Showing papers in "European Journal of Immunogenetics in 1991"

HLA-DR, DQ and DP typing using PCR amplification and immobilized probes.

Abstract: A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA-RFLP METHODS and INTERPRETATION SCHEME FOR HLA-DR and DQ TYPING

Abstract: DNA-restriction fragment length polymorphism (RFLP) typing is a well established and standard technique for identification of HLA-DR and DQ allotypes. We present a detailed review of methods for RFLP analysis and a scheme for interpretation of results. This scheme permits the routine identification of HLA-DR and DQ allotypes using the restriction endonucleases TaqI and either HindIII or MspI.

NON‐RADIOACTIVE OLIGOTYPING FOR HLA‐DR1‐DRw10 USING POLYMERASE CHAIN REACTION, DIGOXIGENIN‐LABELLED OLIGONUCLEOTIDES AND CHEMILUMINESCENCE DETECTION

Abstract: We describe a rapid non-radioactive DNA typing of the serological types DR1-DRw10 using polymerase chain reaction (PCR)-amplified DNA and 15 sequence-specific oligonucleotides (SSO) which are labelled enzymatically at their 3' end with one digoxigenin (DIG). The hybridized SSOs were detected using anti-DIG alkaline phosphatase and Fab fragments and visualization was obtained with the chemiluminescent substate 3-(2'-spiroadamantan)-4-(3''-phosphoryloxy)-phenyl-1,2-di o xetan (AMPPD). The results were identical with those of the previously used 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/4-nitrobluetetrazolium chloride (NBT) system. The use of AMPPD is more rapid and allows the repeated rehybridization of the membrane-bound DNA.

The Eurotransplant HLA-DRB oligonucleotide typing set.

Abstract: SUMMARY Several sequence-specific oligonucleotides (SSO) were designed based upon available sequences of HLA-DRB 1 genes which are associated with HLA-DR serotypes. Targets for these SSO were polymerase chain reaction (PCR) products using primers specific for the second exon of DRB genes. In view of practicality, a hybridization system was developed which was independent of temperature or composition of the hybridization buffers, hence uniformly applicable to all SSO, yet allowing discrimination based upon one nucleotide mismatch only. The SSO typing results were compared with HLA serological typing and a high correlation was observed (99%).

HLA-DRB and DQB typing by a combination of serology, restriction fragment length polymorphism analysis and oligonucleotide probing.

Abstract: SUMMARY DNA sequencing has revealed extensive polymorphism within the HLA-DRB and DQB genes of the major histocompatibility complex. At least 43 alleles of the DRB loci and 13 alleles of the DQB1 locus are currently recognized. Identification of these alleles can be performed phenotypically by cellular and serological techniques or genotypically by restriction fragment length polymorphism (RFLP) analysis and sequence-specific oligonucleotide (SSO) probing of DNA amplified by the polymerase chain reaction (PCR). However, each method has its technical limitations so the tissue typing laboratory must therefore choose from the range of available techniques to maximize the efficiency and accuracy of HLA typing. In this paper we describe a scheme for the accurate determination of all the serologically defined DRB and DQB allotypes using a combination of serology, RFLP analysis and PCR-SSO probing. The efficiencies of serology versus RFLP analysis for the DR typing of 800 individuals, and RFLP analysis versus PCR-SSO probing for the DQB typing of 317 individuals, are compared and the merits of each technique discussed. This scheme, which types for both HLA-DRB and DQB with an accuracy approaching 100%, is now routinely employed in all HLA studies of disease and transplantation in our laboratory.

An additional family of VH sequences in the channel catfish.

Abstract: SUMMARY A heavy chain variable region cDNA sequence of the channel catfish (Ictalurus punctatus) prototypical for a new VH family (approximately 20 or more members) is presented. The nucleotide and inferred amino acid sequences differ by 32–52% and 41–68%, respectively, from those for the already described catfish VH families.

PCR-SSO typing for HLA-DRB alleles.

Abstract: SUMMARY Polymerase chain reaction (PCR) amplification of the second exon of DRB genes from genomic DNA is described. Sequence-specific oligonucleotide (SSO) probes have been designed to allow for the typing of DRB alleles. The methods are described in detail and the results of a trial on both homozygous typing cells and restriction fragment length polymorphism (RFLP)-typed local caucasoid controls are given. The ease of use of this form of DRB typing is emphasized and potential complications are discussed.

