A new H-2.1-PositiveD region allele,D dx,controlling two molecules,H-2Ddx and H-2Ldx

The inbred strains GRS/A and LIS/A carry the haplotypeH-2 ^dx , which had earlier been shown to have theK ^d ,I ^f ,S ^f , andG ^f alleles and a previously unknownD region allele,D ^dx . We show here that theD ^dx allele determines a new private specificity, H-2.63, is H-2.28 negative, and determines at least one public specificity of the H-2.1 family. It is thus a second example (afterD ^k ) of a H-2.1-positive H-2.28-negativeD region allele. Capping experiments show that the D^dx product comprises two molecules: H-2D^dx bearing the private specificity H-2.63, and H-2L^dx, which is H-2.63-negative but reacts with sera against the H-2.1 family of specificities. SDS gel electrophoresis of detergent-solubilized immunoprecipitated D^dx products shows that the H-2L^dx antigen has a molecular weight of approximately 45,000 daltons and is associated with a smaller polypeptide (mol. wt. 12,000).     

Evidence for clustering of H-2K, H-2D, and the Fc receptor on the membranes of B cells.

Alloantisera to H-2K, H-2D, and Ia antigens markedly inhibited the binding of EA but not FITC-IgG by the B cell Fc receptor. EA rosette formation approached normal levels when masked H-2 but not Ia specificities were allowed to cap on the membranes of B cells. beta2-mu coated SRBC were bound by the Fc receptor, and high concentrations of soluble beta2-mu were found to moderately inhibit EA rosette formation while lower concentrations enhanced binding. The data support the concept of Fc/Ia identity, and they suggest that H-2K, H-2D, and the Fc receptor may be closely grouped on the membranes of B cells. Further, these observations suggest that the beta2-microglobulin associated with H-2 could serve to link T cells with the Fc receptor of B cells during the inductive phase of antibody synthesis.     

Isolation and antigenic characterization of the product of a third polymorphic H-2 locus, H-2L.

The serology, immunochemistry, and genetics of the product(s) of a third H-2 locus, H-2L (previously designated D') have been studied by using an antiserum raised in BALB/c H-2db mutant mice against tissues from the wild type strain, BALB/c. Genetic mapping studies and sequential immunoprecipitation experiments both indicate that this antiserum reacts specifically with L molecules. These results imply that an H-2L product is antigenically undetectable in BALB/c-H-2db mice and that the lesion in this mutant is confined to the H-2L and not the H-2D locus. Two new specificities, H-2.64 and H-2.65, are defined by the reactivity of anti-L serum on allogeneic cells, and the strain distribution of these specificities suggests the existence of at least three H-2L alleles. This third H-2 locls is therefore polymorphic and in view of this and other similarities to the H-2K and H-2D loci, it must be considered in any evolutionary models dealing with the origin of multiple subloci of the major histocompatibility complex.     

Herpes-Simplex-virus-specific, H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2k) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected taraget cells expressing the Dd alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2Dk determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for Dd alloantigen could be positively selected on H-2d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing Dd alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Herpes-simplex-virus-specific,H-2Dk-restricted T lymphocytes bear receptors for H-2Dd alloantigen

Cytotoxic T lymphocytes generated in the course of an HSV-infection of CBA (H-2 ^k ) mice not only lyse syngeneic, virus-infected target cells but also cross-react with noninfected target cells expressing the D^d alloantigen. On the effector cell level, this alloreactivity is mediated by virus-specific CTL's that are restricted to H-2D^k determinants. On the prekiller cell level, the anti-HSV-reactive T cells exhibiting cross-reactivity for D^d alloantigen could be positively selected on H-2^d spleen-cell monolayers. After differentiation into cytolytic effector cells, target cells expressing D^d alloantigens and syngeneic HSV-infected target were lysed with equal efficiency. The results imply that the phenomenon of H-2-restricted versus nonrestricted T-cell reactivity is not due to distinct T-cell subsets, but rather is dependent on the antigeneic determinants recognized.     

