F1 hybrid-anti-parental H-2b cell-mediated lympholysis: genetic control of target antigens by the H-2D region

The immunogenetic specificity of (C57BL/6 X DBA/2)F1 anti-parental C57BL/6 cytotoxic T lymphocytes (CTL) induced in primary mixed spleen cell cultures was determined in direct lytic and competitive inhibition assays. A large panel of peritoneal exudate cells (PEC) bearing nonrecombinant and recombinant H-2-Tla haplotypes was the source of target and inhibitor cells. All PEC of H-2b, H-2bc, H-2j, and H-2ja types, irrespective of background genetic constitution, were as susceptible to direct lysis as C57BL/6 PEC, but PEC of H-2a, H-2d, H-2k, H-2q, H-2s, and H-2u types were not. The possible involvement of the Tla region in controlling target antigens was excluded by testing PEC obtained from 4 H-2/Tla or intra-Tla recombinant mouse strains. The genes controlling target antigens were mapped to the D region with the aid of 9 intra-H-2 recombinants; for target PEC to be lysed it was necessary and sufficient that Db antigens be part of the H-2 phenotype. These results were confirmed by competitive inhibition assays. Resident peritoneal cells not exposed to fetal bovine serum were also lysed by F1 anti-parental H-2b CTL, a demonstration that target antigens are expressed on normal cells.     

H-2 Antigen variants in a cultured heterozygous mouse leukemia cell line

H-2 antigen variants, derived from a heterozygous mouse Friend leukemia cell line by selection with anti-H-2 antisera and complement, were tested for susceptibility to cell-mediated cytolysis, using T-lymphocytes directed against individual H-2 antigens. The cytotoxic cells were generated in the BALB and B10 backgrounds by a combination of in vivo and in vitro immunizations. The phenotypes of the variants inferred from the patterns of resistance or susceptibility to CML were consistent with those presumed from earlier assays using antisera. The one exception was the variant cell line which was H-2D(d-) in assays using antisera, but was H-2D(d+) by CML.     

H-2RIIBP expressed from a baculovirus vector binds to multiple hormone response elements.

H-2RIIBP is a member of the nuclear hormone receptor superfamily that binds to the region II enhancer of major histocompatibility complex class I genes. Based on its homology with Drosophila XR2C/CF1, H-2RIIBP may play a role in development. By using a baculovirus expression system, a large amount of recombinant H-2RIIBP was produced. The recombinant protein accumulated in the nucleus of insect cells. A series of monoclonal antibodies reacting with the recombinant H-2RIIBP was then generated. A DNA-protein immunoprecipitation assay was developed with these antibodies, enabling the DNA-binding specificity of H-2RIIBP to be distinguished from that of an endogenous region II binding factor expressed in uninfected insect cells. We show that H-2RIIBP binds to estrogen response elements with an affinity comparable to that for the region II enhancer. H-2RIIBP also bound to some, but not all, thyroid hormone response elements and retinoic acid response elements, albeit at a lower affinity. Binding to these elements was demonstrated without exogenous addition of a ligand. The H-2RIIBP binding specificity determined by this assay was in agreement with the specificity assessed by Southwestern and gel mobility shift assays. Furthermore, methylation interference assays indicated that H-2RIIBP recognizes the conserved hormone response motif GG(T/A)CA. Taken together, these data demonstrate that H-2RIIBP is capable of binding to hormone response elements of a variety of genes. They suggest that H-2RIIBP may exert a pleiotropic function.     

Relationship between surface H-2 concentration, size of different target cells, and lysis by cytotoxic T cells

We have examined the parameters of target cell cross-sectional area and surface H-2 concentration in relation to their susceptibility to cytotoxic T-cell-mediated lysis, using a series of commonly used murine target cells of the H-2k haplotype. We used a sensitive immunoferritin labeling technique and electron microscopy to estimate relative cell diameter and H-2 concentration combined with standard 51Cr-release assays for cytotoxicity. We found that susceptibility to cytotoxic T-cell lysis was not related consistently to either factor alone, but was related to a combination of the two, such that above a certain value for the product of the two factors, no further increase in cytotoxic T-cell efficiency was seen. The information presented here should be of value to workers seeking to select a target cell type that will maximize the sensitivity of 51Cr-release-based assays for the detection of cytotoxic T cells.     

Human lymphoblastoid cells as hosts for parvoviruses H-1 and rat virus.

A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.     

