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Partial N-terminal amino acid sequences of the Ia molecule encoded by theI-E orI-C (I-EC) subregion of theH- 2d haplotype are presented. Several homology relationships are apparent when these sequences are compared to the gene products of the mouseI- ECk andI-A subregions and to their human and guinea pig counterparts. Theβ polypeptide differs from the EC β k polypeptide in two of the seven positions at which they can be compared and shows moderate homology with its human p29 counterpart. Theα polypeptide is identical to the EC α k polypeptide in the eight positions which can be compared and is highly homologous to the human p34 polypeptide. The genetic implications of these observations are discussed.

Immune response (Ir) gene products control immunologic function at several critical sites. We administered in vivo a monoclonal antibody reactive with I-Ak to F1 mice with the genotype H-2k/b. These treated mnice made a markedly reduced antibody response to antigen (H,G)-A--L, under the control of I-Ak, but not to antigen (T,G)-A--L, under the control of I-Ab. This relative specificity was lost if the antigen was given in complete Freund's adjuvant rather than aqueous solution. The monoclonal antibody reduced the antibody titer in an ongoing, secondary response as well. Several potential mechanisms can be postulated for this effect. This haplotypic specificity might ultimately be relevant to human disease.

Resistance to radiation leukemia virus-induced leukemogenesis is associated with the H-2D region of the H-2 complex, or with closely linked loci. The H-2Dd allele confers resistance ot the disease, while the H-2D-Q and H-2Ds alleles are associated with susceptibility. It is not clear whether Ir genes, or an alternative mechanism are responsible for the observed H-2-linked resistance to the disease.

Homozygous lymphoblastoid cell lines representing various Dw subtypes of DR2 were examined for polymorphism at the DQ β locus by molecular and cellular techniques. The subtypes studied included Dw2, Dw12, and a group heterogenous by cellular typing that we shall refer to as non-Dw2/non-Dw12. Restriction fragment length polymorphism analysis of cell lines representing these subtypes revealed DQ β-specific patterns consistent with cellular typing. Two-dimensional gel electrophoresis of DQ molecules from representative cell lines revealed a structural polymorphism of DQ β among the three subtypes. The DQ β chain migrated to a position that was unique to each subtype and was consistent among various representative cell lines of each subtype. Nucleotide sequence analysis of cDNA clones of DQ β from Dw2, Dw12, and non-Dw2/non-Dw12 lines confirmed that the variability resided at the genetic level. Variability was found in the form of numerous scattered nucleotide substitutions throughout the first domain of these alleles. The DQ β gene of the non-Dw2/non-Dw12 cell line AZH was further found to be almost identical with the DQ β gene of a DR1 line (Bell et al. 1985b), implicating a common evolutionary origin of these alleles. The only difference between these two sequences was due to an apparent gene conversion event at amino acid 57. T-cell cloning experiments resulted in the derivation of Epstein-Barr virus-specific, DQw1-restricted clones that proliferated against only those cell lines that exhibited the DQ β gene common to AZH and the DR1 cell line. Thus, the polymorphism among DQ β alleles within DR2 results in subtype-specific restriction.

The nonimmune adult spleen contains at least two B-cell subpopulations. The majority of primary B cells express cell surface Ia determinants and have the capacity to give rise to IgG antibody-producing clones after T-cell dependent antigenic stimulation. There is also a small subpopulation of primary B cells which are, by definition, Ia negative, since their activity is not eliminated by negative selection with anti-Ia serum and complement. The Ia-negative B cells give rise to clones that produce only IgM antibody. These B-cell subsets may form a continuum in B-cell maturation, or they may exist as discrete B-cell lineages. Since the cellular expression of Ia antigens appears to correlate with the ability of the B cell to generate IgG-producing clones, it is speculated that Ia molecules may have a role in the IgM to IgG B-cell switch mechanism.

Direct cytotoxic tests and absorption studies demonstrated thatI-region associated antigens (Ia) are not restricted to lymphocytes. Ia was found on spermatozoa, macrophages, and on epidermal cells, whereas Ia was absent from brain, liver, kidney, and fibroblasts. The possible biological meaning of these observations is discussed.

Treatment of splenic T lymphocytes with anti-Ia antiserum inhibits the binding of antigen-antibody (AgAb) complexes to the majority (less than 50%) of Fc receptor-positive (FcR+) T cells. A similar inhibition was observed with anti-H-2D and anti-H-2K sera but not with anti-Thy 1.2. Despite the presence of Ia determinants on peripheral T cells, as established by the inhibition of AgAb binding, Ia could not be detected on peripheral T cells by immunofluorescence assays. Data obtained with the AgAb-binding inhibition assay indicate that determinants controlled by loci mapping in the I-A and I-C, S, or G regions are present on the FcR+ T cells. Evidence is presented that subpopulations of T cells within the FcR+ T-cell population may be distinguishable on the basis of which I-region-controlled determinant is expressed. The data are discussed in terms of phenotypic and functional heterogeneity of T lymphocytes.

