Molecular cloning of a T cell-specific adapter protein (TSAd) containing an Src homology (SH) 2 domain and putative SH3 and phosphotyrosine binding sites.

Adapter proteins link catalytic signaling proteins to cell surface receptors or downstream effector proteins. In this paper, we present the cDNA sequence F2771, isolated from an activated CD8+ T cell cDNA library. The F2771 cDNA encodes a novel putative adapter protein. The predicted amino acid sequence includes an SH2 domain as well as putative SH3 and phosphotyrosine binding interaction motifs, but lacks any known catalytic domains. The expression of the gene is limited to tissues of the immune system and, in particular, activated T cells. The protein expressed by F2771 cDNA in transfected COS cells is localized in the cytoplasm. A polyclonal antiserum raised against an F2771-encoded peptide reacts with a tyrosine-phosphorylated 52-kDa protein expressed in phytohemagglutinin-stimulated peripheral blood mononuclear cells. The gene is localized to chromosome 1q21, a region often found to be aberrant in lymphomas. The T cell-specific expression and the rapid induction of mRNA expression upon receptor binding, as well as the lack of catalytic domains in the presence of protein interaction domains, indicate that the F2771 gene encodes a novel T cell-specific adapter protein (TSAd) involved in the control of T cell activation.

HLA matching of unrelated bone marrow transplant pairs: direct sequencing of in vitro amplified HLA-DRB1 and -DQB1 genes using magnetic beads as solid support.

Sequencing of HLA genes can offer complete information on the HLA class II genes relevant for the outcome of bone marrow transplantation (BMT). Genomic HLA matching of unrelated BMT patient/donor pairs is often based on PCR-SSO typing of HLA class II alleles. Typing a small number of samples by this approach is both expensive and time-consuming, due to the large number of SSO probes required to perform a complete class II typing. Moreover, only polymorphisms explicitly tested for will be found. We now provide the first report of the use of direct sequencing of HLA-DRB1 and -DQB1 genes, using PCR-amplified genomic DNA attached to magnetic beads, for clinical routine HLA matching. Sequencing ladders obtained by this procedure are easily readable, the patterns can be interpreted in HLA homozygous as well as heterozygous individuals, and sequence differences or similarities between the BMT donor and recipient can be directly identified. This genomic typing method is informative, relatively fast and therefore well-suited for the small number of samples usually analyzed in matching of BMT pairs. Furthermore, this technique has the potential for automation.

T cell receptor usage by HLA-DQw8-specific T cell clones.

To investigate whether T cells recognizing the same HLA molecule may demonstrate homology in parts of their TCR, five different HLA-DQw8-specific T lymphocyte clones (TLC) were studied. The TCR alpha and -beta genes of four alloreactive, HLA-DQw8-specific and one antigen-specific TLC were sequenced. All TLC used different V alpha and V beta genes. However, four of the TLC shared a certain CDR1 beta motif and all five used either J beta 2.3 or -2.5. In addition, two used the same J alpha. The results indicate a possible preferential usage of certain TCR structures by T cells specific for DQw8.

Partial DNA sequences derived from two previously unknown HLA class I genes.

Genomic DNA of HLA class I gene segments was amplified by the polymerase chain reaction (PCR). The amplified 0.8 kb gene segment encompassed sequences corresponding to an exon 2 (alpha 1 domain), an intron 2 and an exon 3 (alpha 2 domain). The PCR product was cloned in M13 and sequenced. Two previously unknown sequences were found. One of them (DAN2) had an open reading frame and intact intron splice sites, but we did not find evidence for transcription. Best homology (87.5%) was found with HLA-BeWo C.1, a recently described HLA-C gene. The other sequence (DAN4) is derived from a pseudogene, because the putative 3' splice site of intron 2 was changed from AG to ATG and the sequence corresponding to exon 3 had a shift in reading frame resulting in a stop codon.

Distribution of HLA class II alleles among Norwegian Caucasians.

We report genomic HLA class II typing of 181 randomly selected Norwegian controls. Seventeen DRB1, 7 DQA1, 10 DQB1, 2 DPA1, and 16 DPB1 alleles were found in the tested population. HLA class II antigen and allele frequencies are given, as well as the distribution of DRB1, DQA1, DQB1 haplotypes. Linkage disequilibrium between some DPB1 alleles and DRB1 and/or DQB1 alleles are also reported.

Use of DNA amplification (PCR) and direct DNA sequencing in the characterization of C4 alleles.

A procedure for detailed characterization of individual C4 alleles has been developed. DNA containing the two polymorphic clusters of C4 was amplified in the polymerase chain reaction (PCR). Direct DNA sequencing of amplified DNA was then performed by a modification of previously described techniques. The results were confirmed by M13 sequencing. Single C4A3 and C4B1 allele sequences were in accordance with previous reports. An individual typed C4A3B1 revealed double bands in the autoradiogram in the positions corresponding to the polymorphic nucleotides. We did not find the reported thymine in position 3641 specific for the C4A4 allele in an individual typed C4A4B2.