PCR-SSO typing for HLA-DPB alleles.

Abstract: Accurate HLA typing is essential for the matching of donor-recipient pairs in allogeneic transplantation and in the genetic analysis of predisposition to various autoimmune diseases. HLA class I and class I1 typing is routinely performed by serology (Terasaki et al., 1978). while DNA-restriction fragment length polymorphism (RFLP) typing for DR and DQ is now commonplace (Carlsson et al., 1987; Bidwell et al., 1988; Howell et al., 1989). HLA-DP antigens are expressed at much lower densities on the cell surface than DR and DQ and were initially defined at the cellular level by primed lymphocyte typing (PLT) (Wank & Schendel, 19841. More recently, monoclonal antibodies and DNA-RFLP analysis have been used to detect certain HLA-DP alleles as defined by PLT (Bodmer et al., 1987; Hyldig-Nielsen et al., 1987; Maeda et al., 1988). All of these methods are subject to various limitations. Suitable PLT reagents are difficult to generate and the technique is time consuming. In addition, approximately 40% of the DP alleles cannot be typed by the PLT assay (Baur et al., 1984). DP allele-specific monoclonal antibodies are still few in number, while DP RFLP patterns are complex to interpret, requiring DNA digestion with several restriction enzymes. and often reveal little more identifiably functional DP polymorphism than is seen by the PLT assay. These limitations in DP typing techniques have limited our knowledge of the possible role of HLA-DP in disease association. population genetic and transplantation matching studies. However, as a viable and superior alternative, DP typing is now feasible using polymerase chain reaction (PCR) DNA amplification and sequence-specific oligonucleotide (SSO) probing and this approach reveals extensive DPB 1 allelic polymorphism (Bugawan et al., 1988; Angelini et al., 1989; Howell er al.. 1990). PCR-based analyses of HLA-DPB alleles have become possible as a result of extensiye gene mapping and, more particularly, nucleotide sequencing studies of the DP region. This region consists of two A-B gene pairs, DPA1-DPB1 and DPA2-DPB2, located approximately 700kb from the HLA-DR genes at the extreme centromeric end of the HLA gene complex. Only the DPA1-DPBl gene pair is expressed, since both DPA2 and DPB2 are pseudogenes, containing frameshift mutations and lacking correct RNA splicing sites. DPAl and DPBl are orientated 'head to head', unlike the other expressed class I1 A-B gene pairs, and their promoter regions are separated by 2.3 kb (Carol1 et al., 1987; Dunham et al., 1987; Kelly & Trowsdale, 1985). Both

Phagocytosis of fluorescent latex microbeads by peritoneal macrophages in different strains of mice: a flow cytometric study

Abstract: The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited macrophages was analysed by flow cytometry. The percentage of phagocytosing macrophages and the number of internalized microspheres per cell was determined from cell size and fluorescence histograms. Results were corrected for the adherence of microbeads to the cells in the presence of sodium azide in the medium. Human C3b- or murine monoclonal IgG-coated microspheres were applied to assess receptor-mediated phagocytosis in different inbred strains of mice. Phagocytic activity of thioglycollate-elicited macrophages was consequently higher than that of resident macrophages. A decreasing gradient of C3b and Fc receptor-mediated phagocytosis was established in the following order: B10.BR, B10, C3H/Di and C3H.SW strains. Our results indicate that the phagocytic function of murine macrophages is under control of both the somatic (non-H-2) and H-2 genes.

HLA-DR/Dw matching by PCR fingerprinting: the origin of PCR fingerprints and further applications.

Abstract: SUMMARY Polymerase chain reaction (PCR) fingerprinting, a new method for the rapid matching of HLA-Dr/Dw allotypes, involves the visual comparison of polymorphic HLA-DRB gene second exon PCR products, resolved in non-denaturing polyacrylamide gels (Bidwell & Hui, 1990). We show here that the satellite DNA bands within PCR fingerprints originate by heteroduplex formation between heterologous DNAs co-amplified by a common PCR primer set. We also present two further applications of the technique which permit discrimination between unrelated HLA-DR/Dw allotypes with similar PCR fingerprints.

