Generation of stable monocyte-derived dendritic cells in the presence of high concentrations of homologous or autologous serum: influence of extra-cellular pH.

Recent studies have highlighted the high degree of differentiation of monocytes. Indeed, dendritic cells (DC) can be generated from monocytes, in the presence of appropriate cytokines. However, human serum is usually avoid in such cultures. Here, we report that human serum does not inhibit generation of mature DC from blood monocytes, but rather that extra-cellular pH may play an important role in the regulation of monocyte differentiation. Indeed, monocytes cultured at pH 7.4 in the presence of high concentrations of human serum developed efficiently into mature DC, as opposed with monocytes cultured at pH 7. These pH 7.4 cultured DC presented features characteristic of mature DC, at the phenotypical, functional and morphological levels. In addition, these DC were stable, with respect to their sustained expression of CD83 and CD86, upon withdrawal of cytokines. Finally, when autologous plasma was used instead of homologous serum, differentiation of monocytes into mature DC was efficient, as well. Thus, altogether, our data show the importance of extra-cellular pH on differentiation of monocyte-derived DC in the presence of human serum, which should be maintained at plasma levels.

Sequence of a new HLA-DR4 allele with an unusual residue at position 88 that does not seem to affect T-cell allo recognition.

New HLA alleles can be identified by unorthodox patterns observed during low-resolution typing performed with sequence specific oligonucleotide probes (SSOP). One of the best examples is locus DRB1, where allelic subtypes are characterized by a combination of a limited number of residues located in three hypervariable regions of exon 2. HLA-DR oligotyping analysis of a female caucasoid bone marrow donor led to the identification of an individual that typed as DRB1*11, DRB3*02, DRB4*01, DQB1*0301-0302. This donor was, however, typed by serology as DR11 DR4, DR52, DR53, DQ7 DQ8. PCR-SSP typing for DR4 subtype revealed an amplification pattern typical for DRB1*0404. After sequencing the entire exon 2, a new DRB1 allele was identified: DRB1*04var that is identical to DRB1*0404, except for one nucleotide at codon 88 resulting in a Ser-->Arg exchange. This mutation had prevented amplification with the DR generic primers. Cellular typing by three HTCs-DRB1*0404/DW14 from the 9th Workshop showed that this DRB1*04var typed exactly like a DW14 cell. This suggests that residue 88 does not affect T cell recognition.

Implication of HLA-DR residues at positions 67, 71, and 86 in interaction between HLA-DR11 and peptide HA306-320.

To get further insight into the role of three polymorphic DR residues located in one alpha-helix of the HLA-DR binding groove, we studied how natural substitutions at positions 67, 71, and 86 on DR11 molecules influence MHC binding and/or T cell recognition of peptide HA306-320 and of monosubstituted peptide analogues. Our results show that: 1) Reactivities of all HA306-320-specific T cell clones tested are decreased by DR substitution at position 86 and can even be lowered by additional substitutions at position 71, and at positions 71 plus 67, indicating that these three residues are functionally important. 2) The functional effects of substitutions at positions 67, 71, and/or 86 cannot be explained by a decreased affinity of HA306-320 for the substituted DR11 molecules, as determined in binding assays. 3) More likely, they are explained by modifications of the conformation, orientation, or location of the peptide once bound in the HLA groove, because each individual DR substitution at positions 86, 71, and 67 differentially affects the binding ability of the same panel of 50 monosubstituted analogues. 4) This interpretation is reinforced by the identification of a small set of monosubstituted analogues that can compensate the functional effects of DR substitutions at positions 86, 86 plus 71, or 86 plus 71 plus 67, and thus restore T cell reactivities. All together these results strongly suggest that residues 67, 71, and 86 play a key role in interactions with HA306-320, probably by modifying the way the peptide is bound within the binding groove of HLA-DR11. Using the same DR11.1-restricted clones, we identified putative T cell and DR contact residues of HA306-320 by comparing DR binding and T cell-activating capacity of the peptide analogues. This analysis suggests that: 1) Residues 310, 311, 312, 313, and 316 are putative TCR contacts. 2) Peptide HA306-320 anchors to DR11.1 molecules mainly via residue Y-309, possibly at the vicinity of DR residue 86, whereas peptide residues 315 and 317 constitute minor aggregotopes that would be at the vicinity of DR residues 71 and/or 67. 3) Finally, residues 308, 310, and 314 might also be on the MHC side of the DR-peptide-TCR complex.

