Localization of the disulfide bridges of human histocompatibility antigens.

Both papain-solubilized and detergent-solubilized human histocompatibility antigens have been treated with NTCB (2-nitro-5-thiocyanatobenzoic acid) which cleaves these molecules at cysteine residues. A study of the fragment produced has made it possible to deduce the size and location of the two disulfide loops in these molecules. The sizes of the two loops in HLA-B7 and in the mixture HLA-B7 + 12 are about 5100 and 6600 daltons, a size similar to that of the disulfide loops found in immunoglobulins. The disulfide loops in HLA-A2 may be smaller in size. The two loops are located in middle regions of these molecules; neither the N-terminal nor the C-terminal regions contain disulfide loops.     

Identification of murine H-2Db histocompatibility antigens in cells transfected with cloned H-2 genes

Clones of mouse L-cells transformed with 21 cosmids containing 15 major histocompatibility complex class I genes of C57BL10 (H-2b) sperm cell DNA were analyzed for the expression of their transfected H-2 and Qa/Tla genes. Three cosmids contained a single gene, mapping to the H-2D region. This gene encodes the H-2Db alloantigen: mouse L-cells transfected with cosmids containing this gene reacted with monoclonal antibodies and alloantisera specific for the H-2Db antigen and expressed a 46-kd H-2 heavy chain associated with beta 2-microglobulin in their cell membranes. Furthermore, these transfected cells were stimulators of, and targets for, anti-H-2Db cytotoxic T-lymphocytes. Eighteen cosmids contained 14 different genes mapping to the Qa and Tla regions. L-cells transfected with these genes did not express class I genes reacting with alloantisera or monoclonal antibodies against Qa2, Qa4 or TL differentiation antigens. In particular, the Qa2,3 gene of C57BL10 was not identified.     

Partial purification and characterization of chicken immune-associated antigen in serum coded for by the major histocompatibility complex

Chicken serum contains 3 molecular species of immune-associated (Ia) antigen coded for by the major histocompatibility complex (MHC). These molecules are named B-L alloantigens [1]. They vary in electrophoretic migration velocity and molecular size [2]. The aim of this study was to characterize one of the antigen species - the low molecular size form. Therefore, we performed a partial purification by: (i) affinity chromatography; and (ii) ammonium sulfate precipitation of serum B-L antigen from the chicken plasma. Since the MHC-antigens were known to be glycoproteins, the purification was based on lectin affinity chromatography, as previously used for the membrane-bound MHC-antigens [3]. Electro-immunochemical analysis using rabbit antibodies against the chicken lymphocyte plasma membrane and the partially purified antigen were employed to monitor the purification and to characterize the different molecular forms of the Ia molecules. The partially purified preparation was then analyzed to elucidate the biochemical structure of the serum B-L antigen. Finally, rabbit antiserum was raised against this preparation to evaluate its level of purity and to follow further purification of this molecule.     

In vitro production of a hybrid monoclonal antibody that preferentially binds to cells that express both HLA-A2 and HLA-B7

A hybrid mouse monoclonal IgGl having one low affinity combining site for HLA-A2 and one low affinity combining site for HLA-B7 was made by the chemical method of Nisonoff and Palmer (Science 143:376,1964). This involved selective reduction of interchain disulphides, a splitting of the IgGl into half molecules at low pH and ionic strength, and reassociation of the half molecules by neutralization. Serologically active hybrids were separated from parental IgGl by an absorbtion procedure and recovered in about 10% yield. The hybrid discriminated between cells that express either HLA-A2 of HLA-B7 from cells that express both A2 and B7. This is because it could bind bivalently to the cell with both A2 and B7 but could only bind with a single combining site to cells expressing A2 or B7. The consequence of these different modes of attachment was to give up to sevenfold greater binding to the cell expressing A2 and B7 in comparison to the cell expressing only A2 or B7.     

Monoclonal antibody (Tü48) defining alloantigenic class I determinants specific for HLA-Bw4 and HLA-Aw23,-Aw24 as well as -Aw32

A monoclonal antibody (Tii48) was prepared against human lymphocytes. Immunochemical analysis indicated that Tii48 binds to a fraction of HLA molecules. Tii48 was tested on 398 HLA-A,B,C/DR-typed normal blood donors in the microcytotoxicity assay and was found to detect a “supertypic” determinant on molecules bearing the HLA specificities -Bu4. Au23(9). -Au24(9), and -Au32. Among the HLA-Bu4-positive individuals, negative or weak reactivity of Tii48 was found with about one-fourth of normal donors heterozygous for the HLA antigen Bw44. This points to the possibility that the antigenic determinant detected by Tii48 is not expressed on all HLA-molecules carring the HLA-Bu4 specificity on the cell surface. Alternatively, weak or no expression of the Tii48 antigenic determinant on some HLA-Bu44 bearing molecules might be explained by the existence of molecular variants of glycoproteins with this HLA specificity. The concept of “supertypic” HLA specificities is discussed with regard to the expression of a monoclonal antibody-defined epitope on both certain HLA-A and B molecules.     