The partial N-terminal amino acid sequences and tryptic peptide maps of I-A subregion products from the H-2b, H-2d, H-2k and H-2s haplotypes demonstrate that haplotype-associated differences are present in both alpha and beta polypeptides. The genetic and evolutionary implications of these multiple amino acid substitutions and the homology relationships among Ia molecules from human, guinea pig and the I-E subregion of the mouse are discussed.

An immunochemical analysis of the kinetics of appearance of Ia antigens during embryonic development was performed. Ia antigens first appear on the surface of embryonic cells 11 days postconception and their expression between days 11 and 16 of gestation is confined to the fetal liver. Ia antigen synthesis by fetal liver cells is detectable at day 14. Ia seems to precede Ig as a surface marker of embryonic liver cells, since Ig cannot be detected until day 16 of gestation. H-2 antigens may be immunoprecipitated from day 10 whole embryo cells. F9 primitive teratocarcinoma cells are Ia negative and H-2 negative.

The experiments presented in this study define the molecular basis of the bm 12 mutation. Initial characterization of an alloreactive T cell clone, 4.1.4, showed this clone to recognize an allodeterminant present on the E beta b and A beta bm12 chains, but not on the bm 12 parent A beta b chain. To define the extent of sequence shared between the I-E beta product and the mutant I-A beta product, we isolated a cDNA clone of the E beta b gene and determined its nucleotide sequence. Comparison of the nucleotide sequences of E beta b, A beta b, and A beta bm12 shows the the A beta bm12 gene to be identical to the E beta b gene in the region where it differs from its A beta b parent. We predict that the bm 12 mutation arose by gene conversion of this region, which spans 14 nucleotides between amino acid residues 67-71 of the mature A beta chain, from the E beta b locus to the corresponding position at the A beta b locus. Recognition of this region, which spans one of the previously defined E beta allelic "hypervariable" regions, by an alloreactive T cell clone provides the first direct evidence of the functional importance of these hypervariable regions in T cell stimulation. The identification of a gene conversion event involving one of these allelic variable regions implicates conversion as a mechanism that acts on class II beta genes to create sequence diversity in regions of Ia molecules that interact with foreign antigen or a T cell receptor, regions where protein sequence polymorphism would presumably be selected for by the expanded ability it affords the organism to mount effective immune responses against a wider variety of foreign antigens.

The genetic and molecular requirements for cell-surface expression of Ia antigens precipitated by anti-I-E subregion sera have been examined. Inbred mice f the d, k, p, and r haplotypes synthesize and express on their lymphocytes the two I-region products normally found in anti-I-E-subregion immunoprecipitates, E alpha and Ae (E beta). Cells from mice of the b and s haplotypes fail to synthesize E alpha chains but do synthesize Ae chains, which remain in the cytoplasm as partially glycosylated precursors. Cells of the f and q haplotypes fail to synthesize either the Ae or E alpha polypeptide chains, as shown by both genetic complementation tests and analyses of total cell proteins by two-dimensional polyacrylamide gel electrophoresis. The patterns of expression of the intact E alpha: Ae complex are consistent with the theory that both the Ae and E alpha polypeptide chains must be present in the cells for either chain to be expressed in normal amounts on the cell surface. The implications of these observations for the genetics of I-region-controlled functions are discussed.

A soluble allogeneic effect factor (AEF) was produced by using H-2 congenic mouse strains and a serum.free cell culture medium. An AEF derived from untreated activated responder cells and irradiated stimulator cells provided helper cell function in a primary and secondary antibody response for both T-cell-depleted responder B cells and stimulator B cells. This interaction may be determined by genes situated in the I-A and I-B regions: additional K-region control was not excluded. Ia antigens, but neither H-2 nor Ig determinants are molecular constituents of AEF. The active components of this AEF consist, in part, of Ia antigens derived from both the activated responder cell population and irradiated stimulator cell population. An AEF derived from Ia negative responder cells and irradiated T-cell- depleted stimulator cells helps a secondary antibody response of T-cell- depleted stimulator B cells but not responder B cells. This genetically restricted AEF contains Ia antigens determined by the stimulator haplotype but not the responder haplotype. The priming antigen, DNP- keyhole limpet hemocyanin, is not a component of restricted AEF. The data suggest that restricted AEF may be a product of a stimulator B cell and/or macrophage. They support the hypothesis that the recognition by allogeneic T cells of Ia antigens on B cells activates the B cell to IgG antibody production.

