HLA-D typing in multiple sclerosis: Israelis tested with European homozygous typing cells

The association of A3, B7, Dw2 and DR2 histocompatibility (HLA) markers with multiple sclerosis (MS) is well established among Northern Europeans and Caucasoids in the United States. We showed previously that A3 and B7 were not increased among Israelis with MS, and in a preliminary study Dw2 as well. An association of A3 and B7 is also lacking in Italian, Jordanian and Japanese MS patients. In Black American MS patients, the B7 frequency is slightly increased but Dw2 is still significantly associated with MS.
For the HLA-DR antigen series DR2 is shown to have a stronger association to MS than A3 and B7. Conceivably, this antigen could be associated with MS even in populations where an association with A3 or B7 is lacking. Therefore, a study of HLA-A, B, C, DR and D antigens was carried out in Israel. No significant excess or deficiency of HLA antigens was found in MS.
Possible explanations for these results are as follows: (1) the relevant HLA-D alleles in the Jewish population arc not detected by the homozygous typing cells (HTCs) used, since they were derived primarily from European sources; (2) in contrast to the Caucasoid populations the genetic factor predisposing for MS is not associated with HLA alleles in the Israeli population; (3) MS is an heterogeneous disease and in Israelis, an environmental factor is sufficient to cause the disease.     

HLA-D typing in Austria. A panel study using 26 newly defined homozygous typing cells

A panel of 120 HLA-A, -B, -C and -DR typed Austrians has been typed for HLA-D by the use of 26 Homozygous Typing Cells (HTC). The new Austrian HTC, partly defined by the 9th International Histocompatibility Workshop (9WS), partly by a checkerboard experiment with internationally well defined reference HTC, type for HLA-Dw1 to -Dw7 and an obviously new, so far unknown HLA-DR2 related HLA-D determinant. Associations of HLA-DR and HLA-D antigens in Austria and their frequencies are determined. Antigen frequencies in Austria are compared to frequencies in other Caucasoid populations.     

Population studies of HLA-linked SB antigens and their relative importance in primary MLC typing. Analysis of HLA-D homozygous typing cells and normal heterozygous populations

SB phenotyping was undertaken on 96 HLA-D homozygous typing cells (HTCs) and 129 normal unselected heterozygous donors in the German population, using Interleukin-2-propagated primed lymphocyte typing (PLT) reagents. The results showed that the SB antigens in the normal population behave as a system of alleles at a single locus in Hardy-Weinberg equilibrium (p approximately equal to 0.20). Estimated gene frequencies in the German population appeared to be significantly different (p less than 0.002) from the North American Caucasian population: the principal differences were increased frequencies of the specificities SB1 and SB4, and decreased frequencies of blanks. Of HLA heterozygous donors 41% typed for two distinct SB specificities; 57% typed for one; and 2% were blank. In the HTC group, 20% typed for two specificities; 68% typed for one; and 12% were blank. Thus, a significant proportion of HLA-D homozygous test cells were, nonetheless, heterozygous for HLA-linked SB antigens. Performance of checkerboard mixed leukocyte cultures (MLCs) between 16 SB typed HLA-Dw3 HTCs, however, did not indicate that the observed mutual or one-way responses were influenced in any simple way by SB antigens; neither heterozygosity nor assumed homozygosity for SB antigens appeared to influence the frequency of MLC typing responses of HLA-Dw3-positive donors on these HTCs. These results add further confirmation of the genetic and functional independence of the SB gene product(s) and the HLA-D/DR gene product(s).     

Generation of HLA-D specific primed lymphocyte typing (PLT) cells and cross-reactions of PLT-cells primed with homozygous typing cells

An approach for the selection of HLA-D specific primed lymphocyte typing (PLT) cells is described. The responder cells were primed with homozygous typing cells. Reproducible extra reactions were found and were analyzed in relation to HLA-D antigens defined by homozygous in cells (HTC's). The secondary response of 105 different PLT-cell combinations generated by 29 different primary responders against 19 different homozygous typing cells of the specificifies Dw1 to Dw8 and the local specificity "H" were tested in secondary PLT toward 17 different homozygous typing cells and 10 heterozygous cells. Cross-reactions were defined as reactions equal to or higher than the lowest HLA-D specific reaction observed. The entire experimental design and data analysis gave rise to a conservative definition of cross-reactivity. Two main groups of cross-reacting HLA-D determinants seem to exist: (i) Dwl, 3, 4, 7, and the local specificity "H", and (ii) Dw2, 5, 6, 8, and "H". The primary pairwise cross reactions were in group (i): Dw1-3, Dw1-"H", Dw3-4, Dw3-7, Dw7-"H", and in group (ii): Dw2-6, Dw2-8, Dw5-8, and Dw5-"H". The existence of such cross-reactions is likely to interfere with the results of PLT-typing and should be taken into account when attempts are made to develop HLA-D specific PLT-cells.     