Interferon-induced expression of class II major histocompatibility antigens in the major histocompatibility complex (MHC) class II deficiency syndrome

Class II antigens encoded by genes of the major histocompatibility complex (MHC) are expressed by a variety of cell types and have a vital role in the cellular interactions required for an effective immune response. We have analyzed the regulation of HLA-DR, DP, and DQ class II antigen expression on cells of different lineage from an immunodeficient patient with the MHC class II deficiency syndrome. T and B lymphocytes, monocytes, and fibroblasts, which initially expressed no class II antigens, were treated with inductive stimuli that normally lead to enhanced expression of class II antigens. Monocytes, but not fibroblasts, cultured for 48–96 hr in the presence of recombinant gamma interferon expressed all three types of class II antigens. In contrast, T lymphocytes did not express class II antigens following their exposure to a variety of stimuli, including activation with phytohemagglutinin and culture in the presence of interleukin-2, transformation by the retrovirus HTLV-1 or HTLV-2, or exposure to the demethylating agent 5-azacytidine. Similarly, class II antigens were not induced on B cells by cross-linkage of surface immunoglobulin molecules with anti-mu, exposure to Epstein-Barr virus, or treatment with soluble factors secreted by activated T cells. These results demonstrate that the regulation of class II MHC antigen expression by monocytes and lymphocytes is dissimilar and suggest that different regulatory genes are involved in the control of class II antigen expression by cells of different lineage.     

Structural studies on H-2Db and H-2Kd molecules indicate that murine major histocompatibility b and d haplotype alloantigens share structural features

Structural properties of the H-2D^b and H-2K^d murine major histocompatibility complex (MHC) antigens were examined by radiochemical methods. Radiolabelled preparations of the H-2D^b and H-2K^d antigens were obtained by indirect immune precipitation of NP-40 lysates of the lymphoid tumor cell lines EL-4 (H-2^b) and C14 (H-2^d), respectively. After preparation of the 37,000 molecular weight papain fragment the antigens were cleaved with CNBr. The H-2K^d antigen yielded four major CNBr fragments whereas the H-2D^b molecule provided six. These CNBr fragments were subjected to partial NH₂-terminal amino acid sequence analysis and aligned by homology to the H-2K^b glycoprotein. Comparison of the structural properties of the H-2K^d and H-2D^b molecules with previously published data on the other known major transplantation antigens of the b and d haplotypes (H-2K^b, H-2D^d and H-2L^d) reveal a marked structural similarity. First, the data show that certain methionine residues have been highly conserved and that cleavage by CNBr at these positions provides an initial strategy for the study of these molecules. Secondly, disulfide-linked peptides obtained after CNBr cleavage could be aligned and the data suggest the presence of disulfide bridges in homologous positions. Third, after CNBr cleavage both the H-2K^d and H-2D^b molecules yielded two glycopeptides which were homologous to glycopeptides from the H-2K^b molecule. Fourth, overall homology for a limited number of comparable positions is about 81% between the H-2K^b and H-2K^d gene products and 88% between the H-2K^b and H-2D^b gene products.     

Complexity of minor histocompatibility loci

Allografts can be rejected as a result of major histocompatibility antigen disparity or as a result of differences at any of a number of minor histocompatibility antigens. In many cases, rejection due to multiple minor histoincompatibility is as difficult to control as that induced by major histoincompatibility. Although an understanding of the molecular, biochemical, and functional parameters of the major histocompatibility loci and their products is increasing at an exponential rate, little is known about these same facets of minor histocompatibility loci and their products. It is generally accepted that minor histocompatibility loci in the murine model have a degree of polymorphism similar to that of H-2K or H-2D. This conclusion was based on typing alleles by the classic F1-skin graft test. Based on these allelic assignments, numerous unexpected findings of CTL specificity were made. Therefore, a systematic analysis was made comparing CTL specificity, F1-complementation, and allograft rejection. Based on these three parameters, the data presented using strains of mice that were bred to, and therefore presumed to, differ only at H-3 indicate that the antigen disparity of these congenic strains and the parental B10 strain as defined by CTL specificity and skin graft rejection is much more complex than originally described. One especially interesting chromosomal region is H-3/beta 2-microglobulin in the fifth linkage group of chromosome 2. Using CTL, ten specificities are defined, three of which appear to be specific for beta 2-microglobulin-A, -B, and -C. These findings raise the question of whether any minor histocompatibility locus is polymorphic or is instead a composite of multiple minor H-loci which are masquerading as a single locus.     

