Activity and phenotype of natural killer cells in peptide transporter (TAP)-deficient patients (type I bare lymphocyte syndrome).

In this paper we describe the function and phenotype of natural killer (NK) lymphocytes from HLA class I-deficient patients. These cells are, as has been previously reported, unable to lyse HLA class I- K562 cells, but are able to perform antibody-dependent cellular cytotoxicity (ADCC), although with lower efficiency as compared to NK cells from normal individuals. Transporter associated to antigen processing (TAP)- NK cells proliferate when cultured in the presence of lymphoblastoid B cells (B-LCs) and interleukin 2 and develop a spectrum of cytotoxicity similar to that of activated normal NK cells. Importantly, activation of the TAP- NK cells induces strong cytotoxicity to autologous B-LCs. Analysis of the phenotype of circulating TAP- NK lymphocytes showed them to display a normal diverse repertoire of HLA class I-specific NK receptors. These receptors were expressed at normal levels, apart from the CD94-NKG2A complex, which appeared to be overexpressed. This latter finding could reflect an adaptation to the low expression of HLA class I molecules. Finally, functional analyses indicated that the inhibitory receptors in TAP- individuals can transduce inhibitory signals. Our results suggest that in vivo, the NK cells of TAP- patients could participate in immune defense, at least through ADCC, but upon activation, may be involved in autoimmune processes.

HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1.

The transporter associated with antigen processing (TAP), which is composed of two subunits (TAP1 and TAP2) that have different biochemical and functional properties, plays a key role in peptide loading and the cell surface expression of HLA class I molecules. Three cases of HLA class I deficiency have previously been shown to result from the absence of a functional TAP2 subunit. In the present study, we analyzed two cases displaying not only the typical lung syndrome of HLA class I deficiency but also skin lesions, and found these patients to be TAP1-deficient. This defect leads to unstable HLA class I molecules and their retention in the endoplasmic reticulum. However, the absence of TAP1 is compatible with life and does not seem to result in higher susceptibility to viral infections than TAP2 deficiency. This work also reveals that vasculitis is often observed in HLA class I-deficient patients.

Limiting dilution analysis of human IL-2 producing T-cell precursors. Determination of hierarchical alloantigenic potential of human leukocyte antigens and evaluation of alloimune responses to single HLA-DP molecules.

Twenty nine healthy unrelated individuals were carefully selected and divided into three groups according to their HLA (Human Leukocyte Antigens) phenotypes. A sensitive and reproducible limiting dilution analysis (LDA) based bioassay using CTLL-20 cells for detection of human IL-2 was set up and used to assess the hierarchical impact of highly polymorphic HLA molecules on individual's alloreactivity. Our main interest was to evaluate the role of HLA-DP molecules in this process. By calculating frequencies of IL-2 producing helper T cell precursors (HTLp) and amounts of IL-2 produced in each experiment, we were able to confirm that HLA-DR molecules are the most potent alloantigens. In 29 different combinations where a single HLA-DP mismatch between stimulating and responding cells was evaluated, some were reasonably tolerant, while the other ones evoked moderate to relatively strong alloimune responses. Finally, two groups with statistically significant difference in alloimune responses to stimulating HLA-DP molecules carrying D,E,A,V or G,G,P,M amino acid sequences at positions 84,85,86 and 87 in the sixth variable region F of the molecule could be formed, according to HTLp frequencies and amounts of IL-2 detected. Data presented are of great importance for the selection of unrelated as well as related bone marrow donors for haematological patients.

Heterogeneity in the structural basis of the human complement C4A null allele (C4A Q0) as revealed by HindIII restriction fragment length polymorphism analysis.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes. C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes, 21-OHA and 21-OHB, and is also remarkable in the high frequency of the 'null' alleles, C4A Q0 and C4B Q0. The molecular basis for the C4A Q0 allele was studied in 26 families through restriction fragment length polymorphism (RFLP) analysis with C4 and 21-OH cDNA probes after digestion of the DNA with the endonuclease HindIII. The individuals expressing the extended haplotype HLA-A1 (of A2) Cw7 B8 C2C BfS C4AQ0B1 DR3 have a large deletion taking off the C4A and 21-OHA genes.