Analysis of Ly5 chromosome 1 position using allelic differences and recombinant inbred mice.

Abstract: SUMMARY Differences between the mouse Ly5a and Ly5b alleles can be distinguished on the basis of polymerase chain reaction (PCR)-restriction enzyme analysis and differential monoclonal antibody reactivities. To more precisely map the Ly5 gene on the mouse chromosome 1, analytical DNA and protein tests were performed on recombinant inbred strains of mice prepared from SJL/J (Ly5a) and B ALB/cke (Ly5b) progenitor strains. Each recombinant inbred strain was characterized to determine whether it carried the Ly5a or Ly5b allele. Both assays, DNA-PCR and protein-immunofluorescence, yielded identical results for each strain examined. Placement of the Ly5 gene with respect to other characterized markers of mouse chromosome 1 for these recombinant inbred mouse strains shows a gene order of Idh-1.Ity:Pep3:/Ly5, CfhJ.

Hla dpb1 alleles and susceptibility to rheumatoid arthritis

Abstract: SUMMARY HLA-DPB1 genotypic and phenotypic frequencies were investigated in a series of 35 adult rheumatoid arthritis (RA) patients and 42 controls. No significant associations between DPB1 alleles and susceptibility to RA were demonstrated, although some non-significant differences in DPB1*0301 and 0401 allele frequencies between patients and controls were observed.

PCR-SSO typing in HLA-disease association studies.

Abstract: Associations between a large number of diseases and markers within the major histocompatibility complex (MHC) have been described. In particular, susceptibility to several autoimmune disorders, including type I diabetes mellitus and rheumatoid arthritis, is linked to genes within the MHC and strong population associations are demonstrable between certain HLA class II alleles and these conditions. Genetic mapping of HLA susceptibility loci has traditionally relied on the use of phenotypic markers defined by alloantisera, cellular typing reagents and biochemical analysis of histocompatibility antigens. Polymerase chain reaction sequence-specific oligonucleotide (PCR-SSO) typing combines the ability to define the finest of HLA specificities, by analysis of the corresponding DNA sequences, with the possibility of study large populations of normal and affected individuals. The applications of this technology to characterizing precisely the MHC loci associated with susceptibility to autoimmune diseases such as rheumatoid arthritis, type I diabetes mellitus, coeliac disease and pemphigus vulgaris are reviewed here.

A DR7 specific monoclonal antibody TAL13.1, raised against a transfectant detects IL-4 upregulated antigen on peripheral B-lymphocytes.

Abstract: A monoclonal antibody TAL13.1 was raised against mouse L cells transfected with the human HLA-DRB1*0701 gene. This antibody was found to be polymorphic recognizing a determinant expressed by the DR7, DRB1*0701 and DRB1*0702 gene products. Four polymorphic sites unique to this specificity have been identified within the DR beta 1 domain. These are residues 11-14, 25, 30 and 71-74, one or a combination of which is postulated as being responsible for conferring the specificity of the antibody. In Western blot analysis TAL13.1 was found to react with the DR alpha beta dimer, but not with the free alpha or beta chains. However, in flow cytometry it failed to bind a DR alpha/DQ beta mixed pair transfectant confirming that it recognizes an epitope on the DR beta not the DR alpha chain. Although TAL13.1, a low affinity antibody is negative or only weakly positive on resting peripheral blood lymphocytes (PBLs), we have demonstrated that by interleukin-4 (IL-4) stimulation we can up-regulate the levels of antigen already present and gain a level of binding comparable to that found on B lymphoid cell lines (B-LCLs) where it has been found to be a valuable reagent in their characterization.

PCR‐SSO TYPING FOR DR4‐Dw SUBTYPES: APPLICATION TO UNRELATED BONE MARROW TRANSPLANT DONOR SELECTION

Abstract: SUMMARY Thirty-seven DR4-positive patient-unrelated bone marrow donor pairs previously DR/DQ restriction fragment length polymorphism (RFLP) typed and tested in mixed lymphocyte culture (MLC), have been DR4-Dw subtyped retrospectively using sequence specific oligonucleotide probes. We found that DR4-Dw subtyping substantially increased the accuracy of pre-MLC matching and could potentially accelerate donor searches by avoiding unnecessary MLC tests on Dw-mismatched donors.

