveric donors contained only insulin-deficient islets (14 of 20). The remaining six patients (30%) had numerous insulin-positive cells within at least some islets, with two different histological patterns. Pattern A (which we would associate with type 1A diabetes) had lobular retention of areas with 'abnormal' beta cells producing the apoptosis inhibitor survivin and HLA class I. In pattern B, 100% of all islets contained normal-appearing but quantitatively reduced beta cells without survivin or HLA class I. CONCLUSIONS/INTERPRETATION: Our data demonstrate that C-peptide secretion in long-standing diabetic patients can be explained by two different patterns of beta cell survival,possibly reflecting different subsets of type 1 diabetes.

Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.

Avian leukosis virus induces lymphoma in chickens after proviral integration within the c-Myc gene, and subsequent expansion of Myc-overexpressing lymphocytes within transformed bursal follicles. The clonal expansion of these follicles allowed us to examine how Myc influences cell differentiation, growth, and apoptosis in lymphoid progenitors soon after the onset of Myc overexpression. Immunohistochemical analysis of developmental markers established that Myc overexpression consistently blocks lymphocyte differentiation at a late embryonic stage. Myc-transformed follicles also grow much more rapidly than normal follicles. This rapid growth is not mediated by suppression of apoptosis, as normal and Myc-transformed follicles showed similar rates of cell death by TUNEL immunohistochemical analysis of cells undergoing DNA degradation. Measurements of DNA synthesis and mitotic index showed modest effects of Myc to increase lymphocyte proliferation, as normal lymphocytes already divide rapidly. The major mechanism mediating rapid growth of transformed follicles instead involved failure of myc-overexpressing lymphocytes to emigrate from transformed follicles, while normal lymphocytes actively emigrate after hatching, as measured by BrdU pulse-chase labeling and immunohistochemical measurements. This failure to undergo the normal program of differentiation and subsequent bursal retention of lymphocytes accounts for most of the growth of transformed follicles, while Myc-induced proliferation makes a smaller contribution.

Heterophile antibodies indicate progression of autoimmunity in human type 1 diabetes mellitus before clinical onset.

We previously reported serum cytokines in a group of long term non-progressors to Type 1 diabetes; this reactivity detected in ELISA is now identified as heterophile antibody in some sera. Here, we characterize heterophile antibody activity. A 14 kDa-polypeptide from heterophile antibody containing serum bound to an anti-IL-4 column, but IL-4 was not detected by Western blot or by MS/MS sequencing. However, in 2/13 heterophile antibody positive sera, T-cell growth was potentiated and was blocked by an anti-human immunoglobulin. To examine the relationship between low affinity heterophile antibody presence and disease progression, 1100 archived serum samples were analyzed with two pairs of antibodies from 443 diabetes-free first degree relatives of Type 1 diabetes mellitus patients for heterophile antibody; 95 individuals developed diabetes on follow-up. Twenty-two individuals, whose serum was heterophile antibody positive with the second pair of antibodies (but negative with the first pair of antibodies), had a significantly higher incidence of developing diabetes after five years. Thirty-seven individuals with heterophile antibody reactivity with the first pair of antibodies, regardless of reactivity with the second pair of antibodies, had a significantly lower incidence of developing diabetes. While we cannot exclude the presence of genuine cytokine in all sera, these data indicate the presence of distinct groups of heterophile antibodies in patients at high risk to develop diabetes. Thus, anti-Ig heterophilic antibodies with different immunochemical reactivities are linked to the progression of or protection from Type 1 diabetes autoimmunity.

Multiple differences in gene expression in regulatory Valpha 24Jalpha Q T cells from identical twins discordant for type I diabetes.

Quantitative and qualitative defects in CD1d-restricted T cells have been demonstrated in human and murine autoimmune diseases. To investigate the transcriptional consequences of T cell receptor activation in human Valpha24JalphaQ T cell clones, DNA microarrays were used to quantitate changes in mRNA levels after anti-CD3 stimulation of clones derived from identical twins discordant for type 1 diabetes and IL-4 secretion. Activation resulted in significant modulation of 226 transcripts in the IL-4 secreting clone and 86 in the IL-4-null clone. Only 28 of these genes were in common. The differences observed suggest both ineffective differentiation of diabetic Valpha24JalphaQ T cells and a role for invariant T cells in the recruitment and activation of cells from the myeloid lineage.

Noncanonical Valpha24JalphaQ T cells with conservative alpha chain CDR3 region amino acid substitutions are restricted by CD1d.

Human NKRP-1A (CD161)+ T cells include members of a family of CD4+ or CD4-/CD8- lymphocytes that utilize an invariant alpha chain in the T-cell receptor (TCR). The alpha chain consists of the Valpha24 segment joined to Jalpha18 (JalphaQ) (TCRAV24/AJ18). These families of T cells rapidly produce both interleukin-4 and interferon-gamma upon TCR cross-linking, and are restricted by CD1d. To determine the spectrum of allowable V/J rearrangements in the Valpha24+ CD4-/CD8- family, TCR Valpha24 chain transcripts derived from the total CD4-/CD8- population in peripheral blood mononuclear cells were sequenced. A second invariant rearrangement, Valpha24Jalpha45, was found in two donors. In addition, a subset of 15 clones with single amino acid substitutions in the CDR3 were identified and used to define CD1d restriction. All 15 variant clones were indistinguishable from invariant clones on the basis of surface phenotype and response to CD3 cross-linking. However, CD1d was the restriction element only for those clones with the conservative substitution of threonine or asparagine for serine at the V/J junction. Thus, the family of human CD161+ T cells can be extended to include a subset of Valpha24-JalphaQ rearrangements with a single amino acid substitution that defines a Valpha24 CDR3 residue critical for CD1d restriction.