H-2 antigen variants in a cultured heterozygous mouse leukemia cell line

An (H-2k/H-2d)F1 sarcoma cell line was subjected to immunoselection using ascites fluid from a mouse growing a hybridoma secreting an anti H-2Kk antibody.One hundred random clones were picked from the surviving population and screened by direct cytolysis using the hybridoma antibody or alloantisera against H-2Kk and H-2Dk. Fifty-nine clones were resistant to all three antisera, indicating that they no longer expressed the entire H-2k haplotype. Thirty-two were resistant to the ascites and to the anti H-2Kk alloantiserum, but sensitive to the anti H-2Dk serum, indicating that they had lost H-2Kk antigen, but retained H-2Dk. Nine clones were sensitive to the alloantisera, but resistant to the hybridoma, indicating that, though they retained the product(s) recognized by the alloantiserum against H-2Kk, they had lost the site(s) that bound the hybridoma antibody. Quantitative absorption assays using lymph-node cells from young BALB.K (H-2Kk) mice as targets show that one representative clone from the last group absorbs the anti H-2Kk activity in the alloantiserum. This implies that the sensitivity of the variant clone to the alloantiserum is not due to contaminating anti C-type virus antibodies in the serum. The possible implications of these data are discussed.     

Synergistic interactions between lymph node and thymus cells in response to antigenic differences limited to selected regions of the H-2 complex.

Thymus and lymph nodes from the A.TL recombinant line were utilized as sources of responding cells in MLR (mixed lymphocyte response) assays to MHC-determined (major histocompatibility complex) antigenic differences. Cells from both sources were stimulated to proliferate by antigenic determinants controlled by the H-2K region alone, H-2D region and the H-2I-H-2S regions. Nylon-fiber-adherent splenic cells from each of the stimulating cell strains stimulated T-cell-dependent responses. Synergistic interactions between A.TL thymus and lymph node cells were initiated by antigenic products limited to single H-2 regions. Antigenic differences determined within the H-2I region were not required for synergistic responses to H-2K-controlled products or for the generation of cytotoxic killer cells to H-2D-associated antigens. The H-2I-region-associated products also were very effective in stimulating T-cell synergy. These data demonstrate that the two responsive T-cell subpopulations can both be stimulated by alloantigens coded within a single known H-2 region.     

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).     

Mouse Hepa-1 tumor is rejected by H-2Db-restricted CTL despite decreased MHC class I antigen expression

It has recently been hypothesized that tumor cells with reduced levels of MHC class I antigens are more susceptible to NK-mediated lysis and are rejected by NK cells, whereas tumor cells with normal levels of class I are rejected by tumor-specific CTL. We have tested this hypothesis using a mouse hepatoma system. The Hepa-1 tumor is a spontaneous H-2Kb loss variant that arose from the BW7756 tumor, when BW7756 was adapted to growth in culture. Our studies have shown that despite the loss of H-2Kb antigen, Hepa-1 is not more susceptible to NK lysis than its H-2Kb-transfected variants. These studies also suggested that NK cells were not responsible for rejection of the Hepa-1 tumor. The Hepa-1 tumor, therefore, appears to contradict the hypothesized linkage of MHC levels and NK susceptibility. Because NK cells are not involved in immunity to this tumor, we have sought to identify the effector cell responsible for Hepa-1 rejection. Cytotoxic T lymphocyte assays demonstrate that in vitro, Hepa-1 cells are lysed by Hepa-1-specific H-2Db-restricted CD4-CD8+ T lymphocytes. Footpad assays demonstrate that in vivo, Hepa-1 rejection requires CD4+CD8- and CD4-CD8+ Hepa-1-primed splenocytes. These results indicate that immunity to Hepa-1 is T cell mediated. Hepa-1 is therefore an example of an unusual tumor in that down-regulation of MHC class I antigen expression is associated with increased CTL susceptibility.     

Cell-mediated cytotoxicity against allogeneic mutant H-2 antigens: restricted and nonrestricted responses