T lymphocyte recognition of a celiac disease-associated cis- or trans-encoded HLA-DQ alpha/beta-heterodimer.

The HLA-associated susceptibility to develop celiac disease may to a large extent be attributed to the combination of particular DQA1 and DQB1 genes, i.e., the DQA1*0501 and DBQB1*0201 alleles, located either in cis position (on the DR3DQw2 haplotype) or in trans position (DR5DQw7/DR7DQw2 heterozygous individuals). We report three alloreactive T lymphocyte clones that recognize an HLA-DQ alpha/beta heterodimer both when the DQA1*0501 and DQB1*0201 alleles are located in cis or in trans position. Thus, the celiac disease associated DQA1 and DQB1 genes encode a functionally expressed DQ alpha/beta heterodimer.

Patients with multiple sclerosis carry DQB1 genes which encode shared polymorphic amino acid sequences.

Of 61 Norwegian multiple sclerosis patients tested, 59, i.e., 97%, were positive for at least one of the HLA specificities DR2, DR4, or DRw6. Typing with sequence-specific oligonucleotide probes revealed that the same 59 patients carried DR2-, DR4-, or DRw6-associated HLA-DQB1 genes which encode shared polymorphic amino acid sequences in the membrane-distal part of their HLA-DQ beta chains. This shared DQ beta polymorphism may explain previously reported DR associations and could thus be the primary HLA association in MS.

An increased risk of insulin-dependent diabetes mellitus (IDDM) among HLA-DR4,DQw8/DRw8,DQw4 heterozygotes.

Serological HLA typing of 92 insulin-dependent diabetes mellitus (IDDM) patients and 300 healthy controls was performed by the immunomagnetic typing technique. We found an increased risk of IDDM among DR4/w8 heterozygotes, similar to that seen for DR3/4 heterozygotes. The DQ alleles of these DR4/w8 patients were therefore established. When hybridized with a cDNA DQB probe, BamHI-digested DNA from eight out of the nine DR4/w8 patients revealed only one single 10.8-kb DQB1-specific fragment typical of DQw4 and DQw8. DNA from all DR4/w8 patients also hybridized to an oligonucleotide probe corresponding to amino acids (aa) 23-30 of the beta chain of DQw8. However, using the oligonucleotide probe, the staining intensity was found to be only half of that seen when DNA from DQw8 homozygotes was used instead, suggesting that the eight DR4/w8 patients had DQw8 in a single dose and carried the DQw4 allele at the DRw8 haplotype. Therefore, eight of nine DR4/w8 IDDM patients seemed to be DR4,DQw8/DRw8,DQw4, which, thus, may be associated with susceptibility to develop IDDM. One common explanation of IDDM susceptibility for DR4,DQw8/DRw8,DQw4 and DR3,DQw2/DR4,DQw8 heterozygotes may be that they share similar DQ alpha beta epitopes encoded by DQA and DQB genes in trans.

Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer.

Typing of DNA from 94 unrelated children with celiac disease (CD) with HLA-DQA1 and -DQB1 allele-specific oligonucleotide probes revealed that all but one (i.e., 98.9%) may share a particular combination of a DQA1 and a DQB1 gene. These genes are arranged in cis position on the DR3DQw2 haplotype and in trans position in DR5DQw7/DR7DQw2 heterozygous individuals. Thus, most CD patients may share the same cis- or trans-encoded HLA-DQ alpha/beta heterodimer.

HLA-DQw3.1 and DQw3.2 associated exon polymorphisms detected by oligonucleotide probes.

HLA class II polymorphisms have been analyzed on the genomic level by two oligonucleotide probes corresponding to the DNA sequence coding for the amino acids 23-30 in the first domain of the beta chain of DQw3.1 and DQw3.2. Specific hybridization to single endonuclease fragments of DNA from some HLA homozygous cells was detected. Here we report the distribution of these DNA exon sequences among different DQ alleles, and demonstrate that the probes may be used to type for DQw3.1 and DQw3.2 respectively.

Direct immunomagnetic quantification of lymphocyte subsets in blood.

A method is described where superparamagnetic polymer microspheres coated with monoclonal antibodies (MoAb) are used for the direct and fast quantification of the absolute number of cells of various lymphocyte subsets in blood. Blood samples were incubated with microspheres coated with a subset specific MoAb. Using a magnet the microsphere-rosetted cells were isolated and washed. Following lysis of the cell walls to detach the microspheres, the cell nuclei were stained with acridine orange and counted in a haemocytometer using an immunofluorescence microscope. With MoAb specific for CD2, CD4, CD8 and CD19, reproducible absolute counts of the corresponding lymphocyte subsets were obtained which correlated closely with those obtained by an indirect quantification method.