Monoclonal antibody detection of carbohydrate-defined and protein-defined H-2Kk antigens

Three different monoclonal anti-H-2K^k antibodies, 27R9, 30R3 and 11-4 were examined for the biochemical nature of the antigenic determinants they recognize. When these were compared on the basis of their sensitivity to pronase and various glycosidases, 27R9 was shown to bind to protein-defined H-2K^k antigens, while 30R3 and 11-4 bound to H-2 antigens defined by carbohydrate. From sugar inhibition studies, and treatments with specific glycosidases, d-mannose appears to be the immunodominant sugar involved in the antigenic site recognized by 30R3, while several sugars, namely sialic acid, d-mannose and α-and β-linked d-galactose would appear to be components of the antigenic site bound by 11-4. The carbohydrate determinants appear to be present on glycolipid molecules, since both the 30R3 and 11-4 antibodies could be inhibited by glycolipid extracts from spleen cells of the appropriate H-2 haplotype, as well as from several other strains of mice previously shown to be cross-reactive targets for these antibodies. This finding is supported by evidence that the molecule carrying the protein-defined antigen is distinct from that carrying the carbohydrate-defined antigens. The results are discussed in the light of current information on the nature of glycolipid Ia antigens, as well as the role of H-2 antigens in T-cell interactions.     

Characterization of major histocompatibility antigens on trinitrophenyl-modified cells.

Cell membrane proteins encoded for by the major histocompatibility complex (MHC)¹ are associated with the antigenic determinant(s) recognized on trinitrophenyl (TNP)-modified cells by syngeneic murine cytotoxic T lymphocytes and by hapten-reactive guinea pig T cells. To investigate the relationship of the TNP moiety on TNP-modified cells to these major histocompatibility antigens, peritoneal exudate cells or splenocytes from two inbred guinea pig strains and one inbred murine strain were TNP-modified, radioiodinated and lysed in detergent. TNP-derivatized proteins were then isolated using an anti-TNP immunoabsorbent, and the presence on TNP-derivatized histocompatibility antigens in the eluted proteins was determined by immunoprecipitation experiments and SDS-polyacrylamide gel electrophoretic analysis. Whereas most of the various histocompatibility antigens examined were found to be TNP-derivatized in amounts proportional to the degree of membrane protein derivation as a whole, only small amounts of TNP-modified strain 2 guinea pig Ia antigens were found, and no hapten-modified strain 13 guinea pig Ia antigens were detected. It is concluded that, in contrast to most MHC gene products, strain 13 Ia antigens are not derivatized on TNP-modified cells and, thus, represent an important exception demonstrating that histocompatibility antigens need not be directly TNP-derivatized for T cell recognition and activation.     

Abnormal differentiation of immunoregulatory T-lymphocyte subpopulations in the major histocompatibility complex (MHC) class II antigen deficiency syndrome

The major histocompatibility complex (MHC) class II deficiency syndrome is a rare immunodeficiency disease associated with defective expression of class II MHC antigens. We have examined the consequences of this defect for the differentiation and functional capabilities of immunoregulatory T-cell subpopulations in an affected patient. Although the number of circulating T cells was normal, there was a striking reduction in the number of CD4+ T cells. Furthermore, purified CD4+ cells from the patient were unable to provide help for antibody secretion. This defect in helper function appeared to be due to the abnormal differentiation of the few CD4+ cells present, virtually all of which expressed the CD4 + HB11 + phenotype characteristic of immature virgin T cells. Abnormal development of immunoregulatory CD8+ T cells was also observed. Although increased numbers of CD8+ T cells were present, virtually none had phenotypic properties of suppressor cells (i.e., CD3+/CD8+/9.3-granular lymphocytes that coexpress the Leu-15 or Leu-7 antigens), and purified CD8+ cells from the patient had no suppressor activity. Thus, the absence of class II MHC antigens profoundly disrupts the development of immunoregulatory T cells. We propose that these effects occur by the following mechanisms: (1) the absence of intrathymic class II antigens results in deficient production of CD4+ cells, (2) the CD4+ cells that do emerge from the thymus do not undergo postthymic maturation into CD4+HB11- cells with helper capabilities, and (3) the absence of CD4+HB11- effector cells results in abortive development of suppressor cells involved in feedback suppression.     

