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To improve the feasibility of in vivo monitoring of autoreactive T cells in the diabetogenic process, we generated T1 and T2 doubly transgenic non-obese diabetic (NOD) mice in which transgenic human CD90 (hCD90) is simultaneously expressed on IFN-gamma-producing cells or murine CD90.1 (mCD90.1) is expressed on IL-4-producing cells. These transgenic NOD mice develop diabetes with the same kinetics and incidence as wild type NOD mice, permitting the physiological characterization of CD4(+)hCD90(+) cells, which represent T(H)1 cells in lymphoid organs and at the site of insulitis. CD4(+)hCD90(+) cells had a higher capacity to secret IFN-gamma than CD4(+)hCD90(-) cells in an autoantigen-specific manner. Transgenic mice treated with GAD65 plasmid were protected from autoimmune diabetes, and had a lower number of CD4(+)hCD90(+) cells, confirming the pathogenic role of CD4(+)hCD90(+) cells in autoimmune diabetes. To further investigate the effect of IL-12 on the development of T(H)1 cells in autoimmune diabetes, we crossed these doubly transgenic mice to IL-12p35-deficient NOD mice. Despite severe disturbance of diabetes in p35(-/-) mice, the frequency of T(H)1 cells in these mice was slightly lower than in wild type mice. These data support the pathological role of IL-12 in autoimmune diabetes and suggest the existence an IL-12-independent pathway of T(H)1 development.

Systemic injection of antigen is one of the approaches that reproducibly induces effective antigen-specific hyporesponsiveness. Here, Roland Liblau and colleagues discuss the cellular and molecular bases of such tolerance, review the current use of this therapeutic strategy in experimental organ-specific autoimmune diseases and analyse what steps are necessary to make this approach suitable for clinical use.

Experimental autoimmune encephalomyelitis (EAE) serves as a rodent model of the autoimmune disease multiple sclerosis. In mice, EAE is induced by immunizing with spinal cord homogenate, components of the myelin sheath, such as myelin basic protein (MBP) or proteolipid protein (PLP), or peptides derived from these components. EAE can be induced in H-2u or (H-2u x H-2s)F1 mice with the N-terminal peptide of MBP, Ac1-11. Coimmunization with Ac1-11 and Ac1-11[4A], an analog in which lysine at position four is substituted with alanine, prevents EAE. The mechanism of inhibition has not been elucidated, but probably does not work through MHC blockade, T cell anergy or clonal elimination of encephalitogenic T cells. We have isolated T cell clones and hybridomas from (PL/J x SJL/J)F1 mice immunized with either Ac1-11 alone or Ac1-11 and Ac1-11[4A] and analysed these cells for differences in their T cell receptor repertoire and in vitro response. Although T cells elicited by coinjection of Ac1-11 and Ac1-11[4A] expressed TCR that used V alpha and Vbeta gene elements similar to those elicited by Ac1-11 alone, they differed in the sequences of the junctional region of the alpha chain. Most of these T cells also responded less well to Ac1-11 in vitro, suggesting that coinjection of Ac1-11 and Ac1-11[4A] preferentially activates T cells bearing TCR of different affinity for Ac1-11 bound to I-A(u), and which may therefore be less encephalitogenic. Furthermore, our results show that a more diverse repertoire of V alpha and Vbeta genes are elicited by Ac1-11 in (PL/J x SJL/J)F1 mice compared to PL/J and B10.PL mice, providing further evidence that a restricted TCR repertoire is not required for the development of autoimmune disease.

The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg–+/+ or TCRtg–lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CD4+ peripheral T cells in TCRtg–lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg–+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