Lymphocytotoxic and monocytotoxic antibodies in the serum and cerebrospinal fluid of multiple sclerosis patients.

Serum cold cytotoxic antibodies (CA), detected at 15 degrees C using a microcytotoxicity technique, were present in 12 of 21 multiple sclerosis (MS) patients, weak or absent in 6 neurological patients without MS and present but weak in 5 out of 32 healthy controls. In MS, these cold CA were directed against 3 distinct cellular populations: total lymphocytes, B lymphocytes and monocytes; certain antibody tests were positive at 37 degrees C; no correlation between CA and clinical disease was observed. Cerebrospinal fluid (CSF) antibody levels were high in both MS and non-MS patients and at 37 degrees C produced lysis of monocytes in the absence of complement. These antibodies may be normal CSF constituents. Our results suggest that there may be 3 different antibodies and that they may play a role in immunomodulation, especially in MS.

DP epitope mapping by using T-cell clones.

To determine whether a correlation exists between the genomic HLA class II DP DNA polymorphism and cell surface expression and to detect the DP epitopes responsible for alloreactivity, anti-DP T-cell clones were generated against new PLT blank RFLP DPa and DPb-defined specificities. The clones were tested on the 10th IHWS B-LCLs and on local panel cells. Oligotyping of the tested cells made it possible to (a) correlate the DPa specificity with the DPB1*0402 specificity and (b) split DPb into DPB1*1001 and DPB1*1401. By comparing DNA sequences of the second exon to panel reactivity, the epitopes responsible for DPB1*1001 and 1401 were defined and attributed to beta-chain residues contributing to peptide selection inside the HLA groove. However, DNA sequences could not explain anti-DPa allospecificity, indicating that another structure not yet definable may be involved.

Complexity of the HLA-DP region: RFLP analysis versus PLT typing and oligotyping.

A total of 84 individuals were DP typed in parallel with the restriction fragment length polymorphism (RFLP) analysis and the primed lymphocyte test (PLT). Whereas 80% of the cases gave concordant results, the other 20% showed discrepancies for one of the two alleles carried by the typed individuals. Oligotyping, PCR-RFLP and sequencing confirmed the results found by PLT. The 20% discordant results obtained with RFLP led us to conclude that RFLP typing cannot replace PLT typing. From a more general point of view, the RFLP analysis revealed the DP region to be more complex than expected since for each given PLT DP defined specificity, more than one RFLP DP haplotype could be determined. These were possibly induced by crossing-overs or gene conversion events.

New human MHC class I antigens segregating with HLA-A antigens detected on some lymphocyte subpopulations.

Recent genetic studies of the murine chromosome 17 have demonstrated that many genes encode class I antigens, most of which are still not detected serologically; most of these genes belong to the Tla region. Five human alloantisera were selected from 383 female sera and were further studied using a panel of peripheral blood lymphocytes (PBL), B lymphocytes (BL), and PHA activated lymphocytes (PHA-L) from the same blood donors. After intensive platelet absorption, the five sera still reacted positively by a complement-dependent cytotoxicity technique with PHA-L, but negatively with PBL and BL. The antigens detected by these antibodies segregated with an HLA-A allele and were assumed to belong to the class I antigen series as they could be blocked by a turkey anti-beta 2 microglobulin serum. They were found on some lymphocyte populations: PHA-L, common acute lymphoblastic leukemia (cALL) cells, and (preliminary results) a small subpopulation of PBL cells (mostly NK cells), but were not found on chronic lymphocytic leukemia (CLL) cells, T and early T acute lymphoblastic as well as myeloblastic leukemia cells. Kinetic studies showed that several hours of culture with PHA were necessary for the antigen to be expressed. These results show that the antigens described do not belong to the classic HLA antigen series but could be considered to belong to the human Qa-like antigens or to be the human counterpart to the second murine H-2K locus antigens.