Cell membrane polymorphisms coded for in the HLA-D/DR region I. Relation between D and DR

The relation of HLA-D and -DR determinants was studied in Dutch Caucasoids. The recognition of subgroups of DR4, DR5, and DR7, and the specificities LB12 and LB13 are described. Phenotype and gene frequencies and a Hardy-Weinberg analysis of DR and local (LB) B-cell groups are given. Excellent correlation between D and DR typing was obtained when HTCs were studied by selected B-cell antisera. When the same sera were used to type a panel of D typed cells, the correlation was decreased (with the exception of DR3 and Dw3). In the case of discrepancies the DR specificity, but not the corresponding D specificity, always could be found and not the other way around.

The data fit best the assumption that HLA-D and -DR are carried by the same molecule, although they might be different determinants on this molecule. A number of possible explanations for the observed discrepancies has been given.     

HLA-D-DR relationships: PLT studies of HLA-D specificities associated with DR1, DR2, and DR4

The primed lymphocyte test (PLT) detects gene products of the HLA-D-DR region which activate the secondary (memory) response of MLC stimulated T cells. In the present study attempts were made to determine whether different HLA-D alleles associated with the same DR, such as DR1, 2, and 4 can be discriminated by PLT typing. PLTs were generated by using, as responders and primary MLC stimulators, HLA-D different HTCs which shared all DR groups (major DR, supertypic MT and second locus MB) or only the MT or MB groups. As secondary stimulators, lymphocytes from an HLA-D selected panel of 72 individuals were used. PLTs raised in DR identical responder-primary stimulator combinations were able to discriminate between the different HLA-D antigens associated with the same DR. In contrast, when priming was performed in combinations differing for the major DR group, the restimulation response was highly associated with the DR specificity of the primary stimulator, regardless of whether or not this was compatible with the responding HTC for the MT or MB groups.

This data indicate that the specificity of primed lymphocytes largely depends on the combinations used for priming and that the memory response can be activated by both HLA-D and DR antigens. The dissociation of HLA-D from DR by PLT typing might provide a useful tool for further analysis of this HLA region.     

Lack of shared lymphocyte-stimulating determinants between H-2I and HLA-D/DR using mouse primed lymphocyte typing cells

A number of anti-H-2 alloantisera containing antibody reactive with I region gene products (Ia) of the major histocompatibility complex cross-react with determinants expressed by human peripheral blood B lymphocytes. Such data have led to the conclusion that Ia and DR antigens share cross-reacting determinants. We have attempted to generate mouse primed T lymphocyte populations specific for defined I region gene product determinants which concomitantly recognize DR determinants on human peripheral blood leukocytes in primed lymphocyte typing (PLT) analysis. Mouse PLT cells were generated in primary MLC using strain combinations identical to those in which positive mouse/human cross-reacting antisera have been obtained. The resulting PLT cells exhibited strong, yet specific, secondary MLC responses against mouse cells expressing the Ia determinants used as the primary stimulus. In contrast, when examined on panels of human peripheral blood leukocytes, no reactivity was detected. This lack of cross-reactivity suggests that mouse T cells primed toward Ia determinants do not regularly recognize cross-reacting determinants of DR or D-associated antigens expressed on human PBLs. Consequently, mPLT cells are not a useful reagent in defining HLA-D region polymorphism.     

HLA-D clusters associated with DR2 and the definition of HLA-D"AZH": a new DR2 related HLA-D specificity in Israel

In order to investigate the HLA-D clusters associated with DR2 in Israeli Jews, 40 DR2 positive unrelated individuals were studied with a panel of DR2 associated homozygous typing cells (HTC's) which detect the lymphocyte defined specificities HLA-Dw2, Dw12, Dw9 and D-WJR. The results confirmed the existence of two distinct HLA-D clusters associated with the same serologically defined DR2. Of 40 individuals 22.5% (9/40) were Dw2 and 50% (20/40) were Dw12 carriers. Yet, no HLA-D specificity could be assigned to the remaining 11 DR2 positive individuals. In the present study we have defined a unique DR2-associated Dw specificity, HLA-D"AZH". The donor of the HTC was of Moroccan origin and an offspring of a first cousin marriage. This cell was not typeable with the known DR2-associated homozygous typing cells nor with other HTC's which define the well established HLA-Dw1 to Dw11 specificities. It was shown to segregate with DR2 positive HLA haplotypes in family analysis and in a population study, typed out 7 of 11 unrelated DR2 positive, Dw blank individuals, thus identifying a unique and new HLA-D cluster provisionally designated D"AZH".     

