Abraham Fuks

Insulin-Dependent Diabetes Mellitus Is Associated With Genes That Map to the Right of the Class I RT1.A Locus of the Major Histocompatibility Complex of the Rat

Previous studies have demonstrated that the presence of at least one u-haplotype of the rat major histocompatibility complex (MHC), RT1, is a necessary but not sufficient condition for the development of overt diabetes mellitus. The present studies were undertaken to determine which portion of the RT1 gene complex is necessary for the occurrence of diabetes. We crossed hooded diabetic rats (RT1 .AaBuDu) with PVGr8 rats (RT1 .AaBuDu). F1 animals were mated to give 82 F2 animals and backcrossed with the hooded diabetics to produce 41 backcross animals. Diabetes occurred in animals with all three possible RT1.A genotypes. The diabetes was similar to that seen in BB rats and in hybrid strains developed from them. An immunoregulatory defect was marked by decreased percentage of peripheral blood lymphocytes staining with w3/25 monoclonal antibody, by an increased percentage of peripheral blood lymphocytes binding a mouse ascites control protein, and by decreased responsiveness of peripheral blood lymphocytes to stimulation by conconavalin A. We conclude that the u-allele of the class I A-locus gene product is not necessary for susceptibility to the development of diabetes in the rat. Therefore, either genes coding for the class II products of the uhaplotype or genes in linkage disequilibrium with these genes and mapping to the right of the A locus provide the permissive condition. Furthermore, the data suggest, but do not prove, that the u-haplotype derived from a strain remote from the BB rat can confer this susceptibility to the development of diabetes. We can also demonstrate that neither the susceptibility to diabetes nor the presence of peripheral blood lymphocyte characteristics, which mark the immunoregulatory defect, segregates with albinism.

IDDM in BB Rats: Enhanced MHC Class I Heavy-Chain Gene Expression in Pancreatic Islets

Modulation in major histocompatibility complex (MHC) gene expression correlates with the inflammatory reactions that occur during graft rejection and autoimmune disease. We analyzed the expression of class I and II MHC genes in the pancreatic islets of prediabetic and newly diabetic BB rats by immunohistochemistry of tissue sections and Northern blotting of RNA extracted from isolated islets. We show that enhanced levels of MHC class I heavy-chain RNA are present in pancreatic islets before overt inflammation and the onset of insulin-dependent diabetes mellitus (IDDM) in the spontaneously diabetic BB rat. Immunohistochemical analysis revealed enhanced class I antigen expression throughout the pancreatic islets of newly diabetic animals but no induction of class II antigen on endocrine cells within the islet. Varying degrees of inflammatory infiltrate were observed in the sections exhibiting enhanced class I antigen expression or in nearby serial sections. Southern blot analysis revealed no restriction-fragment-length polymorphism or amplification of the endogenous class I heavy-chain genes compared with those of seroidentical disease-resistant Wistar-Furth rats. I-Aα and I-Eα hybridizing RNA appeared de novo before overt diabetes, although concomitantly with T-lymphocyte-receptor β-chain and interferon-γ gene hybridizing RNA and after MHC class I heavy-chain RNA enhancement was observed. These data indicate the possibility that enhanced class I heavy-chain gene expression plays a role in the progression of IDDM.

Isolation of T-Lymphocyte Lines with Specificity for Islet Cell Antigens from Spontaneously Diabetic (Insulin-dependent) Rats

T-lymphocyte lines specific for islet cell antigens were isolated from the spleen and pancreas of newly diabetic BB rats or from the related strain BBUF. These cell lines were grown in continuous culture with inter-leukin-2 (IL-2) containing medium for >60 days. Such T-lymphocytes responded by proliferation and IL-2 secretion in the combined presence of islet cell antigens and major histocompatibility (MHC)-matched antigen-presenting cells. By fluorescence-activated cell sorter (FACS) analysis the cells were W3/13+, W3/25+, and 0X8−. Thus, both functionally and by cell-surface-marker analysis they appear to be of the T-helper phenotype. The long-term growth and study of anti-islet T-lymphocyte lines will permit a detailed analysis of the role of T-lymphocytes in the pathogenesis of IDDM.