TCR beta chain gene expression of individual T cell clones that share the same MHC class II restriction and similar fine specificity for the encephalitogenic NH2 terminus of the autoantigen myelin basic protein (MBP) has been examined. TCR V beta expression was examined by FACS analysis with mAbs specific for the V beta 8 subfamily of TCR beta chain genes. 14 of 18 (78%) NH2-terminal MBP-specific clones examined express a member of the TCR V beta 8 subfamily. Southern analysis was used to identify which member(s) of the TCR V beta 8 subfamily is expressed by these clones. Each of four clones examined uses V beta 8.2, though two different V beta 8.2-J beta 2 combinations were identified. Our findings indicate that there is restricted TCR V beta usage in the autoimmune T cell response to the dominant encephalitogenic NH2-terminal epitope of the MBP. The use of an mAb to the antigen-specific TCR in the prevention of T cell-mediated autoimmune disease has been investigated. Our results demonstrate that in vivo administration of a TCR V beta 8-specific mAb prevents induction of autoimmune encephalomyelitis.

The I region of the murine major histocompatibility complex (H-2) encodes two cell surface glycoprotein complexes (Aα:Aβ and Eα:Eβ) which exert profound control over many immunologic reactions including humoral and cellular responses to a variety of antigens, i.e., Ir gene effects, stimulation in mixed lymphocyte reaction, graft versus host disease, antigen presentation to T cells by macrophages, collaboration between T and B cells and determination of T cell help or suppression [1-13]. In the past few years, several laboratories have reported that antibodies directed against I region gene products (anti-la) are effective in modulating immune responses both in vitro and in vivo [14-32, 48-51]. The ability of anti-la antibodies to alter immune responsiveness was attributed to their interaction with macrophage la antigens, thus masking or sterically hindering la epitopes involved in antigen presentation [16,17, 33-35]. However, other reports have suggested that additional mechanisms may be involved [28, 36-...

One hundred seventy-two members from 27 randomly selected multiple case Caucasian families of patients with insulin-dependent diabetes mellitus (IDDM) were studied at the DNA level to ascertain the reliability of codon 57 of the HLA-DQ beta-chain gene as a disease protection/susceptibility marker. The analysis was carried out by polymerase chain reaction amplification of DNA encoding the first domain of the DQ beta chain and by dot blot analysis of the amplified material with allele-specific oligonucleotide probes. One hundred twenty-three randomly selected healthy Caucasian donors were also tested. The results demonstrated that haplotypes carrying an aspartic acid in position 57 (Asp-57) of their DQ beta chain were significantly increased in frequency among nondiabetic haplotypes (23/38), while non-Asp-57 haplotypes were significantly increased in frequency among diabetic haplotypes (65/69). Ninety-six percent of the diabetic probands in our study were homozygous non-Asp/non-Asp as compared to 19.5% of healthy unrelated controls. This conferred a relative risk of 107 (chi 2 = 54.97; P = 0.00003) for non-Asp-57 homozygous individuals. Even though the inheritance and genetic features of IDDM are complex and are not necessarily fully explained by DQ beta chain polymorphism, this approach is much more sensitive than HLA serolog in assessing risk for IDDM.

In independent studies, 51% and 36% of patients with rheumatoid arthritis have been shown to possess HLA-Dw4 compared to 7% and 13% of controls. In one study Dw4 positive patients more frequently had rheumatoid factor in their sera than did Dw4 negative patients. In order to determine if the Dw4 specificity is associated with the disease or with the presence of rheumatoid factor, the frequency of this HLA antigen has been determined in 24 healthy women known to have rheumatoid factor (median titer 1:160). Only 3 were found to have the Dw4 specificity, suggesting that this specificity is not associated with rheumatoid factor in the absence of rheumatoid arthritis.

(NZB x NZW)F1 mice spontaneously develop an autoimmune syndrome characterized by a fatal immune complex glomerulonephritis. Administration of monoclonal antibodies specific for an I region gene product (I-Az) of the H-2 haplotype associated with susceptibility to glomerulonephritis in these animals produced a remission in female mice with established renal disease. The results demonstrated that anti-I-A therapy stabilized the level of proteinuria and increased the 1-yr survival rate from 10% to greater than 90% in treated animals relative to control mice. These findings may ultimately have therapeutic potential for the treatment of systemic lupus erythematosus.

Sequential immunoprecipitation analyses of Ia antigens were performed with 125I-labeled B10, B10.A(3R), and B10.A(5R) spleen cell lysates. The results suggest the presence of one I-region associated (Ia) locus (Ia-1) in the I-A subregion. In addition, they indicate that two Ia loci map between the I-J and S regions. One locus, Ia-5, maps in the I-E subregion. The second locus, most likely Ia-3, maps in either the I-E or I-C subregion. Taken together, the data demonstrate that at least three Ia loci in the I-region of the H-2 complex code for molecules detectable by immunoprecipitation.

The (NZB X NZW)F1 mouse is recognized as an important animal model of the human disease systemic lupus erythematosus (SLE). Groups of NZB/W F1 mice were treated either with IFN-gamma or with PBS. The results demonstrate that IFN-treated animals have accelerated development of fatal immune complex glomerulonephritis relative to age-matched controls. On the other hand, administration of mAbs specific for IFN-gamma to such mice from 4 to 7 mo of age resulted in significant remission. Development of both proteinuria and anti-DNA antibodies were delayed and survival at 11 mo was increased from less than 20% to 95% in treated mice relative to controls (p less than or equal to 0.001). These findings may have therapeutic implications for the treatment of SLE.