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex.

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (BamHI, EcoRI, HindIII, PvuII and TaqI), and successively hybridized with the two radiolabelled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.

Functional dissection of the serological DR LYGUE and genotypic DRB1*1303 specificities using a tetanus toxin-specific T-cell clone.

The DRB1 sequence of the homozygous cell line HAG (DR13-DwHAG-DQ7) represents a new DRB allele assigned DRB1*1103, whereas its DRB3 sequence corresponds to the previously described DRB3*0101 (DR52a) allele. The DRB1*1303 gene product is undetectable by current sera used in routine serology typing. We report here direct evidence that the MHC molecule encoded by the DRB1*1303 gene is functional in antigen presentation and in T-cell restriction. We describe a T-cell clone specific for tetanus toxin whose restriction pattern strictly follows the DRB1*1303 allele, as defined by oligonucleotide typing. It also follows the serologic reactivity with the serum LYGUE and also the DwHAG MLC-defined specificity pattern, with one exception. The potential functional sites for the DRB1*1303 gene product involved in T-cell restriction were deduced from sequence comparisons between DRB1*1303 and closely related DRB1 alleles. The relevant as substitutions were located within close proximity to each other on the HLA class II structural model. Our results demonstrate that 1) DRB1*1303 is functional in antigen presentation and T-cell restriction 2) the functional region involved in antigen presentation and T-cell restriction by DRB1*1303 can be defined structurally.

Cytomegalovirus infection--an etiological factor for rejection? A prospective study in 242 renal transplant patients.

The study aimed at analyzing the role of CMV infection as a risk factor for rejection occurring after CMV infection because of the clinical consequences of the prevention of CMV infection that might lead to the decrease in rejection episodes. Two hundred forty-two consecutive renal transplant patients were prospectively checked for the occurrence of CMV infection. CMV infection was defined virologically by a positive viremia or/and a positive viruria or/and a seroconversion or/and a significant rise of the anti-CMV antibody titers. Viremia, viruria, and serology were performed weekly for the first month and then at day 90, day 180, and every 6 months, and moreover if clinical symptoms related to a viral infection occurred. Rejection episode was defined by a creatininemia rise of 25%, after cyclosporine nephrotoxicity and urological complications had been discarded, and by the response to the antirejection therapy, steroids, or OKT3 in case of steroid-resistant rejection. The outcome factor was rejection episode occurring from day 4 after the diagnosis of CMV infection. A patient undergoing "a rejection episode after CMV infection" could also be exposed to other potential confounding factors that can be considered as risk factors of rejection among our patients. Rejection occurring before CMV infection was the main factor because it was linked both to CMV infection itself and to "rejection after." Thus infected and noninfected patients were randomly paired off. To the noninfected patient of the pair was attributed the date of a fictitious CMV infection that was the date of the CMV infection of the infected member of the pair. Therefore, "rejection after" and "rejection before" were defined in infected and noninfected patients of the pair according to the time of onset of CMV infection of the infected member of the pair. The incidence of CMV infection was 65%, 157 of the 242 patients were infected, and 85 not infected. Thus 85 pairs of infected-noninfected patients were studied. The incidence of "rejection after" the diagnosis of CMV infection was significantly higher in the group of patients with CMV infection: 45% among infected (38/85) versus 10.60% among noninfected (9/85) (P < 0.0001). Among the 85 pairs, 48 pairs were concordant in which patient of the pair evinced the same outcome factor: 43 showed no rejection after, and 5 showed one.(ABSTRACT TRUNCATED AT 400 WORDS)

[Lymphoproliferative syndromes associated with Epstein-Barr virus in transplantation]. / Syndromes lymphoprolifératifs associés au virus d'Epstein-Barr en transplantation.