Application of polymerase chain reaction (PCR) to HLA-C locus typing.

Abstract: Polymerase chain reaction/oligonucleotide typing was used to identify HLA-Cw*0601 (Cw6) in patients with psoriasis and psoriatic arthritis. The assignment of HLA-Cw*0601 was established by the concordant presence of codons for alanine (position 73), lysine (position 80) and tryptophan (position 97). The frequencies of all three codons were increased in the patient groups.

Combined use of RFLP and PCR-ASO typing for HLA-DR-Dw and DQw typing.

Abstract: SUMMARY Due to some limitations of restriction fragment length polymorphism (RFLP) analysis in HLA-DR-DQ typing, we present a combined use of RFLP and polymerase chain reaction (PCR)-allele-specific oligonucleotide (ASO) typing. This scheme consists in selectively amplifying the few RFLP ill-defined genes (DR1/DR ‘Br’ and DR4-Dw subsets) using PCR with allele specific primers to avoid cross-hybridization.

Optimizing PCR conditions for HLA class II SSO typing.

Abstract: SUMMARY The polymerase chain reaction (PCR) has made the technique of sequence-specific oligoncucleotide (SSO) typing fast, accurate and very sensitive. These combined techniques are an ideal tool for analysing the complex patterns of polymorphism seen throughout the HLA complex. The success of the technique relies heavily on accurate and specific amplification of the DNA under study. This paper considers the principles behind the PCR amplification technique and discusses the factors which lead to optimal amplification. Primer design is discussed and a variety of sources of target DNA considered. Precautions designed to prevent contamination are discussed. Reaction components are considered both in isolation and as part of the complete reaction. Finally, a complete PCR protocol is suggested. The paper is illustrated with examples of HLA class II amplification.

Discrimination between HLA-DR1 (DRB1*0101) and DR'Br' (DRB1*0103) using sequence-specific oligonucleotide probes.

Abstract: Serological identification of the HLA-DQw1(w5)-associated or HLA-DQw3(w7)-associated DR'Br' (DRB1*0103) allele cannot be accomplished in the presence of a second DQw1(w5)-positive or DQw3(w7)-positive haplotype, respectively. DNA-restriction fragment length polymorphism (RFLP) analysis assists in identification of DR'Br', though not in the presence of DR1. We describe an alternative or complementary method for identification of DR'Br' using two oligonucleotide probes which target HLA-DRB1 gene HV3 regions.

Extended HLA-DQw2 haplotypes: molecular analysis.

Abstract: The HLA-DQw2 specificity, homogeneous in serology, is strongly associated to two HLA-DR specificities: DR3 and DR7. These alleles are found mainly on DQw2 bearing extended haplotypes with strong linkage disequilibrium. We describe, with BamHI, HindIII and RsaI, two restriction fragments length polymorphisms (RFLP) for the A gene of DQw2. These two subtypes correlated with the DR3 and DR7 specificities. Interestingly, by non-equilibrium pH gradient electrophoresis (NEPHGE), two DQ alpha chains were also found, respectively correlated with the same DR specificities. In addition, HincII polymorphism allowed us to distinguish several patterns of B genes for (DR7) DQw2 haplotypes but without any detectable association with another HLA marker. However, only one DQ beta chain was found by NEPHGE in the (DR7) DQw2 haplotype. Furthermore, HincII discriminated the B genes of the two extended haplotypes: (B8, DR3) DQw2 and (B18, DR3) DQw2. The same result was found by NEPHGE: two DQ beta chains were described, corresponding to the same extended haplotypes. The use of exon-specific DQB probes showed that the genomic polymorphism in DQw2 haplotypes is located, at least, at the 3' end of the gene. These data add new characteristics to the different DQw2 extended haplotypes.