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.

Genetic control of the immune response linked to the major histocompatibility (H-2) complex in the mouse has been described for synthetic polypeptide antigens and for low doses of native proteins. The phenomenon is well documented(1,2). Extensive screening of intra-H-2 crossover-derived recombinant strains has localized H-2-linked immune response (Ir) genes to the I-immune response region of the H-2 complex (3). For most antigens, Ir genes are autosomal, dominant, and they segregate as single loci. It is not known whether these crossover-defined loci respresent single genes with multiple alleles or clusters of tightly linked genes (4). In 1972, Stimpfling and Durham (5) postulated that two interacting loci within the H-2 complex were required for the response to the alloantigen, H-2.2 (6), and, in 1975, Dorf et. al. (7) observed a responder phenotype in a recombinant derived from two strains which were nonresponders to the synthetic linear terpolymer, L-glutamic acid, L-lysine, L-phenylaline (GLPhe). Analysis of additional recombinants and complementation tests with F(1) hybrids clearly demonstrated that genes in two intra-I-region loci controlled the immune response to GLPhe. Subsequently, requirement for genes mapping in two intra-I-region loci were reported for porcine LDH(B)(8), the alloantigen Thy-1.1 (9), and for the synthetic terpolymers L-glutamic acid, L-lysine, L-tyrosine and L-glutamic acid, L-lysine, L- leucine (6,10). Demonstration that responses to both synthetic polypeptide and native protein antigens can be controlled by genes in two distinct I-region loci prompted speculation that the phenotypic expression of two I-region genes is a general phenomenon which may provide the key for understanding the mechanism of Ir gene function and cellular collaboration in the immune response. Benacerraf and Dorf (10) have shown that Ir gene complementation is often more effective in the cis than in the trans configuration. This concept is further supported by the data reported for GLPhe (10-12) which indicate that both of the complementing genes must be expressed in each of the cell types participating in the interaction. Failure to detect complementation for the majority of antigens under H-2-linked Ir-gene control might be attributed to the limited number of available intra-I- region recombinant strains.

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.

The molecular analysis of the Class II region of the MHC of mice and of humans has to date led to some important conclusions. These regions encode sizeable families of related loci, at least 2 alpha and 6 beta in mice and 6 alpha and 7 beta in man. In addition to the sizeable number of potential molecules that could be expressed by the loci of this region, all the beta chains and both A alpha and DC alpha have extensive amounts of polymorphism. This polymorphism is strikingly similar at Class II loci in mouse and man. The majority of the variability is found in the first domain and consists of discrete regions of variability. These variable regions appear to be important functionally in presenting antigen, as evidenced by the bm12 mutant. The allotypy of these molecules is complex and is presumably generated by a combination of gene conversion and point mutation followed by selection. Final definition of the range of allelic variability and further insights into the mechanism by which it was generated are still to be resolved, as in a complete map of all the Class II loci in both mouse and man. The data available at present pose certain questions that now need to be addressed. The precise functional role of individual regions or residues of the Class II molecules can now be analyzed using the available sequence data and site specific mutagenesis. Fine structural analysis of these molecules is impossible at present using only sequence data, and X-ray crystallography will be required to answer fundamental questions about 3-dimensional structure. Finally, disease-specific sequences may be uncovered on particular haplotypes which permit the production of disease-specific probes. The molecular revelations of the last few years have resolved many questions about the polymorphisms of Class II products, but many new questions need to be resolved before these molecules and their extensive polymorphism can be properly understood.

I-A beta-chain cDNA clones from mice of the q, k, u, s, and f haplotypes have been isolated and sequenced. Nucleotide sequence comparisons among these five A beta chains show considerable allelic variation in the region encoding the first external (beta 1) domain of the mature A beta protein. The beta 1 domain variability is clustered into three discrete regions, two of which divide the A beta chains into subgroups, suggesting an evolutionary history for the separation of alleles in inbred strains of mice. The amino acid sequences of these five chains are compared to each other and to previously published I-A beta chains. Correlations are made between the primary structural differences and the serologic and immune response characteristics mapping to the I-A subregion.

It has previously been shown that J.H., a human leukocyte antigen (HLA)-Dw2 homozygous multiparous woman, fails to respond in a mixed lymphocyte reaction (MLR) to her Dw1 homozygous husband W.H., and that her T cells suppress the responses of HLA matched responders to W.H. The present studies take advantage of the observation that J.H. suppressor cells resist a dose of gamma-irradiation which functionally eliminates her MLR responder cells. J.H. cells, depleted of alloreactive cells, suppress the responses of Dw2 heterozygous or homozygous cells to W.H., regardless of their associated HLA-A or B antigens. Only when W.H. or a few other cells are present as the irradiated stimulator is J.H. suppression of Dw2 responses detected. Thus, the J.H. suppressor T cell recognizes determinants in the irradiated stimulator cells as well as D locus products in the responder.