Polygenic Nature of Spontaneous Diabetes in the Rat: Permissive MHC Haplotype and Presence of the Lymphopenic Trait of the BB Rat Are Not Sufficient to Produce Susceptibility

We describe the phenotypic characteristics of animals in the fifth backcross-intercross generation of a breeding program in which the RT1 u haplotype and the phenotypic trait responsible for the T-lymphopenia of BB rats have been transferred to the ACI background. In this generation of animals, 24% were lymphopenic with decreased numbers of PBL expressing CD5, TCRα, and RT6. The PBL of the lymphopenic animals had a decreased mitogenic response to ConA. All of the nonlymphopenic animals were homozygous for RT6.2. Phenotypic analysis of intestinal IEL revealed that this was also the case for the lymphopenic animals. Moreover, IEL of the lymphopenic animals exhibited a pattern of staining (increased numbers of TCRαβ+CD4+CD8+ and decreased numbers of TCRαβ+CD4−CD8+) similar to that of BB DP animals. The ACI.1U(BB)-lymphopenic animals, although having two of the genetic traits associated with the expression of spontaneous diabetes mellitus, uniformly fail to develop diabetes. Breeding studies in which these animals were crossed with BB and hBB rats suggest that other genes are necessary for development of overt diabetes.

The role of p23,30-bearing human macrophages in antigen-induced T lymphocyte responses☆

Abstract The effect of anti-p23,30, a rabbit antiserum to the human Ia-like antigen p23,30, on two macrophage-dependent T-cell functions, proliferation in response to soluble antigens, and production of lymphocyte mitogenic factor (LMF) was studied. T cells depleted of macrophages neither proliferate nor secrete LMF, and these functions are restored by addition of as few as 0.5% adherent macrophages. Treatment of macrophages with anti-p23,30 and C, however, abolishes their capacity to reconstitute these T-cell functions. In contrast, treatment of T cells with anti-p23,30 and C did not affect their capacity to respond in the presence of untreated adherent cells. We conclude that the presence of p23,30-bearing macrophages is critical for the expression of these antigen-induced T-cell responses.

Association of Susceptibility to Spontaneous Diabetes in Rat With Genes of Major Histocompatibility Complex

This study was designed to map the diabetes susceptibility gene(s) associated with the rat major histocompatibility complex (MHC) RT1. We have crossed spontaneously diabetic male rats bearing the recombinant RT1r8 haplotype with female rats of the ACI. 1r4 congenie strain. Three diabetic rats were determined to be homozygous for the r4 haplotype by serotyping. The absence of recombination within the MHC was confirmed by inspection of restriction-fragment-length patterns of the diabetic animals and the parental strains. In conjunction with previous breeding studies, this study maps the diabetes susceptibility gene to the right of the RT1-A locus and to the left of the RT1-C locus. A low incidence of diabetes in the F2 (4.5%) emphasizes the multifactorial nature of the susceptibility. The presence of depressed responsiveness of peripheral blood lymphocytes to concanavalin A stimulation increases the prevalence of the overt disease. An unusual feature of the diabetic syndrome in this study is the sparse or absent pancreatic lymphocytic inflammatory response, with true insulitis being a rare finding.

Major Histocompatibility Complex Restriction of T-Lymphocyte Responses to Islet Cell Antigens in IDDM Rats

BBUF rats, derived from BB rats, spontaneously develop a form of insulin-dependent diabetes mellitus (IDDM) associated with infiltration of the islets of Langerhans by lymphocytes (insulitis). BBUF rats bear the RT1U major histocompatibility complex (MHC) haplotype that we have shown to be necessary for the expression of this form of IDDM. A T-lymphocyte line obtained from the pancreas of a diabetic rat (UPCC.5) and three T-lymphocyte hybridomas derived by fusing T-lymphocytes of BBUF rats (MUS1.2, MUS1.13, and MUP3.21) respond to islet cell antigens in an MHC-restricted way. UPCC.5 responds to a combination of islet cell antigens (ICAg) and antigen-presenting cells by proliferation, whereas the T-hybridoma responses are detected on the basisof IL-2 production in a similar assay. This study reveals that an antiserum against μhaplotype MHC antigens or a monoclonal antibody against the product of the D class II subregion of the rat MHC could inhibit ICAg recognition. A monoclonal antibody against the product of the B class II MHC subregion of the rat was not inhibitory. These results suggest that RT1.D antigens (analogous to human DR and mouse I-E) restrict islet cell recognition in this rat model of spontaneous IDDM.