Phenotypical and functional analysis of B lymphocytes of two siblings with combined immunodeficiency and defective expression of major histocompatibility complex (MHC) class II antigens on mononuclear cells

Phenotypic and functional analysis of B lymphocytes in two siblings with combined immunodeficiency associated with defective expression of class I and class II major histocompatibility complex (MHC) antigens on mononuclear cells is described. The results of the analysis of the membrane phenotype of the B cells performed at the age of 1 and 5 years, respectively, by the use of monoclonal antibodies against class I (HLA-A, -B, -C) and class II (HLA-DR, -DP, -DQ) MHC antigens showed a decreased expression of class I antigens and a complete lack of class II antigens. Class I antigen expression consistently remained of the same magnitude during follow-up. Class II antigen expression remarkably had been positive early in life on B cells and activated T cells, whereas monocytes were negative for class II from birth onward. B lymphocytes of both patients respondedin vitro to polyclonal activation withStaphylococcus aureus Cowan I staphylococci (SAC) with the production of IgM-type immunoglobulins only. This neonatal type of response was in agreement with the membrane immunoglobulin phenotype of the B cells since a high sIgM/sIgD ratio characteristic of neonatal B cells was present. However, the expression of the FMC7 antigen on B cells of both patients was comparable to that on B cells of normal adults. We hypothesized that the lack of MHC antigen expression may impose a resting state on the lymphocytes in these patients due to ineffective cellular interactions. In this view the high sIgM/sIgD ratio reflects the activation state of the B cells rather than the maturational state of the cells. Furthermore, the B cells of these patients did not produce antipolysaccharide antibodies upon simultaneous stimulation with SAC and pokeweed mitogen (PWM). In the youngest child (age 1 year) this may reflect the unresponsiveness to polysaccharide antigens usually observed in children less than 2 years old. In the eldest child the nonresponsiveness might reflect a more general B-cell defect as a result of waning B-cell function increasing with age and imposed by lack of effectivein vivo activation.     

DR antigens on melanoma cells: analysis with monoclonal antibodies

Two monoclonal antibodies (691-13-17 and 37-7) can precipitate DR antigens from radioiodinated, detergent solubilized melanoma cells. However, after complete depletion of lysates with one antibody (691-13-17) alpha-like chains can be still be precipitated from some melanoma cells by the other antibody (37-7). Antibody 37-7 also precipitates an additional distinct antigen from SK-MEL 37 but not from any five other melanoma cell lines.     

HLA serological cross-reactivity: HLA-B15 has two public antigens

On the basis of their serologic cross-activity, HLA antigens can be organized into cross-reactive groups of CREG's. We have recently defined immunochemically two public alloantigenic determinants X and Y which can account for the serological cross-reactivity of the B7-CREG and B5-CREG, respectively. One of the smaller of these CREG's consists of HLA-B15 and B17. Using microcytotoxicity testing, a fluoresceinated Protein A binding assay, and chemical immunoprecipitation techniques, we have defined a new public alloantigenic determinant, tentatively designated “Z,” which is present on the 44,000 dalton glycoprotein chains of HLA-B15 and HLA-B17, but distinct from the B15 and B17 determinants. Since HLA-B15 is also a member of the B5-CREG, and therefore bears allodeterminant Y, this report constitutes the first immunochemical demonstration of two public determinants. Y and Z, on a single HLA-B molecule, HLA-B15.     

HLA-DR7 in Association with Chlorpromazine-induced Lupus Anticoagulant (LA)

The presence of anti-phospholipid antibodies (aPL) has been associated with the major histocompatibility complex (MHC) genes. These autoantibodies occur in individuals with infections such as that produced by the human immunodeficiency virus 1 (HIV-1) or with syphilis, but they can also occur in drug-induced lupus-like syndromes. In the present study, we analysed the presence of aPL (detected as lupus anti-coagulant) and its relationship with the MHC markers in 93 Caucasian psychiatric patients chronically treated with chlorpromazine. Forty-one out of 93 patients were positive for LA, and the HLA-DR7 antigen was significantly increased in LA-positive patients as compared to normal controls or LA-negative patients (PC=0.024, RR=2.12 and P=0.05, RR=1.57, respectively). Likewise, we noted a significantly increased frequency of HLA-B44 in LA-positive patients as compared to normal controls (PC=0.024, RR=2.12), but not when compared to aPL-negative patients. No significant differences were found among any other class I, II or III MHC antigens. Haplotype analysis showed that DR7 was mostly part of the HLA-B44-DR7-FC31 and B7-DR7-SC31 haplotypes. These results suggest that the HLA-DR7 antigen might be playing a role in the production of aPL in chlorpromazine-treated patients.     