Early attempts to prevent IDDM typically relied on immunosuppressive drugs (cyclosporine) or drugs that indiscriminantly inhibit cell proliferation (imuran), often leading to serious side effects. Therefore, a great deal of effort has focused on selectively targeting those T cells involved in the disease process. One general approach has been to employ monoclonal antibodies specific for molecules expressed by the effector T cell population. Monoclonal antibodies specific for CD4 (Shizuru et al. 1988xImmunotherapy of the nonobese diabetic mouse; treatment with an antibody to T helper lymphocytes. Shizuru, J.A, Taylor-Edwards, C, Banks, B.A, Gregory, A.K, and Fathman, C.G. Science. 1988; 240: 659–662Crossref | PubMedSee all ReferencesShizuru et al. 1988) and CD3, a component of TCRs (Chatenoud et al. 1993xAnti-CD3 antibody induces long-term remission of overt autoimmunity in nonobese diabetic mice. Chatenoud, L, Thervet, E, Primo, J, and Bach, J.F. Proc. Natl. Acad. Sci. USA. 1993; 91: 123–127Crossref | Scopus (456)See all ReferencesChatenoud et al. 1993), have been shown to be effective in the prevention and treatment, respectively, of diabetes in NOD mice. Similarly, prediabetic NOD mice are protected from disease when treated with antibodies that interfere with antigen recognition (anti-class II, Boitard et al. 1988xPrevention of diabetes in nonobese diabetic mice by anti- I-A monoclonal antibodies (transfer of protection by splenic T cells) . Boitard, C, Bendelac, A, Richard, M.F, Carnaud, C, and Bach, J.F. Proc. Natl. Acad. Sci. USA. 1988; 85: 9719–9723Crossref | PubMedSee all ReferencesBoitard et al. 1988; anti-TCR, Sempe et al. 1991xAnti-α/β T cell receptor monoclonal antibody provides an efficient therapy for autoimmune diabetes in nonobese diabetic (NOD) mice. Sempe, P, Bedossa, P, Richard, M.F, Villa, M.C, Bach, J.F, and Boitard, C. Eur. J. Immunol. 1991; 21: 1163–1169Crossref | PubMedSee all ReferencesSempe et al. 1991), cellular activation (anti-B7; Lenschow et al. 1995xDifferential effects of anti-B7-1 and anti-B7-2 monoclonal treatment on the development of diabetes in the nonobese diabetic mouse. Lenschow, D.J, Ho, S.C, Sattar, H, Rhee, L, Gray, G, Nabavi, N, Herold, K.C, and Bluestone, J.A. J. Exp. Med. 1995; 181: 1145–1155Crossref | PubMed | Scopus (528)See all ReferencesLenschow et al. 1995), and homing to the pancreas (anti–L selectin and anti-VLA-4; Yang et al. 1993xInhibition of insulitis and prevention of diabetes in nonobese diabetic mice by blocking L-selectin and very late antigen 4 adhesion receptors. Yang, X.D, Karin, N, Tisch, R, Steinman, L, and McDevitt, H.O. Proc. Natl. Acad. Sci. USA. 1993; 90: 10494–10498Crossref | PubMedSee all ReferencesYang et al. 1993). Finally, antibodies targeting cytokines associated with Th1 activity (anti-IFNγ, anti-TNFα, and anti-IL-12; Rabinovitch 1994xImmunoregulatory and cytokine imbalances in the pathogenesis of IDDM. Therapeutic intervention by immunostimulation. Rabinovitch, A. Diabetes. 1994; 43: 613–621Crossref | PubMedSee all ReferencesRabinovitch 1994) have been able to prevent disease in prediabetic NOD mice. In general, however, the applicability of antibodies specific for these “immune–related molecules” to human IDDM is limited by the side effects of chronic administration, such as immunogenicity, and the lack of selectivity.An alternative approach is to devise protocols in which immunomodulation can be selectively applied through the use of a specific antigen/peptide. Recently, it has been demonstrated that insulin, when adminstered prior to the onset of diabetes, can delay or prevent disease in individuals at high risk for IDDM (Keller et al. 1993xInsulin prophylaxis in individuals at high risk of type I diabetes. Keller, R.J, Eisenbarth, G.S, and Jackson, R.A. Lancet. 1993; 341: 927–928Abstract | PubMed | Scopus (261)See all ReferencesKeller et al. 1993). The precise mechanism by which protection is mediated is not known. Both metabolic and immunologic factors may contribute to the effectiveness of this form of therapy. Nevertheless, multicenter trials of subcutaneous insulin prophylaxis to individuals at high risk for developing diabetes have recently been initiated.In general, antigen-specific tolerance can be induced via two distinct processes: clonal deletion/anergy and induction of regulatory T cells. Clonal deletion/anergy has been shown to be effective in acute experimental autoimmune diseases where the inciting autoantigen/peptide is known. However, the high degree of specificity associated with this approach might be limiting in IDDM, in which the inciting autoantigen is not known, and where spreading of the autoimmune response to a number of epitopes within a single autoantigen and targeting of other autoantigens occur. Despite these reservations, administration of GAD, insulin, or HSP60 (but not carboxypeptidase H or peripherin) to NOD mice appears to result in the induction of antigen-specific regualtory T cells (Th2) that effectively suppress the disease. These regulatory T cells are thought to suppress the effects of nearby diabetogenic T cells through the antigen-stimulated secretion of IL-4, IL-10, and TGFβ. The advantage of this approach is that knowledge of the inciting β cell autoantigen (if only one such antigen truly exists) is not required. However, it is still unclear whether regimens can be devised that effectively induce a long lasting form of active suppression with no deleterious side effects in a clinical setting. For example, oral administration of antigen appears to be nontoxic, but its effects are variable and dose specific. This does not appear to be the case with systemically administered antigen. However, the possibility exists that systemic administration of antigen might have an immunizing effect and exacerbate disease.Although antigen-specific immunotherapy appears to be a promising method to prevent IDDM, it is most likely that a combination of approaches may prove to be more generally effective. Thus, active suppression by antigen-induced regulatory T cells may be enhanced in concert with antibodies targeting cytokines required for Th1 development and function. Furthermore, as additional β cell autoantigens are identified and shown to have a role in the disease process, therapy might employ a number of autoantigens to target the polyclonal population of autoreactive T cells, thereby increasing the likelihood of successful treatment.Even if safe, effective, and long lasting immunotherapies are developed, their application is a formidable challenge. Only 15% of new cases of IDDM occur in families with a previous case in the kindred. Overt diabetes develops only when β cell destruction is nearly complete, and the patient is asymptomatic for months or years until that point is reached. Immunotherapy thus must be preventive, which requires inexpensive, accurate genetic, autoantibody, and T cell screening techniques. Given the large number of islet cell autoantigens now available and the rapid progress in identifying genetic susceptibility markers, such screening techniques should soon be feasible. Hopefully, effective methods of prevention will promote widespread population screening and the application of preventive therapy.