Interferon-gamma induces transcription and differential expression of MHC genes in rat insulinoma cell line RINm5F.

We have reported that enhanced levels of class I major histocompatibility complex (MHC) antigen are expressed throughout the islets of prediabetic and newly diabetic BB rats and that the endocrine cells of the islet remained class II negative. In this study we investigated the molecular biology of lymphokine-induced expression of the class I and II MHC genes in subclones of the rat insulinoma cell line RINm5F. Treatment of a particular subclone of RINm5F cells (which are normally class II negative, class I low expressors) with crude lymphokine preparation or various doses of recombinant interferon-γ resulted in enhancement of MHC class I antigen expression but no detectable induction of class II antigen expression. This enhancement of class I antigen expression was a dose-dependent phenomenon and was preceded by a dose-dependent increase in class I–specific RNA. Both class I and II genes were induced at the transcriptional level, as determined by Northern blotting and in vitro nuclear transcription assays, but exhibited strikingly different induction kinetics. Supernatants from concanavalin A–stimulated splenocytes had a similar class I–restricted inductive effect on MHC gene expression. This subclone of RINm5F cells, which exhibits a class I lymphokine response–positive, class II response–negative phenotype, 1) mimics the behavior of β-cells in the prediabetic and newly diabetic pancreas and 2) represents a valuable system for probing the similarities and differences in the lymphokine-mediated induction pathways for class I and II MHC genes.

Allospecific monoclonal antibodies recognizing rat class I and class II histocompatibility antigens.

Rat-mouse hybridomas have been developed producing monoclonal antibodies which recognize rat major histocompatibility complex specificities. Splenic lymphocytes from a LEW (RT11) rat alloimmunized against ACI (RT1a) splenocytes were used for hybridoma production. The six clones presented demonstrate two patterns of alloreactivity against ACI tissues: clone 36.3 produces an antibody which agglutinates ACI RBCs and binds to ACI splenic lymphocytes and RBCs. Five clones, 65.6, 79.7.1, 79.7.5, 155.3.1, and 189.3.5, from four independent wells produce monoclonal antibodies which bind to ACI splenic lymphocytes but not RBCs. Screening of these six monoclonal antibodies against a reference panel of RBCs and lymphocytes from 15 standard, congenic, and recombinant rat strains indicated that the monoclonal antibody 36.3 detects a RTI.Aa product (a rat major histocompatibility complex (MHC) Class I antigen) and that 65.6, 79.7.1, 79.7.5, 155.3.1, and 189.3.5 are monoclonal antibodies which recognize a RT1.Ba private specificity (Class II or Ia antigens). These reagents are unique as they are rat monoclonal alloantibodies recognizing private Ia specificities of the rat MHC.

A Class II monoclonal antibody specific for the RT1.B, rather than the RT1.D, product of the rat major histocompatibility complex.

An allospecific monoclonal antibody, 79.7.5., has been shown to be specific for a Class II histocompatibility product of the ACI (RT1.AaBaDa) rat. To further specify the reaction of this antibody to the B or D locus Class II products of RT1, we examined the binding of this antibody to peripheral blood lymphocytes (PBLs) of the WRC rat (haplotype RT1.AnBnDa). Radiolabelled monoclonal antibody 79.7.5. did not bind to PBLs from the WRC rat, but it did bind to PBLs from the WRA rat (RT1.AdBaDa) and the DA (RT1.AaBaDa) rat. These results were confirmed using radiolabelled Staphylococcus protein A in an indirect binding assay. In addition, binding of 79.7.5. could be inhibited by alloantiserum BN anti-BN.1A (DA) (directed against AaBaDa) but not by BN anti WRC (directed against Da). These data demonstrate that monoclonal antibody 79.7.5. reacts with the a allele product of RT1.B rather than RT1.D. This antibody can be used to probe the structures and functional roles of different Class II products of the rat major histocompatibility complex.