Intra-H-2 recombination in the mouse. III. I-Region characterization of haplotypes H-2at2, H-2at3 and H-2a2

Serological characterization of three K-S interval recombinant strains, TBR2 (H-2at2), TBR3 (H-2at3) and AIR 1 (H-2a2) was performed using anti-H-2, Ia, Ss and Slp antisera. The data presented here reveal that the crossover events in both TBR2 and TBR3 occurred between the I-A and I-E subregions. In both cases, the H-2K and I-A subregions were derived fron the H-2t1 of chromosome, while the I-E, S and H-2D regions were derived from the H-2b chromosome (KsAkEbSbDb). The H-2a2 chromosome resulted from a crossover event between the H-2a1 and H-2i9 chromosomes. Ia and Ss typing of AIR 1 suggested that the K to I-E regions originated from H-2a1 and the S and D regions originated from H-2i9 (KkAkEkSbDd).     

Allograft rejection-defined antigens of the B2m,H-3 region

In this report we delineate the production and histocompatibility characteristics of two new B2m, H-3 region congenic strains, B10.SM-a and B10.FS-a, and further characterize previously described strains. Strains C57BL/10 and B10-we are shown to be histocompatible by the exchange of skin grafts, as are strains B10.UW-we,un at, B10.UW we un a, B10.UW-we + a, B10.UW-+ + a and B10.LP-a. (The latter group will be called B10-H-3b when referred to collectively). C57BL/10, B10-H-3b, B10.C-a, B10.KR-a, B10-pa at, B10.SM-a, and B10.FS-a are shown to be histoincompatible by the rejection of exchanged skin grafts, and histoincompatibility between these strains has been localized to the B2m, H-3 region. The histoincompatibility between C57BL/10 and B10.FS-a is of particular significance because of the identity of these strains at the B2m, H-3 region loci B2m and H-3. Thus the B2m, H-3 region histoincompatibility herein described defines a new locus, which we have called H-42, the a allele being assigned to C57BL/10 and the b allele to B10.FS-a. By using cross-immunization techniques, four allograft rejection-defined reactivity patterns (ADR) have been defined that show concordant strain distribution patterns with the CTL-defined reactivity patterns described elsewhere. On the basis of data presented in this report indicating C57BL/10 and B10.FS-a to differ by a histocompatibility gene in the B2m, H-3 region, and data presented by Kurtz et al. elsewhere indicating C57BL/10 and B10.FS-a to possess the same alleles at the B2m and H-3 loci, the presence of at least three B2m, H-3 region loci-defining cell membrane antigens is established.     

Properties of H-2L locus products in allogeneic and H-2 restricted, trinitrophenyl-specific cytotoxic responses.

The role of the recently defined L antigen (a second D region product) in allogeneic and TNP-specific syngeneic primary CML responses has been investigated. The lysis by anti-L specific cytotoxic effector cells was not inhibited when the target cells were pretreated with an antiserum directed against K and D, whereas an antiserum against L completely abrogated this response. Therefore, H-2L products are recognized on the target cell independently of H-2K and H-2D locus products. Both A.SW cells as well as B10 cells were found to respond to Ld alloantigens, in addition to Dd alloantigens when stimulated by cells differing only in the D region. The results of cold target blocking and antiserum inhibition experiments failed to detect cytotoxic cells with specificity of L antigens in association with TNP, under conditions in which TNP-specific effectors to K and D antigens were demonstrable. These findings suggest that there is a more limited involvement of H-2L locus products than the H-2K or H-2D locus products in the induction and specificity of these responses.     

Ontogeny of Histaminergic Neurotransmission in the Rat Brain: Concomitant Development of Neuronal Histamine, H-1 Receptors, and H-1 Receptor-Mediated Stimulation of Phospholipid Turnover