Molecular basis of complete C4 deficiency. A study of three patients.

The highly polymorphic fourth component of human complement (C4) is usually encoded by two genes, C4A and C4B, adjacent to the 21-hydroxylase (21-OH) genes and is also remarkable by the high frequency of the null alleles, C4A*Q0 and C4B*Q0. Complete C4 deficiency is exceptional because this condition appears only in homozygotes for the very rare double-null haplotype C4AQ0,BQ0. This condition in most cases gives rise to systemic lupus erythematosus and an increased susceptibility to infections. The molecular basis for complete C4 deficiency has not yet been established. Therefore we studied the DNA of three previously described C4 deficient patients belonging to unrelated families by restriction fragment length polymorphism analysis using C4 and 21-OH probes. These studies revealed a deletion of the C4B and 21-OHA genes in two patients and no deletion at all in the third patient. Therefore, complete C4 deficiency as a result of homozygosity for the C4AQ0, BQ0 haplotype is not a consequence of a deletion of the C4 genes. The molecular basis of this genetic abnormality is certainly very complex and may vary also from one case to another.

A new DR14 allele (DRB1*1411) containing a short DR11 sequence and its haplotypic association.

In the present work, we describe a new DR14 allele. Sequencing of its second DRB1 exon showed it to be DRB1*1404 from codon numbers 9 to 56 and 61 to 86, and DRB1*11 from codons 57 to 60 inclusive.

A T-cell clone recognizing an MLC stimulating epitope located on the DRw11 beta 1 chain.

An alloreactive proliferative T-cell clone, 6065 WS, was obtained in an intrafamilial cell combination where the stimulator was a homozygous DRw11, DRw52, DQw3 son and the responder his haploidentical mother. Proliferation assays on eight local DRw11 families, 75 homozygous B-cell lines (Tenth Histocompatibility Workshop panel) and blocking assays with monoclonal antibodies showed that clone 6065 WS recognizes an epitope on the DRw11 beta 1 chain. Comparison of the reactivity of clone 6065 WS with cells expressing the three known DRw11 beta 1 amino acid sequences identified two unique amino acids at positions 71 and 86 which contribute to determining the specific recognition by the T-cell clone 6065 WS. Our data suggest that one or both of these amino acids can either be directly involved in the recognition by the T-cell receptor or responsible for critical conformation of the determinants on the DR molecule. Alternatively, they could affect recognition of a self peptide bound to the major histocompatibility complex class II molecule.

CD1 expression is not affected by human peptide transporter deficiency.

Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides.

Reevaluation of the relative risk for susceptibility to celiac disease of HLA-DRB1, -DQA1, -DQB1, -DPB1, and -TAP2 alleles in a French population.

In a population of 46 children with CD recruited in the Paris area of France, an excess of DRB1*03 and DRB1*07 alleles and of DR3/DR7, DR3/DR3 and DR11(or 12)/DR7 phenotypes was found (RRs of 6.3, 9.3, 24.6, 15, and 15.1, respectively), which is reminiscent of the markers of susceptibility observed in southern rather than in northern European celiac patients. More importantly, the highest association with CD was not found in individuals expressing the DQA1*0501-DQB1*0201 heterodimer in single dosage (RR = 24.9) or in homozygous state, but in people co-expressing one copy of DQA1*0501-DQB1*0201 on one haplotype and a second copy of DQB1*0201 on the second haplotype (RR = 35.7). This suggests that in our population either DQB1*0201 or a gene closely linked to DQB1*0201 influences the susceptibility to CD conferred by the DQA1*0501-DQB1*0201 heterodimer. Significant positive or negative RRs conferred by some TAP2 or DPB1 alleles were found. However, they were moderate compared to the RR conferred by the expression of a second copy of DQB1*0201. Moreover, they were no longer significant when patients were compared with HLA-DR matched controls. This suggests that associations of CD with TAP2 and DPB1 alleles are secondary to linkage disequilibria and argues against the contribution of these alleles in resistance and/or susceptibility to CD. Thus the "raison d'être" of a "DQB1*0201 second haplotype effect" in susceptibility to CD remains to be elucidated.