EXPRESSION OF A MHC NON‐CLASSICAL CLASS I GENE, 04, IS SIMILAR TO A CLASSICAL CLASS I GENE, Dp

Abstract: SUMMARY We have investigated the effects of the cell cycle on expression of Q4 mRNA. Q4, the gene encoding the Qb-1 antigen, is transcribed in a wide variety of tissues, unlike many other non-classical class I genes. We have compared the pattern of Q4 transcription in the cell cycle to classical class I, β-2-microglobulin and actin. We found that the pattern of Q4 RNA levels resembles that of the classical class I genes, consistent with the similarity of the 5′sequences of Q4 and K/D. Thus, Q4 mRNA accumulates during the cell cycle along with the total RNA, but does not show specific transcriptional enhancement. This is consistent with a function for Q4 similar to the classical K/D gene products.

INVESTIGATION OF MINK MHC (MhcMuvi) CLASS I MOLECULES BY ISOELECTRIC FOCUSING (IEF)

Abstract: SUMMARY Mink (Mustela vision) class I leucocyte antigens, here abbreviated MhcMuvi according to the proposal given by Klein et al. (1990), were characterized by isoelectric focusing (IEF), using Triton X-114-extracted and neuraminidase-treated membrane proteins from spleen cells, followed by immunoblotting and development with a murine monoclonal antibody raised against denatured major histocompatibility complex (MHC) class I antigens. Muvi class I antigens were investigated in a group of 97 Danish mink (including five families), of six types (Standard, Wild, Pastel, Pearl, Violet and Sapphire), and a group of 110 French ‘Wild’ mink. For each mink type maximally six protein bands in IEF were identified. Standard, Pastel and Sapphire mink only exhibited from two to four bands. This to us indicated restricted polymorphism of the Muvi class I antigens for these mink. The restriction patterns varied from farm to farm. In the family material the Muvi antigens were found to segregate as expected. The French Wild mink were all naturally infected with Aleutian disease virus (ADV), a parvovirus, and their disease status classified as progressive or non-progressive. There were approximately 50% in each group. The mink were grouped into one of five class I Muvi profiles. When the profiles were compared to progressive versus non-progressive disease status, we found that mink with Muvi profile 4 and 5 almost exclusively (13 out of 15) were classified as having progressive Aleutian disease.

THE MOUSE FACTOR H ALLOTYPES WITH MULTIPLE AMINO ACID REPLACEMENT, H.l AND H.2 SHOW INDISTINGUISHABLE CO‐FACTOR ACTIVITY FOR FACTOR I

Abstract: The authors report the functional analysis of the purified mouse factor H allotypes H.1 and H.2, which were clearly distinguished from each other by an immunodiffusion test. Both allotypes acted as a co-factor for factor I in cleaving mouse C3b and we found no significant difference between their activities. The results strongly suggest that the function of mouse factor H for the co-factor activity has been well conserved between two allotypes.

Susceptibility to murine hepatitis virus (type 3)-induced paralysis is influenced by class I genes of the MHC.

Abstract: Using H-2 recombinant congenic strains of mice, genetic analysis of resistance to murine hepatitis virus type 3 (MHV3)-induced paralysis was performed. It appeared that both H-2K and H-2D, two class I gene regions of the mouse major histocompatibility complex (MHC), can play independent significant roles in the establishment of such resistance.

Imbalance of MHC class I expression in 3LL tumour cells.

Abstract: SUMMARY Cell lines and a clone established from the C57BL/6 (H-2b) Lewis lung (3LL) tumour were previously characterized with respect to tumour growth and metastatic spread in vivo, and to the expression of a 3LL tumour-specific antigen (3LL TA) using a monoclonal antibody raised in syngeneic mice immunized with 3LL cells. No correlation was observed between the presence of 3LL TA and the prevention of metastatic spread which suggests that the immune recognition of this tumour antigen requires the presence of a self H-2 molecule absent from these tumour cells. Indeed, radioimmunoassay (RIA) and cytofluorometric analysis using specific monoclonal antibodies have shown that the H-2Kb molecule was not expressed at the cell surface of all 3LL cell lines and clones, while the H-2Db molecule was present at normal levels. This defect, which was not the consequence of a lack Of (32m expression, was accompanied by an absence or a marked reduction of the H-2K mRNA level (which has been reversed in the M4 cell line by in vitro gamma interferon treatment), while the H-2D class I gene was normally transcribed. Another defective transcription was also observed for a gene in the Tla region (gene 37). This low‘37’phenotype was corrected by in vitro treatment of the M4 cell line with gamma interferon, which indicates that this class I gene of the Qa/Tla region has an interferon response sequence in the promoter.