The ontogeny of histaminergic neurotransmission in the rat brain was studied by assessing development of histamine levels in brain regions, along with H-1 receptor binding of [3H]mepyramine and H-1 receptor-mediated cellular events. In the hypothalamus, which is rich in histaminergic innervation, levels of the amine were low at birth, increased sharply at 8 days of age, and reached adult concentrations shortly thereafter; this pattern is typical of most neurotransmitters. In contrast, regions poor in neuronal histamine showed an initially high histamine level and a subsequent decline with development, as is known to occur during general growth of tissues. The developmental profile of H-1 receptor binding sites resembled that of the neuronal histamine pool, and the increases with age resulted from changes in the number of binding sites without alterations in Kd. Cellular responses to H-1 receptor activation were assessed by determining the stimulation of phospholipid turnover evoked by intracisternally administered histamine, a process that has been shown to involve only the neuronal compartment. Again, the developmental profile was superimposable upon that of H-1 receptor binding and that of hypothalamic histamine levels. These studies indicate that ontogeny of histaminergic neurotransmission is a coordinated process, with simultaneous development of neuronal histamine, its key biosynthetic enzyme, and H-1 receptors coupled directly to cellular events.     

Inhibitory effect of H-7, H-8 and polymyxin B on liver protein kinase C-induced phosphorylation of endogenous substrates

Inhibitory actions of 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), N-[2-(methylamine)ethyl]-5-isoquinolinesulfonamide [H-8] and polymyxin B on the calcium-activated, phospholipid-dependent protein kinase (protein kinase C) of rat liver were compared. Using a partially purified liver protein kinase C and an exogenous substrate histone-III S, polymyxin B showed maximum inhibition (IC50, 9.5 microM) followed by H-7 (IC50, 25 microM) and H-8 (IC50, 36 microM). These inhibitors also inhibited protein kinase C-induced phosphorylation of endogenous cytosolic and particulate proteins in a dose-dependent manner though polymyxin B was relatively less effective with the particulate fraction. With the aid of protein kinase-C activators and these inhibitors, seven proteins in cytosolic (Mr 170K, 150K, 43K, 34K, 30K, 25K and 19K daltons) and six proteins in particulate (Mr 150K, 43K, 34K, 25K, 19K and 16K daltons) fractions were identified as probable substrates for protein kinase C in liver. The identity of these proteins remains to be determined.     

Structural analysis of H-2Kf and H-2Kfm1 by using H-2K locus-specific sequences

The H-2Kf allele and the spontaneous mutant Kfm1 have been cloned using locus-specific sequences. The mutation consists of a cluster of four nucleotide changes, resulting in amino acid substitutions at positions 95 (Leu----Ile) and 97 (Val----Arg). This finding has structural, genetic, and technical implications. The amino acid substitutions are located on the beta-strands of the antigen recognition site. Their influence on the allogeneic properties of the Kf glycoprotein is consistent with the hypothesis that alloreactivity results from alterations in the spectrum of peptides presented to T cells. These substitutions would not, however, be predicted to be directly accessible for binding to antibodies. Nonetheless, the fm1 mutant binds anti Kf alloantisera and mAb much less strongly than the parent molecule, suggesting some indirect effect of these residues on serologic phenotype. The mutant is also interesting genetically because the sequence of the mutated region is identical to the sequence of the Df gene. This implies that there is a gene conversion-like mutational mechanism operating in the H-2f haplotype. Finally, the strategy used to obtain these K-locus cDNA should prove generally useful for isolating other MHC alleles.     

Serological detection of H-2K- and H-2D-gene products

Using the fluorescence-activated cell sorter (FACS II), we have analyzed the expression of H-2K- and H-2D-gene products on the membrane of various cellular components of the murine immune system. Using this serological technique we show a basic difference between T and B lymphocytes. Whereas all cellular components analyzed — hydrocortisone-resistant thymocytes, splenic T and B lymphocytes, macrophages and bone-marrow cells — expressed H-2K-subregion-encoded alloantigens at a high density, it seems that the high density expression of H-2D-encoded alloantigens is restricted mainly to B cells and to macrophages. Hydrocortisone-resistant thymocytes, splenic T lymphocytes and bone-marrow cells, on the other hand, showed significant expression of the H-2D alloantigens only at low membrane density. These results, then, provide evidence for the existence of an imbalance in serologically detectable expression of H-2K- and H-2D-region-gene products on the cell membrane of various cells comprising the murine immune system.Abbreviations usedin this paper DTH delayed type hypersensitivity - FCS fetal calf serum - FITC fluorescein isothiocyanate - HrT hydrocortisone-resistant thymocytes - Ig immunoglobulins P. De Baetselier is an EMBO and Euratom postdoctoral fellow