The effect of thymectomy on the genetically controlled murine immune response-1 (Ir-1) to the synthetic polypeptide poly-L(Tyr, Glu)-poly-D, L-Ala--poly-L-Lys [(T, G)-A--L] was studied with both aqueous and adjuvant immunization regimens. Adult thymectomy (combined with irradiation and bone marrow transfusion) did not affect the aqueous antigen-induced (IgM) primary response of either high or low responder mice, but did ablate the (IgG) secondary or tertiary response, a response which is restricted to the high responder strains. Adult thymectomy also blocked the normal high response to (T,G)-A--L in Freund's adjuvant in high responder mice and the high response to methylated bovine serum albumin (MBSA)-(T,G)-A--L in low responder mice. Neonatal thymectomy was also effective in blocking the response to (T, G)-A--L in Freund's adjuvant in high responder mice. These data are consistent with the concept that the Ir-1 gene effect is mediated via thymus cell interaction with antigen and with "B"-cells during the time of induction of IgG antibody formation.

The influence of immunization with (T,G)-A--L on the frequency and characteristics of [125I] (T,G)-A--L-binding cells (ABC) was investigated in high and low responder mice, whose ability to respond to (T,G)-A--L is under control of an H-2-linked immune response gene, Ir-1. Unimmunized high and low responder mice have about the same number of ABC in spleen and lymph nodes (6–12 ABC/104). However, after immunization with (T,G)-A--L in aqueous solution, ABC in high responders increase to a much greater extent than they do in low responders. By inhibition of ABC with class-specific anti-Ig sera, it was demonstrated that in nonimmune and primed mice antigen is bound to IgM receptors, which is in agreement with the exclusive production of 19S anti-(T,G)-A--L antibody in primed animals. In contrast, after secondary challenge with antigen, ABC in high and low responder mice have mainly IgG receptors, although under the conditions used for immunization, low responders are not able to produce detectable amounts of 7S anti-(T,G)-A--L antibody. From these results and from the evidence that low responders very probably have a T cell defect, it is suggested that the switchover from IgM to IgG precursor cells can be induced by antigen itself, without the action of specific T cells. Furthermore, the failure of marked proliferation of ABC in low responders after antigenic stimulation is explained by the lack of stimulation by specific T cells. By independent methods it has been shown that all ABC detected in this study are B cells. Preliminary experiments indicate that purified peripheral T cells bind antigen, but much less per cell than do B cells.

Summary Fluorescent rabbit anti-bovine serum albumin antibody with bright specific and negligible nonspecific staining was prepared from the conjugated antiserum by a single elution from a diethylaminoethyl cellulose column. The yield was 40 to 60% of the labeled precipitating antibody when conjugates with a molar fluorescein to protein ratio of 4 or less were used. The antibody eluted under the conditions employed had a molar fluorescein to protein ratio of approximately 1.0. Absorption and emission studies of a variety of conjugates show that after conjugation to protein a) amorphous and crystalline fluorescein isothiocyanate preparations have the same emission intensity, and b) fluorescein isocyanate has approximately twice the emission intensity of the isothiocyanate.

The immune response to a synthetic polypeptide built on multichain polyproline, poly-L-(Tyr,Glu)-poly-L-Pro-poly-L-Lys [(T,G)-Pro--L], in the offspring of a cross between DBA/1 and SJL mice is under a genetic control superficially similar to the one operating for the immune response to a similar synthetic polypeptide built on multichain polyalanine, poly-L-(Tyr,Glu)-poly-D,L-Ala-poly-L-Lys [(T,G)-A--L], in the offspring of a cross between CBA and C57 mice. In both cases, the genetic control is a quantitative trait in which the major gene(s) is (are) dominant and the trait is not linked to any of the known structural genes coding for mouse immunoglobulin heavy chains. However, the genetic control of response to (T, G)-Pro--L, designated immune response-3 (Ir-3), is qualitatively different from the one operating for (T,G)-A--L [immune response-1 (Ir-1)] in that it is not linked to the histocompatibility-2 (H-2) locus. A study of the immune response to a related polypeptide built on multichain polyproline, poly-L-(Phe,Glu)-poly-L-Pro-poly-L--Lys [(Phe, G)-Pro--L], in the DBA/1 x SJL cross has shown a genetic control of antibody specificity. F1 x DBA/1 backcross anti-(Phe, G)-Pro--L sera segregate in their ability to bind (T,G)-Pro--L, and there is no linkage of anti-(T,G)-Pro--L binding capacity with the H-2s allele of the SJL grandparent. F1 x SJL anti-(Phe, G)-Pro-L sera segregate in their capacity to bind poly-L-(Phe,Glu)-poly-D,L-Ala-poly-L-Lys [(Phe, G)-A--L] and the ability to bind (Phe, G)-A--L is clearly linked to the H-2q allele from the DBA/1 grandparent. Thus, in mice all responding well to a given antigen [(Phe, G)-Pro--L], the specificity of the antibodies produced [i.e., anti-(Phe,G) or anti-prolyl] is genetically determined. Cross-inhibition of binding m (DBA/1 x SJL)F1 anti-(Phe,G)-Pro--L antisera indicates that the anti-(Phe,G) and anti-prolyl specificities are a function of two separate and largely non-crossreacting antibody populations.