Differential responses of invariant V alpha 24J alpha Q T cells and MHC class II-restricted CD4+ T cells to dexamethasone.

NK T cells are a T cell subset in the human that express an invariant alpha-chain (V alpha 24invt T cells). Because of the well-described immunomodulation by glucocorticoids on activation-induced cell death (AICD), the effects of dexamethasone and anti-CD3 stimulation on V alpha 24invt T cell clones and CD4+ T cell clones were investigated. Dexamethasone significantly enhanced anti-CD3-mediated proliferation of V alpha 24invt T cells, whereas CD4+ T cells were inhibited. Addition of neutralizing IL-2 Ab partially abrogated dexamethasone-induced potentiation of V alpha 24invt T cell proliferation, indicating a role for autocrine IL-2 production in corticosteroid-mediated proliferative augmentation. Dexamethasone treatment of anti-CD3-stimulated V alpha 24invt T cells did not synergize with anti-Fas blockade in enhancing proliferation or preventing AICD. The V alpha 24invt T cell response to dexamethasone was dependent on the TCR signal strength. In the presence of dexamethasone, lower doses of anti-CD3 inhibited proliferation of V alpha 24invt T cells and CD4+ T cells; at higher doses of anti-CD3, which caused inhibition of CD4+ T cells, the V alpha 24invt T cell clones proliferated and were rescued from AICD. These results demonstrate significant differences in TCR signal strength required between V alpha 24invt T cells and CD4+ cells, and suggest important immunomodulatory consequences for endogenous and exogenous corticosteroids in immune responses.

Heterophile antibodies segregate in families and are associated with protection from type 1 diabetes.

Markedly elevated levels of serum IL-4 were reported previously in 50% of a small group of type 1 diabetes nonprogessors. To determine the patterns of expression for this phenotype, a larger cohort of 58 families containing type 1 diabetic patients was examined. Analysis of the two-site ELISA assay used to measure serum IL-4 revealed evidence for heterophile antibodies, i.e., nonanalyte substances in serum capable of binding antibodies mutivalently and providing erroneous analyte (e.g., IL-4) quantification. Interestingly, relatives without type 1 diabetes were significantly more likely to have this phenotype than were patients with the disease (P = 0.003). In addition, the trait appears to have clustered within certain families and was associated with the protective MHC allele DQB1*0602 (P = 0.008). These results suggest that heterophile antibodies represent an in vivo trait associated with self-tolerance and nonprogression to diabetes.

Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes.

Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) is a disease controlled by the major histocompatibility complex (MHC) which results from T-cell-mediated destruction of pancreatic beta-cells. The incomplete concordance in identical twins and the presence of autoreactive T cells and autoantibodies in individuals who do not develop diabetes suggest that other abnormalities must occur in the immune system for disease to result. We therefore investigated a series of at-risk non-progressors and type 1 diabetic patients (including five identical twin/triplet sets discordant for disease). The diabetic siblings had lower frequencies of CD4-CD8- Valpha24JalphaQ+ T cells compared with their non-diabetic sibling. All 56 Valpha24JalphaQ+ clones isolated from the diabetic twins/triplets secreted only interferon (IFN)-gamma upon stimulation; in contrast, 76 of 79 clones from the at-risk non-progressors and normals secreted both interleukin (IL)-4 and IFN-gamma. Half of the at-risk non-progressors had high serum levels of IL-4 and IFN-gamma. These results support a model for IDDM in which Thl-cell-mediated tissue damage is initially regulated by Valpha24JalphaQ+ T cells producing both cytokines; the loss of their capacity to secrete IL-4 is correlated with IDDM.

Oral administration of myelin induces antigen-specific TGF-beta 1 secreting T cells in patients with multiple sclerosis.