Interferon-gamma enhances expression of secretory component, the epithelial receptor for polymeric immunoglobulins.

Recombinant interferon-gamma (IFN-gamma) increased in a dose-dependent manner the intracellular pool, the membrane expression, and the shedding of secretory component (SC) in human colonic adenocarcinoma cell line (HT-29). A similar dose-response relationship was observed when we examined the binding of polymeric IgA to HT-29 cells treated with IFN-gamma, thus reflecting expression of functional SC. Because IFN-gamma is produced by T cells during immune responses, activated T cells may be able to promote the external transport of dimeric IgA and pentameric IgM and thereby enhance the efferent limb of the secretory immune system. This is, therefore, the first observation indicating how the secretory transport capacity may be adjusted to increased local immunoglobulin production.

Determination of the HLA-DR profile of an HLA class II negative carcinoma cell line by restriction fragment length polymorphism (RFLP) analysis.

The human colonic adenocarcinoma cell line HT-29 does not normally express HLA class II molecules. By restriction fragment length polymorphism (RFLP) analysis of DNA with a DR beta-probe, we analysed the genomic DR beta polymorphism of this cell-line, and compared it with the RFLP patterns seen in DNA from a reference panel of different DR homozygous and heterozygous cells. The HT-29 cell-line expressed DR beta fragments similar to the sum of the fragments expressed by DR4 and DR7 homozygous cells. The DR4 and DR7 haplotypes of HT-29 was further confirmed by comparing the RFLP patterns of four DR4/7 heterozygotes with that of HT-29. Furthermore, the HT-29 cell line expressed a Hind III 9.3 kb fragment previously found to be strongly associated to DRw53. Following treatment with gamma-interferon, the HT-29 cells could be induced to express class II molecules. Serological typing revealed the presence of the DR4, DR7 and DRw53 antigenic determinants.

Genomic HLA-DQ beta polymorphism associated with insulin-dependent diabetes mellitus. Analysis of possible functional significance.

Twelve insulin-dependent diabetes mellitus (IDDM) patients and healthy controls, who all carried the serologically defined DR3 and DR4 antigens, were compared with respect to other HLA polymorphisms. No significant differences between patients and controls were found by typing for HLA-Dw determinants by homozygous cell typing, nor by studies of their genomic DR beta polymorphism using different restriction enzymes. In contrast, certain DR beta polymorphism using different restriction enzymes. In contrast, certain DR4-associated genomic DQ beta fragments had a significantly different distribution among the IDDM patients than among the controls. Furthermore, when the distribution of all DQ beta-specific fragments which demonstrated polymorphism in our material was taken into account, nine of the 12 DR3, 4 IDDM patients demonstrated a similar DQ beta polymorphism compared with only two out of the 12 DR3, 4 controls (P = 0.006; corrected P = 0.037). Cells from patients and controls who demonstrated this IDDM-associated DQ beta polymorphism stimulated each other significantly less in reciprocal MLC tests, compared with the responses seen when their cells were confronted with cells from the DR3, 4 individuals with other genomic DQ beta polymorphisms.

Induction of various HLA class II molecules in a human colonic adenocarcinoma cell line.

The colonic carcinoma cell line HT-29 had no constitutive expression of HLA class II molecules. Gamma interferon (IFN-gamma) induced expression of HLA class II molecules in a dose-dependent manner with 100 U/ml as an optimal dose. The expression of HLA-DR, HLA-DP, and HLA-DQ molecules seemed to follow different kinetics. While DR and DP molecules were maximally induced after 2 days, DQ molecules appeared later with maximum percentage positive cells after 8 days. Treatment with a prostaglandin synthesis inhibitor (indomethacin) neither induced class II expression nor altered the dose-response curve for IFN-gamma; this indicated that possible endogenous production of prostaglandins in this cell line did not interfere with its class II expression. The lectins phytohaemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and wheat germ agglutinin (WGA) did not induce class II expression.

Inhibition of protein A24 lyase by nitrosoureas.

The protein A24 content of Ehrlich ascites tumor cells increased several-fold following treatment of cell cultures with nitrosoureas, but did not increase when other alkylating agents not containing carbamoyl moieties were tested. The same nitrosoureas and, in addition, 2-chloroethyl isocyanate inhibited an A24 lyase-containing cytoplasmic extract in cleaving protein A24 into histone H2A and ubiquitin. It appears that carbamoylation of A24 lyase by nitrosoureas inhibits the enzyme and is responsible for the measured increases in cellular protein A24 content due to reduced turnover of this protein.