HLA class II restriction repertoire of antigen-specific T cells. I. The main restriction determinants for antigen presentation are associated with HLA-D/DR and not with DP and DQ

In order to study the HLA class II restriction repertoire in antigen presentation to T cells, T lymphoblasts (T-LB) of ten different HLA class II donors were generated by a simple and rapid technique; peripheral blood lymphocytes (PBL) were restimulated in vitro with purified protein derivative (PPD) or tetanus toxoid (TET), and then propagated in interleukin-2 containing conditioned medium (IL2-CM). These T-LB appeared to be antigen specific and devoid of alloreactivity. Antigen was presented to these T-LB by allogeneic irradiated PBL as antigen-presenting cells (APC) in 179 combinations. T-LB proliferative responses were restricted mainly by determinants associated with HLA-DR and not with -DP or -DQ; in 102 fully DR mismatched T-LB/APC combinations matching for DP or DQ determinants had no significant influence on T-LB responses. For PPD, preferential DR1 restriction was observed, and the results suggest a preferential DRw11 vs. DRw12 restriction for TET. Moreover, DRw13 may be associated with low anti-PPD T-LB responsiveness.     

HLA-A,B,C, HLA-D/DR and HLA-D/DQ expression on unfixed liver biopsy sections from patients with chronic liver disease.

The distribution of HLA-A,B,C, HLA-D/DR and HLA-D/DQ molecules was studied by indirect immunofluorescence with an avidin-biotin technique and monoclonal antibodies, in unfixed cryostat sections of liver biopsies from 76 patients with chronic liver diseases of various aetiologies and five normal liver biopsy specimens. In pathological liver, strong cytoplasmic or membrane-like positivity for HLA-A,B,C of hepatocytes was observed in piecemeal necrosis areas in all groups. Cytoplasmic staining was mainly seen in lobular areas in autoimmune, cryptogenic and HBV-related cases with viral replication, while membrane-like positivity was more frequently observed in primary biliary cirrhosis, alcoholic and HBV-related cases without viral replication. A weak cytoplasmic staining for HLA-D/DR was observed in piecemeal necrosis and lobular areas mainly in HBV-related cases with viral replication. While bile duct cells were positive for both HLA-D/DR and HLA-D/DQ, hepatocytes were consistently HLA-D/DQ negative. The increased HLA-A,B,C expression on hepatocytes should allow T cytotoxic cell aggression. Hepatocellular HLA-D/DR expression is definite but weak and probably does not allow direct autoantigen presentation and induction of autoimmunity. Negativity for HLA-D/DQ further supports this hypothesis. Since cytoplasmic staining for Class I and II molecules is greatly lowered by fixing cryostat liver sections, prestaining conditions should be taken into account when comparing different studies.     

Mixed lymphocyte reactivity in chimpanzees. II. Family studies and identification of D locus antigens

A large number of related chimpanzees were tested in mixed lymphocyte cultures against each other. Several similarities among the D locus products coded for by different ChLA haplotypes were observed. Six animals were found to be homozygous for D locus antigens and two of these animals carried the same D specificity. Hence, the available "typing cells" permitted the identification of five D locus antigens of the chimpanzee. So far, no linkage disequilibrium has been found between any of the D locus antigens and ChLA-A or -B locus antigens.     

The major histocompatibility complex of outbred chickens: II, Analysis of the typing response in mixed lymphocyte culture stimulated by homozygous typing cells

MLR phenotypes of outbred and inbred chickens typing B13 of the major histocompatibility complex (MHC) of the chicken were compared. F1 hybrids of outbred and inbred B13 positive chickens were analysed in mixed lymphocyte culture (MLC). The intermediate strength responses of cells from B13 heterozygous outbred chickens stimulated by inbred B13 homozygous chicken cells were not due to minor variations of B encoded lymphocyte activating determinants (Lads). Nor were Lads encoded by genes unlinked to the B complex responsible for these reactions. In contrast, F1 anti-parental type reactions were observed, and these alone are probably responsible for the intermediate strength reactions so often seen in typing of heterozygous outbreds with homozygous typing cells.     

Typing for RhLA-D in rhesus monkeys: I. Characteristics of ten groups of homozygous typing cells

Certain characteristics of 38 homozygous typing cell (TC's) of rhesus monkeys were determined. These TC's define ten RhLA-D locus specificities. Eight of them are associated with established RhLA-DR antigens. Two other groups of typing cells, D9 and D10, were previously considered to be associated with "blank" antigens of the DR series; they now appear to be associated with B-cell antigens which are also likely to be controlled by the DR locus. No influence of RhLA-A or B antigens of MLC reactivity was observed. It was shown, however, that products of at least one locus other than D/DR is responsible for MLC stimulation. Whether those MLC antigens are associated with serologically identifiable B-cell antigens which are not controlled by the DR locus, is not yet clear.     

