Kathryn Haskins

Following a diabetogenic T cell from genesis through pathogenesis

Nonobese diabetic (NOD) mice spontaneously develop a disease very similar to type 1 diabetes in humans. We have generated a transgenic mouse strain carrying the rearranged T cell receptor genes from a diabetogenic T cell clone derived from a NOD mouse. Self-reactive T cells expressing the transgene-encoded specificity are not tolerized in these animals, resulting in rampant insulitis and eventually diabetes. Features of the disease process emphasize two so-called check-points, recognized previously in the NOD and human diseases but easily misinterpreted. Although NOD mice are protected from insulitis and diabetes by expression of the E molecule encoded in the major histocompatibility complex, the transgenics are not, permitting us to exclude some possible mechanisms of protection.

Chromogranin A is an autoantigen in type 1 diabetes

Autoreactive CD4+ T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4+ T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-Ag7 in an atypical manner, occupying only the carboxy-terminal half of the I-Ag7 peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

The mouse T cell receptor: Comparison of MHC-restricted receptors on two T cell hybridomas

The receptors for antigen plus a major histocompatibility complex (MHC) gene product on a T cell hybridoma specific for ovalbumin plus a Class II MHC product were compared with those on another T cell hybridoma, specific for a Class I MHC product. In each case receptor material was identified by a clone-specific monoclonal antibody. The two receptors proved to have very similar gross structures, being 70-85 kd proteins, and reducing to an acidic alpha-chain and a slightly basic beta-chain, each 40-43 kd. The charge of both the acidic and basic polypeptides varied between the two receptors studied, showing that variable amino acid sequences occur in both chains.

T-Lymphocyte Clone Specific for Pancreatic Islet Antigen

A cloned T-lymphocyte line, BDC-2.5, was derived from a nonobese diabetic (NOD) mouse and has been found to exhibit specificity for islet cell antigen in vitro and in vivo. This clone is a CD4+ T-lymphocyte that proliferates and makes lymphokine in response to islet cell antigen- and NOD antigen-presenting cells. In an in vivo transplantation system in which islet grafts were made in the presence or absence of the BDC-2.5 T-lymphocytes, it was found that incorporation of the islet-specific T-lymphocytes into the graft site resulted in complete destruction of the transplanted tissue. Similar grafts made with pituitary tissue were not affected by the T-lymphocyte clone. These results suggest that the islet-specific T-lymphocytes mediate islet destruction in a tissue-specific manner.

The major histocompatibility complex-restricted antigen receptor on T cells in mouse and man: Identification of constant and variable peptides

The variability of the MHC restricted receptor on murine T cells was examined by comparing tryptic peptide fingerprints of the receptor isolated fom three T cell hybridomas and a T cell tumor. Both variable and constant peptides were seen. Constant peptides were most apparent when comparing receptors from the same mouse strain. Peptide fingerprints of receptors from two independent T cell hybridomas with the same idiotype and specificity were identical. We also describe a molecule detected on the surface of a human T cell leukemia whose properties were identical to those reported for the MHC receptor on normal human T cells. The molecule was a dimer of 85,000-90,000 MW containing a 46,000 MW acidic alpha-chain and an unrelated 40,000 MW neutral beta-chain.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Diabetogenic T-Cell Clones

The role of T-cells in the pathogenesis of IDDM has been an area of much interest, and investigators have recently acquired new tools for studies on T-cells with the advent of T-cell clones that are reactive with islet antigens. Derived from NOD mice, diabetogenic T-cell lines and clones have for the most part been CD4+ and T-helper 1 (Th1)-like in their cytokine production. Some CD8+ cytotoxic clones have also been reported, although these have generally not transferred diabetes in the absence of CD4+ T-cells. The T-cell clones that have been described can also be separated on the basis of their antigen reactivity. While many of the T-cell lines and clones described react with islets, isolated islet cells, or islet membrane preparations, others have known antigen specificities, reacting with defined islet cell proteins such as insulin, GAD, and heat shock proteins. Particularly in the case of insulin-reactive clones, diabetogenicity has also been demonstrated. In light of the many possible T-cell reactivities that may arise from the islet lesion, the question of whether there is a dominant initiating antigen is a particularly intriguing one.

Oxidative stress in type 1 diabetes.

Abstract: We have been investigating the effects of preventing oxidative stress on pathogenesis and complications of type 1 diabetes in the NOD mouse model. Our studies have shown that damage caused by oxidative stress is higher in islets and vascular tissue of NOD mice than in nonautoimmune controls or a diabetes‐resistant NOD mouse. In addition, phagocytic function and cytokine production by macrophages are aberrant in the NOD. We have demonstrated that treatment of prediabetic NOD mice for 2 weeks with a metalloporphyrin superoxide dismutase (SOD) mimetic results in marked reduction of oxidative stress in islets and vascular tissue and a reversal of macrophage defects.

Transfer of Diabetes in the NOD-scid Mouse by CD4 T-Cell Clones: Differential Requirement for CD8 T-Cells

Transfer of an interleukin 2/interferon-γ-secreting islet-specific CD4+ T-cell clone, BDC-6.9, in the immunodeficient NOD-scid mouse induces destruction of pancreatic β-cells without help from host B-cells, CD4+ T-cells, or CD8+ T-cells. However, a second islet-specific T-cell clone, BDC-2.5, showing the same cytokine profile and T-cell receptor Vβ expression as BDC-6.9 was not capable of inducing diabetes or insulitis in NOD-scid mice. Even though BDC-2.5 by itself readily induces diabetes in young unmanipulated NOD mice, cotransfer of CD8-enriched T-cells was required to induce disease in NOD-scid mice. Immunohistochemical staining of pancreatic lesions in young NOD mice receiving either BDC-2.5 or BDC-6.9 showed the presence of CD4+, CD8+, Vβ4+, and MAC-1+ cells within the infiltrate, similar to infiltrates in lesions of spontaneously diabetic female NOD mice. In contrast, NOD-scid mice that received BDC-6.9 showed only the presence of CD4+Vβ4+ T-cells and a large population of MAC-1+ cells in islet lesions. NOD-scid recipients of cotransferred BDC-2.5/CD8+ splenic T-cells showed a small population of CD4+ T-cells and a larger population of CD8+ T-cells within the infiltrated islets, whereas no infiltrate was detectable in recipients of CD8+ splenocytes or BDC-2.5 alone. Our results suggest that at least two types of islet-specific CD4+ T-cell clones play a role in diabetes pathogenesis.

Expression of CD40 identifies a unique pathogenic T cell population in type 1 diabetes

Juvenile diabetes (type 1) is an autoimmune disease in which CD4+ T cells play a major role in pathogenesis characterized by insulitis and β cell destruction leading to clinical hyperglycemia. To date, no marker for autoimmune T cells has been described, although it was previously demonstrated that autoimmune mice have a large population of CD4+ cells that express CD40. We show here that established, diabetogenic T cell clones of either the Th1 or Th2 phenotype are CD40-positive, whereas nondiabetogenic clones are CD40-negative. CD40 functionally signals T cell clones, inducing rapid activation of the transcription factor NFκB. We show that autoimmune diabetes-prone nonobese diabetic mice have high levels of CD40+CD4+ T cells in the thymus, spleen, and importantly, in the pancreas. Finally, as demonstrated by adoptive transfers, CD4+CD40+ cells infiltrate the pancreatic islets causing β-cell degranulation and ultimately diabetes.