CD4+ T cells play a key role in regulating immune system function. When these regulatory processes go awry, organ-specific autoimmune diseases may develop. Here, Roland Liblau, Steven Singer and Hugh McDevitt explore the thesis that a particular subset of CD4+ T cells, namely T helper 1 (Th1) cells, contributes to the pathogenesis of organ-specific autoimmune diseases, while another subset, Th2 cells, prevents them.

Type 1 1diabetes is the result of an ongoing autoimmune response to specific proteins expressed by the insulin producing β cells. Recently, a number of β cell autoantigens have been identified. However, their role in mediating the diabetogenic response is not know. Here we assess the relative importance of a panel of β cell autoantigens in the disease process. The approach was to inhibit T cell proliferation to a given autoantigen by either i.t. or i.v. injections, and then determine the effect this had on the diabetogenic response. We show that administering murine glutamic acid decarboxylase (GAD) to 3-week-old NOD females can reduce the frequency of insulitis and prevent the onset of diabetes. In contrast, carboxypeptidase H or peripherin do not induce a similar protective effect, suggesting that GAD has a critical role in the diabetogenic response. These results also suggest that GAD may provide a useful target for antigen-specific immunotherapy.

Selective homing of autoreactive lymphocytes to the pancreatic islets of Langerhans is essential for triggering the cascade of molecular and cellular interactions which culminate in the specific destruction of the insulin-producing beta-cells. Based upon the sequential multistep model of lymphocyte adhesion to the endothelium, we investigated the possibility of preventing the progression of insulin-dependent diabetes mellitus (IDDM) by selectively blocking L-selectin and alpha 4-integrin homing receptors, which function at different stages of the adhesion process. Treatment of NOD mice with mAb specific for L-selectin or alpha 4-integrin resulted in a significant inhibition of lymphocytic infiltration (insulitis). Both spontaneous development and acute transfer of IDDM were completely prevented by administration of anti-alpha 4-integrin antibody and partially inhibited by anti-L-selectin antibody. The protective effect was of long duration. Interestingly, the autoimmune T cell responses to a panel of beta cell autoantigens and the lymphocytic infiltration of salivary glands (sialadenitis) appeared unaffected by anti-L-selectin or anti-alpha 4-integrin treatment. These data suggest that prevention of lymphocyte homing to the pancreatic islets may provide a selective target for prevention/treatment of IDDM in patients.