Membrane markers, karyotypic abnormalities, ultrastructure and functional properties of lymphocytes in a case of 'D-cell' chronic lymphatic leukemia.

D cells are lymphocytes bearing both receptors for the third complement component and the ability to form spontaneous rosettes with SRBC. We report the case of a patient with a D-cell chronic lymphatic leukemia who presented a long evolution without treatment and whose leukemic cell characteristics have been extensively studied. Cytogenetic analysis showed numerous karyotypic abnormalities among leukemic cells; all metaphases were hypodiploid and arranged in four different clones; seven marker chromosomes were present. The cells were found to bear human T-cell specific antigen, the T helper/inducer phenotype, HLA-A and HLA-B determinants, but no HLA-DR antigens. They displayed a high proliferative response to PHA and Con A, no response to PWM stimulation, and possibly the capacity of allogeneic stimulation in the mixed lymphocyte culture system. Assays for cell-mediated cytotoxicity in the CML system, and for K and NK activities were negative.

The dendritic cell lineage: a ubiquitous antigen-presenting organization.

Dendritic cells are specialized antigen-presenting cells with two unique characteristics: the greatest stimulatory potential and the ability to stimulate naive T-lymphocytes. They originate from the bone marrow and reach their destination via hematogenous or lymphatic migration. Their phenotype is characterized by a high expression of major histocompatibility complex class II molecules and a high expression of adhesion molecules (CD25, CD54, CD58, CD72, and CD80). Pulmonary dendritic cells may be investigated by histologic examination, phenotype analysis, and function studies in a mixed lymphocyte reaction. Their isolation requires enzymatic digestion of lung tissue and subsequent steps of cell separation. The complexity of these manipulations makes it difficult to obtain large numbers of viable cells. A close anatomic relationship with alveolar macrophages underlines a functional interconnection: macrophages down-regulate the antigen-presenting function through release of tumor necrosis factor alpha. Dendritic cells most probably play a major role in lung diseases such as histiocytosis, primary and secondary cancers, and both acute and chronic lung graft rejection. Identification of the precise functional pathways might lead to therapeutic use of modulation of dendritic cell function.

Multiple sclerosis, sleep latencies and HLA antigens.

The role of HLA antigens, and HLA-DR2 in particular, in the determination of mean sleep onset latencies (MSOLs) in multiple sclerosis (MS) was studied. It has been suggested that this antigen may play a part in the reduction of MSOLs, since nearly 100% of patients suffering from narcolepsy are DR2-positive. A multiple sleep latency test was performed in 37 patients suffering from MS without spontaneous complaints of sleep disturbances and who were typed for HLA-A, B, C, DR and DQ. The MSOL was reduced in a total of 21 patients, in only 7 of 15 DR2-positive patients and in 12 of 21 DQw1-positive patients. However, it was reduced in 13 of 16 B8- or B14-positive patients. In contrast with this, in the absence of an early sleep onset (MSOL greater than 30 min), no HLA antigens were found to be over-represented when considered individually; only those patients positive for a group of cross-reacting HLA antigens (B5, B15, B18, B21 or B35) had an MSOL greater than 30 min. These results suggest that the genes which code for the DR2 or DQw1 antigens, which are present in nearly 100% of narcoleptics, are not solely responsible for the appearance of an early sleep onset in MS.

HLA antigens in multiple sclerosis in Alsace.