Two "gain and loss" type mutations of the H-2D region, the H- 2bm13 and H- 2bm14 , resulted in the expression of noncross-reactive CML determinants that are unique to each mutation, the Dbm13 gains and Dbm14 gains, respectively. According to the results of direct cytolytic and competitive inhibition assays of in vitro induced primary cytotoxic T lymphocytes, allogeneic responses specific for Dbm13 gains are generated by responders bearing the H-2b ( KbIbDb ) haplotype, but not by responders bearing the H- 2bm14 ( KbIbDbm14 ), KbIbDd , KbIbDk , or KbIb / qDq haplotype. Responses by the non-H-2b responders against Dbm13 are limited to those determinants shared by the Dbm13 and Db molecules. Because congenic mice differing only at the H-2D region are either responsive or nonresponsive to Dbm13 gains, the responsiveness is controlled by gene(s) in the H-2D region. F1 hybrid offspring of responsive (H-2b) and nonresponsive (non-H-2b) parents are invariably responsive, indicating genetic dominance of the responsiveness. In contrast to the response against Dbm13 gains, cytotoxicity specific for Dbm14 gains is generated by responders bearing the H-2b, H- 2bm13 , KbIbDd , KbIbDk , or KbIb / qDq haplotype. These data indicate the existence of two types of allogeneic MHC determinants; one, represented by Dbm14 gains, is the classic type capable of eliciting CML responses in mice of a wide range of H-2 haplotypes, whereas the other, exemplified by Dbm13 gains, elicits CTL responses only in mice of a few related haplotypes. It is proposed that recognition of Dbm13 gains is restricted by structures shared by Db and Dbm13 but missing from other D (or L, R, etc.) molecules, such as Dbm14 , Dd, Dk, and Dq. Availability of various restricting structures in self MHC molecules may thus influence the alloreactive CTL repertoire.     

Expression of H-2Dd and H-2Ld mouse major histocompatibility antigen genes in L cells after DNA-mediated gene transfer

Among the more than 20 H-2-like genes in the BALB/c mouse genome, there are two classical transplantation antigens (H-2Dd and H-2Ld) encoded at the D-end of the major histocompatibility complex. Here we report the identification of a bacteriophage clone that encodes H-2Dd. The H-2Dd gene was identified by nucleotide sequence analysis and by characterization of the new H-2 antigen expressed when the cloned gene was introduced into mouse L cells by DNA-mediated gene transfer. The previously identified H-2Ld gene was then compared with the H-2Dd gene. The two genes appear to have the same general structure, and for the 854 nucleotides that have been compared, the two genes are 89% homologous. The H-2Ld and H-2Dd antigens expressed on mouse L cells after DNA-mediated gene transfer were examined by immunologic criteria. The stably transformed cell lines express apparently normal levels of H-2Dd and H-2Ld on the cell surface as measured by quantitative immunofluorescence by using monoclonal anti-H-2 antibodies. They synthesize H-2Dd and H-2Ld at normal rates as determined by endogenous labeling and immunoprecipitation of cell extracts. They evoke a strong specific serologic response when used to immunize C3H mice. The newly expressed antigens are able to serve as targets for alloreactive T cells. These cloned genes provide good substrates for examining the evolution of two closely linked H-2 antigen genes. Comparison of the structures of these genes provides clues to the basis for the differential expression of these antigens and their different biologic functions.     

DNA sequence analysis of the C3H H-2Kk and H-2Dk loci. Evolutionary relationships to H-2 genes from four other mouse strains

We generated nucleotide sequences for H-2Kk and H-2Dk from the C3H mouse, as well as for a genomic clone of H-2Db, in order to conduct an evolutionary analysis of the H-2 genes from three haplotypes, k, d, and b. H-2Kk from both the C3H and AKR strains, H-2Kd, H-2Kb, H-2Dk, H-2Ld, H-2Dd, H-2Db, and H-2Dp DNA sequences were aligned, and the alignments used to construct phylogenetic trees inferring the evolutionary relationships among the nine genes by two independent methods. Both approaches yielded trees with similar topologies. In addition, the sequence alignments revealed patterns of nucleotide substitutions which implicate both point mutation and recombination in the divergence of the H-2 genes. Future considerations for evolutionary analysis of class I genes are discussed.     

H-2 polymorphisms in outbred strains of mice

We studied H-2 polymorphisms of five outbred strains, CF1, ddY, ddN, ICR and KUNM, using 15 alloantisera to H-2 class I private antigens. All individuals had at least one private antigen at H-2K and/or H-2D. The number of H-2 class I private antigens in each strain were three in ddY, four in ICR, five in CF1, and seven in ddN and KUNM. The number of H-2 phenotypes were five in ddY, six in CF1 and ICR, twelve in ddN and seventeen in KUNM. Each strain had strain specific antigen(s) which discriminated each strain from the other strains. The CF1 strain had the H-2.23 and H-2.32 antigens controlled by the H-2k haplotype at a frequency of about 70%. The ddN strain had H-2.4 (H-2Dd) at a frequency of 37.8%. The H-2.9 antigen (57%) governed by H-2Df was distributed only in the KUNM strain. Both the ddY and ICR strains possessed H-2.17 and H-2.30 antigens (85-97%) which were controlled by the H-2q haplotype. The H-2 locus will be useful to characterize each strain and distinguish between strains, and for monitoring of outbred strains by H-2 gene frequency.     