Oral administration of antigen is a long-recognized method of inducing systemic immune tolerance. In animals with experimental autoimmune disease, a major mechanism of oral tolerance involves the induction of regulatory T cells that mediate active suppression by secreting the cytokine TGF-beta 1. Multiple sclerosis (MS) is a presumed T cell-mediated Th1 type autoimmune disease. In this paper we investigated, in patients with MS, whether oral myelin treatment (myelin containing both MBP and PLP) induced antigen-specific MBP- or PLP-reactive T cells that were either Th2-like (secreted IL-4 or TGF-beta 1), or alternatively whether Th1 type sensitization occurred as measured by IFN-gamma secretion. Specifically, 4,860 short-term T cell lines were generated to either MBP, PLP or TT from 34 relapsing-remitting patients with MS; 17 were orally treated with bovine myelin daily for a minimum of two years as compared to 17 non-treated patients. We found a marked increase in the relative frequencies of both MBP- and PLP-specific TGF-beta 1 secreting T cell lines in the myelin-treated MS patients as compared to non-treated MS patients (MBP, p < 0.001; PLP, p < 0.003). In contrast, no changes in the frequency of MBP- or PLP-specific IFN-gamma or TT-specific TGF-beta 1 secreting T cells were observed. These results suggest that the oral administration of antigens generates antigen-specific TGF-beta 1 secreting T cells of presumed mucosal origin that may represent a distinct cytokine-secreting lineage of T cells (Th3). Since, in animal models, antigen-specific TGF-beta 1 secreting cells localize to the target organ and then suppress inflammation in the local microenvironment, oral tolerization with self-antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not depend upon knowledge of the antigen specificity of the original T cell clone triggering the autoimmune cascade.

Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-beta1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients.

Oral administration of antigen is a long recognized method of inducing systemic immune tolerance. In animals with experimental autoimmune disease, a major mechanism of oral tolerance triggered by oral administration of antigen involves the induction of regulatory T cells that mediate active suppression by secreting the cytokine TGF-beta 1. Multiple sclerosis (MS) is a presumed T cell-mediated Th1 type autoimmune disease. Here, we investigated whether in MS patients oral myelin treatment, containing both myelin basic protein (MBP) and proteolipid protein (PLP), induced antigen specific MBP or PLP reactive T cells that either secreted IL4, TGF-beta1, or alternatively did Th1 type sensitization occur as measured by IFN-gamma secretion. Specifically, 4,860 short-term T cell lines were generated to either MBP, PLP, or tetanus toxoid (TT) from 34 relapsing-remitting MS patients: 17 orally treated with bovine myelin daily for a minimum of 2 yr as compared to 17 nontreated patients. We found a marked increase in the relative frequencies of both MBP and PLP specific TGF-beta1-secreting T cell lines in the myelin treated MS patients as compared to non-treated MS patients (MBP P < 0.001, PLP P < 0.003). In contrast, no change in the frequency of MBP or PLP specific IFN-gamma or TT specific TGF-beta1 secreting T cells were observed. These results suggest that the oral administration of antigens generates antigen specific TGF-beta1 secreting Th3 cells of presumed mucosal origin that represent a distinct lineage of T cells. Since antigen-specific TGF-beta1 secreting cells localize to the target organ and then suppress inflammation in the local microenvironment, oral tolerization with self antigens may provide a therapeutic approach for the treatment of cell-mediated autoimmune disease which does not depend upon knowledge of the antigen specificity of the original T cell clone triggering the autoimmune cascade.

Antigen-specific TGF-beta1 secretion with bovine myelin oral tolerization in multiple sclerosis.

Multiple sclerosis is a presumed autoimmune disease, associated with inflammation in the CNS white matter, mediated by autoreactive T cells. We previously reported that oral myelin tolerization of relapsing-remitting MS patients resulted in fewer attacks, as compared to a placebo-fed group. Here, we examined whether oral tolerization with bovine myelin resulted in altered autoreactive T-cell populations or altered T-cell fraction. We generated 4,620 T-cell lines from 34 relapsing-remitting MS patients (17 were fed bovine myelin daily), and each line was examined for proliferation to MBP, PLP, and TT and for secretion of IL-4, IFN-gamma, and TGF-beta1. The frequency of TGF-beta1-secreting T-cell lines after MBP and PLP stimulation in fed patients was greater than that of nonfed patients. These experiments demonstrate that oral tolerization with autoantigen results in altered cytokine secretion in a human autoimmune disease with the generation of TGF-beta1-secreting T cells that may regulate the inflammatory response at the site of the demyelinating lesions in multiple sclerosis. These data provide the first evidence of antigen-specific modification of cytokine secretion in a human autoimmune disease.

Lymphoma models for B cell activation and tolerance. X. Anti-mu-mediated growth arrest and apoptosis of murine B cell lymphomas is prevented by the stabilization of myc.

Treatment of the WEHI-2131 or CH31 B cell lymphomas with anti-mu or transforming growth factor (TGF)-beta leads to growth inhibition and subsequent cell death via apoptosis. Since anti-mu stimulates a transient increase in c-myc and c-fos transcription in these lymphomas, we examined the role of these proteins in growth regulation using antisense oligonucleotides. Herein, we demonstrate that antisense oligonucleotides for c-myc prevent both anti-mu- and TGF-beta-mediated growth inhibition in the CH31 and WEHI-231 B cell lymphomas, whereas antisense c-fos has no effect. Furthermore, antisense c-myc promotes the appearance of phosphorylated retinoblastoma protein in the presence of anti-mu and prevents the progression to apoptosis as measured by propidium iodide staining. Northern and Western analyses show that c-myc message and the levels of multiple myc proteins were maintained in the presence of antisense c-myc, results indicating that myc species are critical for the continuation of proliferation and the prevention of apoptosis. These data implicate c-myc in the negative signaling pathway of both TGF-beta and anti-mu.