The HLA-DRB1, -DRB3, -DRB4, and -DRB5 alleles of the Guarani and Kaingang Amerindians were characterized. Our previous serologic analyses detected three class II haplotypes among the Kaingang: DR2-DQ3, DR4-DQ3, and DR8-DQ4. In addition to these, the Guarani presented haplotype DR6-DQ3. Individuals typed serologically (67 Kaingang and 34 Guarani) were selected for molecular analyses. Using a set of 23 SSOs for hybridization of PCR products from generic DRB amplification six different haplotypes were identified, of which only three are shared by the two tribes. The oligonucleotide hybridization patterns are compatible, with haplotypes DRB1 ∗ 1602-DRB5 ∗ 02, DRB1 ∗ 0404-DRB4 ∗ 0101, DRB1 ∗ 0802, and DRB1 ∗ 0901-DRB4 ∗ 0101 in the Kaingang tribe, and haplotypes DRB1 ∗ 1602-DRB5 ∗ 02, DRB1 ∗ 0411-DRB4 ∗ 0101, DRB1 ∗ 1413-DRB3 ∗ 0101, DRB1 ∗ 0802, and DRB1 ∗ 0901-DRB4 ∗ 0101 among the Guarani. DRB1 ∗ 1413 is a new allele, most closely related to DRB1 ∗ 1402, which is common among South and North American Indians. At the segments analyzed, they differ solely at position 57, which is GAT (aspartic acid) in DRB1 ∗ 1402 and AGC (serine) in DRB1 ∗ 1413. This allele probably originated in South American Indians, resulting from a single segmental exchange event between alleles DRB1 ∗ 1402 (the acceptor) and DRB1 ∗ 0411.

Peripheral immunological tolerance is traditionally explained by mechanisms for deletion or inactivation of autoreactive T cell clones. Using an autoimmune disease model combining transgenic mice expressing a well-defined antigen, influenza hemagglutinin (HA), on islet beta cells (Ins-HA), and a T cell receptor transgene (TCR-HNT) specific for a class II-restricted HA peptide, we demonstrate that the conventional assumptions do not apply to this in vivo situation. Double transgenic mice displayed either resistance or susceptibility to spontaneous autoimmune disease, depending on genetic contributions from either of two common inbred mouse strains, BALB/c or B10.D2. Functional studies on autoreactive CD4+ T cells from resistant mice showed that, contrary to expectations, neither clonal anergy, clonal deletion, nor receptor desensitization was induced; rather, there was a non-MHC-encoded predisposition toward differentiation to a nonpathogenic effector (Th2 versus Th1) phenotype. T cells from resistant double transgenic mice showed evidence for prior activation by antigen, suggesting that disease may be actively suppressed by autoreactive Th2 cells. These findings shed light on functional aspects of genetically determined susceptibility to autoimmunity, and should lead to new therapeutic approaches aimed at controlling the differentiation of autoreactive CD4+ effector T cells in vivo.

Oligonucleotide probes were used to investigate the role of DQβ molecules in susceptibility to multiple sclerosis. Although shared amino acid and nucleotide sequences in DQβ1 have been suggested to be critical in disease development, we find that the distribution of sequences corresponding to residues 71–77 is not greater in patients versus controls.

Most autoimmune disorders are associated with particular alleles of the major histocompatibility complex (MHC) class II genes. However, only a minority of individuals with these alleles develop autoimmunity. The identification of alleles more closely associated with an autoimmune disorder such as lupus would facilitate screening and diagnosis and perhaps the understanding of mechanisms of disease. Analysis of sequence variation in the polymorphic first domain of the MHC genes HLA DQ beta and DQ alpha has revealed a novel DQ beta allele in two Caucasian lupus patients and no Caucasian controls. The novel DQ beta allele shares polymorphic amino acids at positions 26 to 30 and 57 with other lupus-associated DQ beta alleles. These hypervariable regions may play a role in lupus susceptibility and may provide insights into the molecular mechanism for this susceptibility.

The murine Ia antigens, a group of cell surface glycoproteins, are involved in the control of the immune response. The structure of one of these class II major histocompatibility complex molecules, A alpha, was recently deduced from sequence analysis of a cDNA clone produced from k haplotype mice. We have now isolated and sequenced A alpha cDNA clones from five other mouse haplotypes: d, b, f, u, and q. Sequence comparison revealed a surprisingly high degree of allelic polymorphism. Interestingly, amino acid substitutions were clustered within the first external domain of this polypeptide chain, particularly at a few highly variable positions. Functional implications of A alpha polymorphism and possible mechanisms for its generation are discussed.

When 15 C3H ↔ C57 tetraparental (allophenic) mice were analyzed for coat color, hemoglobin, and immunoglobulin allotype, all but two were shown to be chimeric. These 15 tetraparental mice were immunized with the synthetic polypeptide (T,G)-A--L, and the origin of the (T,G)-A--L-specific antibody produced was determined by using genetic markers (allotypes) on the immunoglobulin heavy chain constant region. Five tetraparental mice were high responders to (T,G)-A--L and had significant amounts of a (low responder) allotype antibody in their total serum. Three of these mice had significant amounts of anti-(T,G)-A--L antibody of the a (low responder) allotype. The antigen binding capacities of the a allotype fractions of these three were 4–5 times higher than the antigen binding capacities of immunized C3H (low responder) control mice. These results are compatible with the hypothesis that the inability of low-responder mice to produce significant amounts of anti-(T,G)-A--L antibody is a function of Ir -1 A gene expression at the level of T cells.