Expression of rabbit Ig allotypes in litters of mothers immunized against a paternal allotype.

Abstract: SUMMARY Sera of successive littermates of mothers producing anti-allotype antibodies (Ab) were analysed for altered a locus or b locus allotype expression. We measured the allotype concentration in sera of 66 individuals (17 litters) of seven mothers producing anti-al Ab, and 63 individuals (15 litters) of seven mothers producing anti-b4 Ab, in an enzyme-linked immunosorbent assay (ELISA). We confirmed that the ability to induce allotype suppression in utero increases with the number of antigen boosts applied to the mother, even though the Ab titre in the maternal serum may be decreased. All individuals of a litter expressed the allotype in about equal concentration. This contrasts the results we obtained when newborn rabbits were injected with anti-allotype antiserum. Injection of the same amount of anti-allotype antiserum into nine offspring of two mothers caused allotype suppression in only five individuals, showing no effect in the others. No suppression was observed when IgG-depleted antiserum was injected into newborn rabbits. As expected, maternal antibodies to a paternal allotype do not affect the Mendelian distribution of the progeny phenotypes.

Analysis of HLA specificity of human monoclonal antibodies by cytofluorimetry and cell ELISA.

Abstract: SUMMARY In this study we report on the characterization of cytotoxic human monoclonal antibodies (HmAb) detecting polymorphic HLA class II specificities using cytofluorimetric analysis in combination with micro cell ELISA. In both techniques, five anti-HLA HmAb were tested against HLA-transfected murine L cells as target cells and the bound antibody was detected, either by cytofluorimetry or by cell ELISA reader, after addition of fluoresceinated or peroxidase-conjugated anti-human IgG+IgM antibodies, respectively. The results demonstrate that HmAb directed against HLA-DR, -DQ and -DP molecules can be efficiently discriminated by cytofluorimetry and cell ELISA, which appear to be highly sensitive and perfectly comparable to the standard cytotoxicity assay.

H-2 influence on the production of real but not of apparent H-2-specific antibodies induced by the association of syngeneic class I heavy chains with bovine β2-microglobulin

Abstract: Immunogenic properties of class I molecules resulting from the association of mouse class I heavy chains with a xenogeneic beta 2-microglobulin (beta 2-m) were investigated by studying the antibody response of mice of injections to syngeneic Con A lymphoblasts, induced in conditions allowing the replacement of endogenous beta 2-m by exogenously added bovine beta 2-m provided by fetal calf serum (FCS-Con A blasts). Lymphocytotoxic antibodies were regularly produced and according to their specificities they could be divided into two types: antibodies whose reactivity was (1) dependent on and (2) independent of the presence of bovine beta 2-m on target cells. Although both types displayed an H-2 dependent polymorphic reaction pattern, only antibodies recognizing class I molecules without bovine beta 2-m can be considered as real H-2-specific antibodies. The others are only apparent H-2-specific antibodies: their polymorphic reaction pattern is dependent both on the presence of bovine beta 2-m on the surface of target cells and on their H-2 haplotype. A comparison of the antibody response of mice with various H-2 haplotypes to injections of syngeneic FCS-Con A blasts showed no significant difference in the induction of bovine beta 2-m-dependent antibodies (apparent H-2-specific) among the mice from all strains tested (H-2b, H-2p, H-2q, and H-2s). Unexpectedly, for most strains more than 60% of the immunized mice produced also beta 2-m-independent antibodies (real H-2-specific), with the exception of H-2q mice, in which only 30% of sera were positive. The real H-2-specific antibody response is of two types: some mice (H-2p and H-2s) produced antibodies only reactive with allogeneic target cells whereas others (H-2b and H-2q) produced in addition antibodies that were reactive with syngeneic cells. Thus H-2 appears to play an important role in the induction and specificity of the lymphocytotoxic H-2-specific antibodies induced upon immunization with cells expressing syngeneic class I heavy chains associated with bovine beta 2-m.