Thymectomized, lethally irradiated mice that normally respond well to the synthetic polypeptide (T, G)-A—L but poorly to (H,G)-A—L were protected with liver cells and a thymus graft from fetal mice of a strain that responds to these antigens in the opposite manner. All chimeras failed to respond to (T,G)-A—L, but 24 of 27 responded well to (H,G)-A—L. Moreover, when thymectomized mice that respond well to (H,G)-A—L but poorly to (T,G)-A—L were lethally irradiated, protected with syngeneic fetal liver cells, and grafted with thymus from newborn mice of a strain that normally responds well to both antigens, the resultant chimeras responded to (H,G)-A—L only. These data indicate that the control of the antibody response to these two antigens is expressed through a cell type(s) present in the liver during embryonic life. while it is apparent that the presence of the thymus was essential for a detectable response to these synthetic polypeptides, the phenotypic expression of the response was independent of the genotype of the thymic epithelial reticulum.

The influence of immunization with (T,G)-A--L on the frequency and characteristics of [125I] (T,G)-A--L-binding cells (ABC) was investigated in high and low responder mice, whose ability to respond to (T,G)-A--L is under control of an H-2 -linked immune response gene, Ir-1 . Unimmunized high and low responder mice have about the same number of ABC in spleen and lymph nodes (6–12 ABC/104). However, after immunization with (T,G)-A--L in aqueous solution, ABC in high responders increase to a much greater extent than they do in low responders. By inhibition of ABC with class-specific anti-Ig sera, it was demonstrated that in nonimmune and primed mice antigen is bound to IgM receptors, which is in agreement with the exclusive production of 19S anti-(T,G)-A--L antibody in primed animals. In contrast, after secondary challenge with antigen, ABC in high and low responder mice have mainly IgG receptors, although under the conditions used for immunization, low responders are not able to produce detectable amounts of 7S anti-(T,G)-A--L antibody. From these results and from the evidence that low responders very probably have a T cell defect, it is suggested that the switchover from IgM to IgG precursor cells can be induced by antigen itself, without the action of specific T cells. Furthermore, the failure of marked proliferation of ABC in low responders after antigenic stimulation is explained by the lack of stimulation by specific T cells. By independent methods it has been shown that all ABC detected in this study are B cells. Preliminary experiments indicate that purified peripheral T cells bind antigen, but much less per cell than do B cells.

Unidirectional mixed lymphocyte reactions (MLR) were performed between mouse strains differing for various segments within the H-2 complex. Thymocytes and purified lymph node T cells and B cells were used as stimulator cells. In three of five combinations studied, differing only within the I region, both T and B cells stimulated in the MLR. This suggests that the region codes for both T- and B-cell surface structures. However, if the difference was restricted to one I subregion (I-C), only T cells stimulated. This finding suggests that some of the I-region genes may be expressed either in T or in B cells.

Publisher Summary This chapter provides information on histocompatibility-linked immune response genes. The genetic study of the capacity to form specific immune responses has revealed that the recognition of antigens as immunogens by individual animals and inbred strains is governed by the product of individual dominant genes located in the genome in close relationship with the genes coding for the molecules bearing the major histocompatibility specificities. These genes are termed as “histocompatibility,” or “H-linked Ir genes.” The presence of relevant genes permit immune responses to be formed, characterized by cellular immunity and antibody synthesis against the determinants on the antigens concerned. Three types of antigens are most useful in the identification of H-linked Ir genes: (1) synthetic polypeptides with limited structural heterogeneity; (2) alloantigens that differ slightly from their autologous counterparts, and (3) complex multideterminants antigens administered in limiting immunizing doses in conditions where only the most immunogenic determinants are recognized. Thus, the discovery of specific H-linked Ir genes depends upon experiments wherein the immunological system is presented with a challenge of highly restricted heterogeneity and specificity. The chapter presents a description of the specific immune responses that are under the control of H-linked Ir genes in guinea pigs, mice, and rats.

The transfer of spleen cells from (C3H x C57Bl/6) F1 mice, capable of responding to (T,G)-A--L, into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen. This localizes the genetic control of the ability to respond to the spleen cell population and indicates that the genetic control is exerted upon a process directly related to antibody formation. Studies with congenic strains of mice and linkage studies in segregating backcross populations show that the ability to respond to (T,G)-A--L and (H,G)-A--L is linked to the H-2 locus and can thus be localized to the IXth mouse linkage group. Note Added in Proof: Of the three possible recombinant animals noted in Tables IV and V, two were infertile. The third animal was not a recombinant, since progeny testing and reimmunization showed that this animal was an H-22/H-2k heterozygote capable of responding well to (T,G)-A--L.