Major histocompatibility complex (MHC) class II restriction,lymphokine production,and IgE regulation of house dust mite-specific T-cell clones

To elucidate the regulatory mechanism of human IgE synthesis, we have cloned house dust mite (Dermatophagoides pteronyssinus; Dp)-specific T-cell clones from three asthmatic children and three healthy individuals. Twelve clones were cloned from each group. All of these clones were CD3⁺, CD4⁺, CD8^–, and HLA-DR⁺. After stimulation with allergen in the presence of antigen presenting cells (APCs), half of the T-cell clones from asthmatic children and one-third of those from normals produced interleukin 4 (IL-4). None of the patients' clones produced interferon r (IFN-r), while 10 of 12 normals' clones did. After stimulation with calcium ionophore A23187 and phorbol myristic acetate (PMA), the production of IL-4 was markedly increased in both patients and normals. However, only 3 of the 12 patients' clones produced IFN-r, while all of the normals' clones did. The T-cell clones of both patients and normals produced comparable IL-2. To study the kinetics of lymphokine productions, a HLA-DRw12-restricted T-cell clone (FYD 3.1) was stimulated, respectively, with a combination of A23187 and PMA, phytohemagglutinin (PHA), or Dp antigen in the presence of APCs. Maximal IL-2 and IL-4 productions were detected 12 hr after A23187 and PMA stimulation, whereas IFN-r could not be detected even 36 hr after stimulation. When stimulated with PHA, the production of IFN-r peaked on the fourth day, but IL-4 was not detected. After stimulation with Dp antigen and APCs, IL-4 and IL-2 were detected on the second and third days, but IFN-r was not detected. The IgE production by autologous purified B cells in the presence of allergen or IL-4 was found to be augmented by the FYD 3.1 T-cell clones. IFN-r was observed to counteract the effects of the T-cell clones and IL-4. Thus, the secretory patterns of lymphokine and kinetics of lymphokine production of allergen-specific T-cell clones can be used to explore the regulatory mechanism of human IgE synthesis.     

The importance of tRNA for the in vitro cell-free translation of messenger RNA isolated from the malaria parasite Plasmodium lophurae

Preliminary studies had indicated the inadequacy of the wheat germ and rabbit reticulocyte cell-free translation systems for the in vitro translation of mRNA isolated from Plasmodium lophurae. To identify the factors which are important for the efficient translation of parasite proteins, an homologous system was established using polysomes, the pH 5 fraction, and tRNA prepared from P. lophurae. For comparison, the same components were isolated from the host duck reticulocytes and tested. The effect of each of these factors was evaluated by analysis of the translation products and by comparison with products synthesized in vivo. The results indicated that P. lophurae tRNA had a marked stimulatory effect on the synthesis of parasite proteins while it inhibited the synthesis of host proteins. Duck reticulocyte tRNA could not be used as a substitute for the parasite tRNA. Based on these findings, a commercially available rabbit reticulocyte system was supplemented with P. lophurae tRNA, which markedly increased the efficiency of translation of P. lophurae proteins by this system.     

Cytofluorometric analysis of major histocompatibility antigens on human monocytes using monoclonal antibodies

The expression of HLA-A, B, C, and DR antigens was investigated on monocyte preparations by flow cytometry using various monoclonal antibodies. Essentially all human monocytes, either freshly isolated or after culture for several days, were stained for HLA-A, B, C, and DR antigens. When monocytes were incubated with Con-A-stimulated lymphocyte supernatants, an increase in HLA-A, B, C, and DR staining was observed. No increase was noted when two other monoclonal antibodies against non-HLA-related monocyte antigens (63D3 and 61D3) were studied under the same culture conditions. These results indicate that soluble factor(s) present in Con-A-stimulated lymphocyte supernatants modulate the expression of the major histocompatibility antigens on the surface of human monocytes.     

Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes

We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins.     

Orientation of major histocompatibility (MHC) genes relative to the centromere of human chromosome 6

Linkage analysis of the data obtained from a three-generation Dutch family segregating for genetic variants of centromeric heterochromatic region in the band pi 1 on chromosome 6 (6ph), major histocompatibility (MHC) genes HLA-A, HLA-B and HLA-C, glyoxalase I (GLO) and phosphoglucomutase-3 (PGMg) showed that the genetic distance between the HLA gene cluster and 6ph is about 6 cM (at an estimated peak lod score of 3.466), that GLO is closer than HLA to the centromere and that PGM₃ is probably not situated on the same chromosomal arm as HLA.