We have isolated and sequenced cDNA and genomic clones of the murine E alpha gene, one of the immune response genes of the major histocompatibility complex. Comparison of our data with those recently reported for the human homolog DR alpha shows an identical intron-exon structure, and a good conservation of the protein-coding sequences, including the amino acids potentially involved in organizing the second external protein domain into an immunoglobulin-like fold. Noncoding sequences are less conserved, with the exception of the promoter region. Finally, we show that differential expression of the gene in various cell types appears to be transcriptionally regulated, and that genomic rearrangements do not seem necessary for expression.

Peptide binding and lymph node T cell activation studies have been used to characterize T cell recognition of an encephalitogenic T cell autoantigen from myelin basic protein in (PL/J x SJL)F1 mice. Amino acids that determine interactions with either the restriction element of the major histocompatibility complex (MHC) or the encephalitogenic T cell receptor are defined. This information enables the design of peptides that bind MHC yet do not cross-react with the autoantigen. A peptide analog of the encephalitogenic epitope is shown to be "heteroclitic" for MHC binding and activation of encephalitogenic T cells in vitro. This analog is not immunogenic for encephalitogenic T cells in vivo and is shown to inhibit disease that is induced by the autoantigen itself.

A simple and sensitive radioimmunoassay using E. coli β-galactosidase as a model protein has been developed for the detection of specific translation products of foreign gene fragments cloned into plasmid or phage vectors. This immunoassay is based upon the coupling to an insoluble matrix of F(ab)′ 2 fragments derived from the specific antiserum by pepsin digestion. The in situ analysis of phage plaques or of bacterial colonies is performed by overlaying the phage plaques or lysed bacterial colonies with a cellulose filter to which F(ab)′ 2 fragments have been chemically coupled. The antigen bound to the filter is detected by subsequent incubations with undigested antiserum and with 125 I-labeled Staphylococcus aureus protein A followed by autoradiography. By coupling the F(ab)′ 2 fragments to the wells of a plastic microtiter plate, liquid cultures can be analyzed quantitatively for the presence of antigen, making possible the analysis of heterogeneous cultures by sib selection. The detection threshold of the microtiter plate assay for liquid culture is shown to be 8 molecules, or about 1 molecule of β-galactosidase per cell. The in situ immunoassay for bacterial colonies, which permits examination of about 1000 clones per plate, can easily detect microcolonies producing about 10 molecules of β-galactosidase per cell, while the in situ phage plaque assay, also capable of screening about 1000 plaques per plate, is even more sensitive, detecting 7 molecules per bacteriophage plaque.

An objective antiglobulin radioimmunoassay for the analysis of anti-spermatozoa antisera has been used to characterize more than 30 alloantisera. Each of these antisera was raised against spermatozoa from mice carrying T/t region mutations. Whilst all of the antisera had considerable activity for mouse spermatozoa, none of them was found to contain antibodies specific for T/t region gene products. A detailed analysis of a single serum revealed that its activity is for antigens which are non-polymorphic, sperm-specific and species-specific. The failure in this study to detect antibodies specific for T/t region gene products is discussed.

This study focused on clinical subsets within systemic lupus erythematosus (SLE) in order to identify more homogeneous patient groups in which to define disease susceptibility gene(s). Analysis of the major histocompatibility complex gene products and genes with major histocompatibility complex class II and class III locus-specific probes and oligonucleotide probes for selected human leukocyte antigen DQ-beta alleles showed significant increases of human leukocyte antigen DR2 and the rare DQ-beta allele DR2-DQw1.AZH in the lupus nephritis patients compared with lupus patients without renal disease (relative risk = 8.3). C4A null was detected in one third of all of the SLE patients. In two thirds of the C4A null patients this was due to a DR3-associated C4A gene deletion. The remaining third may have a regulatory defect and this was DR2-associated. DR4 was significantly decreased in the nephritis patients in comparison with the non-renal SLE patients (relative risk = 0.3). A novel DQ-beta gene has been sequenced from two SLE patients that has not been observed in the normal population. Potential implications of these findings are discussed.