The distribution of HLA antigens A, B, C and DR was studied in 69 native Alsatian multiple sclerosis (MS) patients. Antigen DR2 was high in MS compared to healthy control subjects, as is known. Given antigens were more frequently, and, above all, more closely linked with certain clinical and organic parameters. Antigens A3, B7 and B40 were preferentially associated with progressive forms, and A32 with remitting forms. B7 may be predictive of the prognosis, as it was correlated with disease severity. B7 and DR2 were more frequent in MS patients presenting intrathecal immunoglobulin synthesis. Sex appeared to be a fundamental factor in the clinical expression of MS, interacting closely with the HLA system. These findings confirm the multi-factor etiology of MS. Several MS susceptibility genes may exist near the HLA complex, and their expression may modulate the clinical and organic signs of MS. Studies of this sort should be carried out on ethnically and geographically homogeneous populations.

T-cell repertoire of normal, rejected, and pathological corneas: phenotype and function.

The specific immune mechanisms of corneal graft rejection are not completely understood. Recently, the technique of growing T-cell lines from rejected allografts using recombinant IL-2 has enabled the cells involved in allograft rejection to be recognized. In the present study, this method was applied for the extraction and propagation of T lymphocytes from rejected, normal, and diseased corneas. T-cell lines could successfully be grown from rejected and normal corneas, but not from corneas with keratoconus or pseudophakic bullous keratopathy. The phenotypic repertoire of the grown cells was studied by FACS scan analysis. Rejected corneas were invaded by a mixture of activated CD4+ and CD8+ T-cell lines, with one population being predominant. In normal corneas only activated CD8+ cells with cytotoxic function were cultured. No cells were obtained from diseased corneas. The in vitro function of cell lines was assessed by primed lymphocyte testing. The present study shows that the technique of propagating invading T-cell lines from organ grafts can be applied to human corneas, offering a new approach to understanding the immunological mechanisms occurring inside this immune tissue.

Frequencies of glyoxalase I phenotypes as biological markers in chronic alcoholism.

Biological markers for alcoholism would be a valuable tool for early diagnosis. We have studied the phenotype frequencies of genetically determined erythrocyte enzymes in 397 alcoholics, including two populations with liver disease: steatosis (n = 86) and cirrhosis (n = 128) and a population of alcoholics without apparent liver disease (n = 183) compared to a well selected control population (n = 177). Only for Glyoxalase I (GLO) phenotypes (1,2 and 2-1) were significant differences found between the male controls and the male alcoholics. In the total male alcoholic population the frequency of phenotype 1 was significantly increased (23.2% vs. 11%, p less than 0.02), and the frequency of phenotype 2 was significantly decreased (32.3% vs. 46.3 p less than 0.02) compared to the male control population. For normal women the frequency of phenotype 1 and 2 was significantly different from normal men. (1: +177% p less than 0.001, 2: -45% p less than 0.01), but no significant differences were found between alcoholic and normal women. Our results suggest that in male subjects Glyoxalase I phenotype 1 may provide a marker for predisposition to alcoholism.

[HLA genotyping: indications and limits]. / Génotypage HLA: indications et limites.

The polymorphism of the class I (HLA-A, B, C) and class II (HLA-DR, DQ, DP) antigens was for a long time investigated using serological methods. Today molecular biology methods are available to define the numerous HLA alleles by genotyping [(82 HLA-A alleles, 174 HLA-B, 38 HLA-C, 166 HLA-DRB1, 27 HLA-DQB1, 71 HLA-DPB1) (nomenclature 1996)]. Many different molecular biology methods can be used to define these alleles (PCR- RFLP, PCR-SSOP, PCR-SSP, PCR-SBT), the choice of method depends on the number of genotypes achieved per day and the time required to obtain a result. The resolution degree of results can reach two levels: low resolution: provides results almost identical to those obtained by serological methods. Low resolution is sufficient to find HLA-identical siblings for bone marrow transplantation, to type organ donor-recipient pairs and for diagnosis in most HLA disease associations; high resolution: defines HLA allele subtypes. High resolution is essential to type bone marrow donor-recipient pairs when the donor is unrelated. Molecular biology methods will gradually replace serological methods in the future. The only restriction is that some alleles, defined at the genomic level, are not expressed at the cell surface and are thus not functional.