CTL and serologically defined antigens of B2m,H-3 region

The antigens of the B2m,H-3 region of 13 chromosome 2 congenic strains and seven inbred strains have been studied by using CML and serologic techniques. Nine patterns of cross-reactivity have been defined by CML assays. These results are in agreement with an extend previously described cross-reactivity studies. The reactivities of three monoclonal antibodies previously thought to be reacting with B2M-B are shown to differ: Ly-m11 and J-5 react with cells of strain B10-pa,at and clone 23 does not. Two H-3 region loci are hypothesized on the basis of CML and serologic activity: B2m and H-3. The CTL responses to the B2M antigens are H-2K restricted; the CTL responses to H-3 antigens are H-2D restricted. The restriction of the response to the H-3 antigen requires effector-target identity of the H-2D molecule but not the B2M molecule of the class I antigen. These loci have been separated by recombination from H-42 in the production of the congenic strain B10.FS-a. A gene order of B2m, H-3, H-42 is suggested.     

The cAMP-Dependent Protein Kinase Inhibitor H-89 Attenuates the Bioluminescence Signal Produced by Renilla Luciferase

Background

Investigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA) increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.

Principal Findings

We present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc) variants in a population of cells and also in single cells. Using 10 µM of luciferase substrate and 10 µM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (±15%) and 54% (±14%) of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.

Significance

The identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell-based assay readout before drawing conclusions from the data.     

Regulation of idiotope expression. III. H-2 influences the magnitude and the idiotypy of a T-independent antibody response in mice of certain genetic backgrounds

Antibody response to the phosphocholine (PC) epitope on Streptococcus pneumoniae R36a (Pn), a T-independent Ag type 2, was studied in H-2 congenic mouse strains. The PC-specific antibody plaque-forming cells (PFC) were enumerated in the spleen at various intervals after the primary Pn injection, and the proportion of PFC that produced antibody expressing the AB1-2 idiotope (Id) was determined by using the corresponding monoclonal anti-Id. AB1-2 is a cross-reactive Id, detectable on germline-encoded PC antibody of the T15 family, and on most, but not all, somatic variants of that antibody. The specific PFC responses in BALB/c (H-2d) and BALB.B (H-2b) strains were of comparable magnitude and most, if not all, PFC were ABl-1 Id-positive (AB1-2+). This was not the case in the responses of the B10D2 (H-2d) vs C57BL/10 (H-2b) strains and the D1.C (H-2d) vs D1.LP (H-2b) strains (on DBA/1 background). In each of these pairs, the H-2d mice were high responders, and the response was dominated by AB1-2 Id (greater than or equal to 80% AB1-2+ PFC at the peak, on day 5). The H-2b mice were low responders, and only a minor proportion of PFC (less than or equal to 30%) were AB1-2+; an increase of AB1-2+ was seen later in the response (d.10). The results of PFC assays were confirmed by measuring the PC-binding antibody and AB1-2 Id in the sera of D1.C and D1.LP mice immunized repeatedly with Pn. Moreover, D1.LP mice that had very low levels of AB1-2 Id had higher serum levels of antibody expressing two other T15 Id, B36-82, and B24-44. The B36-82 and B24-44 Id have been previously found on somatic variants of PC antibody expressed independently of the Ab1-2 Id. The concentrations of these two Id in D1.LP mice after repeated immunization approached those in D1.C. These results indicate that 1) the H-2 allelism may have a significant effect on TID antibody response in mice of a certain genetic background, but not in the BALB/c; and 2) the idiotypic repertoire of the response may be influenced by H-2 at the level of clonal variants of PC-reactive cells.     

A novel H-2K splice form: predictions for other alternative H-2 splicing events

A large number of H-2K and H-2D cDNA clones from a C3HfB/HeN spleen cDNA library were extensively characterized. All H-2D^k cDNAs were shown to exhibit the short form of exon 8, consistent with the presence of a single lariat branchpoint site within intron 7. Twenty-five H-2K^km2 cDNAs were found to bear a short exon 8, whereas only two clones were shown to carry the longer form of this exon. In one of the H-2K^km2 cDNAs, a novel pattern of H-2 splicing was identified, in which an extra 15 nucleotides, derived from the 3′ end of intron 5, were inserted between the intact and unaltered exon 5 and exon 6 sequences. Resulting from the apparent use of a cryptic splice acceptor site in place of the canonical intron 5 site, this insertion is predicted to generate an in-frame insertion of five nonpolar amino acid residues within a highly polar region of the intracytoplasmic domain of the H-2K polypeptide. The features of this novel splice form served as the basis for predicting additional rare, alternative H-2 pre-mRNA splicing events that might produce functionally relevant microheterogeneity in the encoded H-2 gene products.