Cell mediated cytotoxicity against HLA-D region products expressed in monocytes and B lymphocytes. III. Inhibition by monoclonal antibodies and study of an HLA-B/DR recombinant

Attempts to further define the antigens recognized by HLA-D region specific cytotoxic lymphocytes were undertaken using monocytes and transformed B cell lines as target cells. Monoclonal antibody against framework determinants of HLA-DR antigens partially blocked cell mediated lysis, suggesting that at least a portion of the D-region specific cytotoxic cells recognized the HLA-DR determinants. The study of a family with an HLA-B/DR recombinant showed that the determinants recognized by allogeneic anti-HLA-D-region cytotoxic lymphocytes are encoded outside of HLA-B. In addition, cytotoxic lymphocytes specific for the HLA-D region could be generated with cells identical throughout the interval from HLA-A to B and disparate only to the left of HLA-B.     

Role of macrophages in T lymphocyte response to Candida allergen in man with special reference to HLA-D and DR.

Activation of naturally sensitized human T lymphocytes to Candida allergen was studied using three HLA-D and DR heterozygote Japanese cells (Dw1.DR1/DYT.DR4, Dw12.DR2/DYT.DR4, DYT.DR4/DEn.DR blank) and four HLA-D and DR homozygote cells (Dw1.DR1, Dw12.DR2, DYT.DR4, DEn.DR blank). In vitro activation of T lymphocytes to Candida allergen was found to require the presence of autologous or allogeneic compatible HLA-Dw1.DR1 and Dw12.DR2 macrophages.     

Ankylosing spondylitis,HLA-B27 and Klebsiella. II. Cross-reactivity studies with human tissue typing sera.

Human monospecific HLA B27 typing sera have been shown to have increased binding activity for klebsiella extracts by haemagglutination (P less than 0.001), radiobinding assay (P less than 0.025) and radiolabelled antigen competition assay (P less than 0.02) when compared to non-B27 tissue typing sera. These observations are in agreement with those of studies using rabbit sera, suggesting that HLA B27 lymphocytes may exhibit partial cross-reactivity with bacterial antigens found in some Gram-negative microorganisms such as klebsiella. It is suggested ankylosing spondylitis may occur as a result of immunological damage following infection by Gram-negative bacteria carrying antigens having stereochemical similarity to self antigens.     

Allotype suppression in the chicken II. Suppression in homozygous chickens with antiallotype antibody and allotype-disparate B cells

Injection of heterozygous (M-1³/M-1^b, G-1^g/G-1ⁱ) B 14-line chickens with antisera directed against either IgM-1a or IgM-1b induced suppression of the relevant IgM-1 and genetically linked IgG-1 allotypes, whereas a mixture of anti-M-1a and anti-M-1b antibodies failed to produce allotype suppression. Injection of anti-M-1 antiserum into M-1 homozygous chickens induced only a transient delay of a few days in the appearance and rise of serum IgM-1 levels. However, suppression of host allotypes was induced by injecting M-1, G-1 homozygous neonatal or embryonal recipients with anti-M-1 antisera together with B locus-histocompatible allotype-disparate spleen, bone marrow or bursal cells. The active cell type were donor B cells, which established chimerism in the injected hosts, whereas peripheral blood T lymphocytes from agammaglobulinemic donors were ineffective. Allotype suppression was attributed to a homeostatic control mechanism which is exerted by normal B cells (but not T cells) over B cell recruitment in anti-M-1 antibody-treated, immature hosts.     

Differences in processing of an autoantigen by DR4:Dw4.2 and DR4:Dw14.2 antigen-presenting cells

Variations in antigen processing can influence class II-restricted T cell responses. We now report a highly significant difference (p < 0.001) between the ability of antigen-presenting cells from three HLA-DR4:Dw14.2 (Arg⁷¹) and six DR4:Dw4.2 (Lys⁷¹) individals to present recombinant or native acetylcholine receptor antigens to a myasthenia gravis T cell clone. The difference was greatest with longer antigens, and not seen with short synthetic peptides, suggesting that it may result from a difference in antigen processing between the two alleles. The results were not related to the presence of myasthenia gravis or of steroid therapy. They could, however, be of relevance in rheumatoid arthritis where particularly severe disease associates with Dw4.2/Dw14.2 heterozygosity.