A synthetic branched polypeptide, (T,G)-A-L, formed complexes with RNA which banded at densities lower than that of pure RNA in cesium sulfate gradients after equilibrium centrifugation. In different experiments, the minor bands appeared at densities ranging from 1.56 to 1.63 whereas pure RNA banded at a reproducible density of 1.68. The presence of minor bands was independent of the addition of (T,G)-A-L, but when (T,G)-A-L was added to cell homogenates prior to extraction of RNA, the minute fraction of polypeptide that remained associated with RNA in the gradients was concentrated in the minor bands. The formation of minor bands was independent of the cell type, since they formed to the same extent when peritoneal exudate cells, HeLa cells or E. coli cells were used. Minor bands were not found when RNA was extracted with hot rather than cold phenol. RNA had a pronounced adjuvant effect in priming CSW (responder) mice for immune responses to (T,G)-A-L, but E. coli RNA was at least as effective in this respect as RNA from peritoneal cells. No support was found for the existence of a ribonucleoprotein fraction unique to macrophages.

Summary A comparison of CBA and C57 antisera to (T,G)-A--L by means of quantitative precipitin curves, antigen-binding capacity assays and radioimmunoelectrophoresis has confirmed the previously described quantitative difference in the ability of these two strains of mice to respond to (T,G)-A--L, and has failed to show any qualitative correlation between the ability to respond and the immunoglobulin type of the antibodies made in the responding animal. In addition, in genetic crosses the ability to respond well was not correlated with the presence of the immunoglobulin allotype of the higher responding (C57) strain, indicating that the ability to respond is not linked to the structural genes coding for the Fc fragments of γG1-, γG2a-, γG2b- and γA-immunoglobulins. It has also been shown that CBA and C57 mice differ in their ability to undergo a secondary response to aqueous antigen.

CBA and C57 mice were tested for their ability to make an immune response to a related series of branched, multichain synthetic polypeptide antigens in which the antigenic determinants on the amino termini of the branched side chains were systematically varied. Neither strain responded to the polyglutamic acid determinant. Both strains responded well and equally to the poly(phenylalanine, glutamic acid) determinants. CBA mice responded poorly, and C57 mice responded well to two different antigens bearing poly(tyrosine, glutamic acid) determinants. CBA mice responded well, and CS7 mice responded poorly to two different antigens bearing poly(histidine, glutamic acid) determinants. The genetic control of the immune response to (H,G)-A--L appears to be dominant and polygenic, as it has been shown to be for (T,G)-A--L. The related antigens used in this study show extensive cross-reactions with antisera against other members of the related series.

The response of inbred mouse strains to two polypeptides derived from multichain polyprolines, (T,G)-Pro--L and (Phe,G)-Pro--L, is different from the response of the same mouse strains to a similar series of polymers built on multi-poly-D,L-alanyl--poly-L-lysine, although the same short sequences of amino acids are attached to the side chains of the polypeptides in the two series. These results indicate that a portion of the side chain (e.g. polyalanine or polyproline) participates in the antigenic determinant. This was confirmed by studying the response of different mouse strains to two kinds of polypeptides: (T,G)-Pro-A--L 717 and 718 and (T,G)-A-Pro--L 719 and 721. Antibody assay of antisera to (Phe,G)-Pro--L with the cross-reacting antigens (T,G)-Pro--L and (Phe,G)-A-L indicates that different inbred mouse strains make antibodies specific for different parts of the same polypeptide. Thus, antibody from DBA/1 mice reacts almost exclusively with the (Phe,G) sequence, while SJL antisera bind only (T,G)-Pro--L and fail to bind (Phe,G)-A-L. The immune responses to the same amino acids on two different polypeptides (i.e. A--L and Pro--L) appear to be under separate genetic control.

Acute necrotizing inflammatory disease after intracerebral injection of LCM virus is largely dependent on the host immune response to the virus and is controlled, in part, by a dominant gene which is closely linked to the H-2 locus. The F 1 hybrid ( H-2 q/k ) from mating a susceptible SWR/J mouse ( H-2 q/q ) to a resistant C3H/HeJ mouse ( H-2 k/k ) is susceptible to LCM virus disease. When such hybrids ( H-2 q/k ) are backcrossed to susceptible parents ( H-2 q/q ), all F 2 offspring ( H-2 q/q , H-2 q/k ) are highly susceptible. In contrast, hybrid ( H-2 q/k ) backcross to resistant parents ( H-2 k/k ) results in half of the F 2 offspring being susceptible ( H-2 q/k ) while the other half are resistant ( H-2 k/k ). Similarly, in congenic H-2 q/q and H-2 k/k mice, H-2 q/q mice are relatively susceptible to acute LCM disease, whereas H-2 k/k are resistant.

Acute anterior uveitis is a relatively common disease of uncertain aetiology. Its association with a wide variety of systemic disorders including ankylosing spondylitis, Reiter's syndrome, Behcet's disease, juvenile rheumatoid arthritis, psoriatic arthritis, inflammatory bowel disease and sarcoidosis and the prevalence of the HLA antigen B27 (ref. 1) in cases without associated disease have been noted but not explained. In general, animal models for uveitis have not clarified the mechanism of human disease. Most require direct intravitreal injection2 or repeated immunizations with retinal or uveal antigens3. Uveitis does occur in adjuvant arthritis, a syndrome that can be produced in rats by the systemic injection of pepti-doglycan from the cell wall of a variety of Gram-positive bacteria4. The disease, which was initially described following the injection of mycobacteria in mineral oil, has many similarities to Reiter's syndrome5, including the development of eye pathology in as many as 40% of the animals6. As Reiter's syndrome is associated with enteric Gram-negative infections such as Shigella, Salmonella and Yersinia, we sought to produce an arthritis in rats by injecting killed Gram-negative bacteria. Although we have not observed clinical arthritis in these animals, careful histological study has shown the near-universal appearance of uveitis. The studies described below indicate that the eye disease is due to a portion of the bacterial cell wall known as endotoxin or lipopolysaccharide (LPS).

THE I REGION of the major histocompatibility complex (H–2) of the mouse1 controls various immunologically related functions including immune responsiveness and T-cell, B-cell and macrophage interactions2,3. The only gene products of the I region which have been identified by immunochemical techniques are the Ia antigens which are encoded by loci mapping to the I–A and I–E subregions of the I region4. Previously there has been ambiguity over whether the I–E and/or I–C subregion(s) control molecules detected by immunoprecipitation. Recent evidence suggests that the former, not the latter, controls these molecules5,6. The Ia antigens are highly polymorphic by serological analysis and are expressed predominantly on B lymphocytes and epidermal cells7. Ia molecules are integral cell-surface glycoproteins and consist of two subunits of approximate molecular weights 35,000 (α) and 28,000 (β) which are noncovalently associated7. A general nomenclature has been agreed upon for the Ia polypeptides8. For example, the α polypeptide from the I–E subregion of a mouse of the H–2d haplotype is denoted Eαd. Partial N-terminal amino acid sequence analyses of Ia polypeptides from the I–E and I–A subregions of mice8–14, humans14,15 and guinea pigs16 have led to several important observations. (1) The Eα polypeptides of mice show striking homology with human α chains (p34) at least at their N-termini. The Eβ polypeptides of mice also demonstrate some homology with their guinea pig and human counterparts. (2) The Ia polypeptides from the I–A subregion of mouse show little apparent homology with their human, guinea pig and I–E-subregion counterparts. (3) The Aα, Aβ and Eβ polypeptides of various haplotypes differ by one or more residues at their N-termini. However, for the Eαd and Eαk polypeptides no amino acid differences have been unequivocally demonstrated. To determine whether the N-terminal differences in the Eβ polypeptides extend throughout other regions of these polypeptides and whether the Eα polypeptides are identical, we have undertaken tryptic peptide analyses of two Ia molecules from the I–E subregion. We report here the application of high-pressure liquid chromatography (HPLC) to the separation of tryptic peptides and present comparative tryptic peptide maps for I–E subregion products from the H–2d and H–2k haplotypes.

Conjugate formation between effector T cells raised in vivo in a BALB/c host and fluorescein diacetate-labeled allogeneic EL4 tumor target cells was observed and scored by using a fluorescence microscope. This conjugate formation was found to be specifically inhibited by plasma membrane vesicles isolated from the EL4 cells.

The (NZB x NZW)F1 hybrid mouse develops a severe autoimmune disease similar to systemic lupus erythematosus in humans. Both the human and murine form of the disease show strong associations with alleles of the major histocompatibility complex (MHC) gene products. The severe form of the disease found in F1 mice is due, in part, to dominant NZW gene(s) mapping with the H-2 complex (the murine MHC). Here we present evidence that the tumour necrosis factor (TNF-alpha) gene, which is located within the H-2 complex (the murine major histocompatibility complex), could be involved in the pathogenesis of lupus nephritis in F1 mice. Thus, a restriction fragment length polymorphism in the TNF-alpha gene correlates with the reduced levels of TNF-alpha produced by NZW mice. Furthermore, replacement therapy with recombinant TNF-alpha induces a significant delay in the development of the nephritis.

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.

SummaryTreatment with interferon which markedly depressed vaccinia virus growth also inhibited the production of new virus DNA. This was indicated by the small number of cytoplasmic DNA grains due to tritiated thymidine found in radioautographic studies of vaccinia virus infected cells pretreated with interferon.