We report 23 cases of lymphoproliferative diseases which occurred among 2,100 patients with kidney or combined kidney+pancreas transplant. Eleven patients developed a severe diffuse disease within the first 3 months post-transplantation; immunoblastic B cells of recipient origin infiltrated the bone-marrow, transplanted organs, liver, spleen, lymph nodes, lungs, and brain; immunoglobulin abnormalities with fever, leuko-thrombocytopenia and liver dysfunction constituted the symptoms; all patients received anti-lymphocyte globulins; 9 patients were also treated with cyclosporin. Three out of 6 tumors analysed were monoclonal. Epstein-Barr virus was present in 3 lesions analysed. Treatment consisted of cessation of immunosuppressive therapy. Nine patients died with lactic acidosis. Five patients had a less severe form. Seven patients had solid tumors involving the tonsils, lungs (2), lymph nodes (2), and bladder, 8 months after transplantation. All patients received cyclosporin; 4 also received anti-lymphocyte globulins and 3 OKT3. Tumor cells were immunoblasts expressing B cells markers at a late stage of B cell differentiation; 4 tumors were monoclonal. C myc was negative. Treatment consisted of cessation of immunosuppressive therapy, antiviral agents, and monoclonal antibodies (mAb): anti-CD21 and anti-CD24 mAb therapy was followed by cure of the lymphoma in 1 patient, by transient remission in a second one and by failure in the third patient. Two patients had a recurrence of the lymphoma and received chemotherapy; 2 patients died of the lymphoma, 1 died of unrelated cause; 4 are alive, 3 of them having a good graft function.

Apparent HLA DR triplet due to the coexpression of a DRB5-encoded molecule on a DR1 haplotype.

HLA DR1 molecules are coded by a single polymorphic DRB1 gene. We have observed rare DR1 cells in one Caucasoid family and three unrelated individuals that also reacted with some anti-DR2 sera. Since the second DR antigen was normally expressed, these cells appeared as triplets. Contrary to serology, the cells were not typed by HTCs defining Dw2, Dw12, and Dw21. Further investigations on these unusual DR1+2* haplotypes were conducted by DNA oligotyping and by sequencing of the DRB first-domain exon. The results showed that these DR1 haplotypes, besides their DRB1*0101 allele, carried also a DRB5*0101 allele.

The bare lymphocyte syndrome.

The bare lymphocyte syndrome is a combined immunodeficiency resulting from the lack of expression of either class I or class II HLA antigens at the cell surface. The main clinical manifestations are infections of the respiratory or the digestive tract. The immunodeficiency involves the absence of antibody formation and the absence of cell-mediated response, to specific antigen, contrasting with virtually normal transplant immunity to allogeneic determinants. The responsible gene(s) is not born by chromosome 6. The best treatment appears, at the present time, to be in utero stem cell transplantation into the sick fetus, and it may, in the future, be gene therapy.

Alloantisera can detect some of the variants of HLA DRw13 identified by oligotyping.

Oligotyping has revealed a considerable polymorphism of HLA DRw13; so far, 5 alleles coded by DRB1 have been identified. An even greater number of haplotypes are apparent when considering the association of DRw13 with alleles coded by DRB3 and DQB1. Serology can now define some of these variants which had formerly escaped detection. We have tested by serology and by oligoprobes 66 genotyped Caucasoid cells comprising the known DR and DQ alleles. Among the 28 DRw6 cells, 10 different haplotype patterns were selected, of which 8 concern DRw13 and 2 DRw14. Four variants of DRw13 were represented: DRB1 *1301, *1302, *1303 and *1305 associated with usual and with uncommon alleles belonging to DRB3 and to DQB1. We have centered our analysis on 19 sera of the XIth Workshop defining DRw6. Two sera (639, 826) were noteworthy since they reacted with certain subtypes of DRw13 and with DRB1 *1102, *1103 only, the other DR specificities remaining negative.

In utero transplantation of stem cells in humans: immunological aspects and clinical follow-up of patients.

Four human fetuses were treated by transplantation of human fetal liver stem cells. Two of them had severe immunodeficiency disease and the two other ones had thalassemia major. Three of these in utero transplants were followed by engraftment. The three patients are now born: the first one is now very healthy thanks to the reconstitution of cell-mediated immunity associated with this transplant, and he lives normally at home; the two other ones, who have been more recently treated, have a significant improvement of their condition and they also live normally at home. This procedure, for the first time used in humans, has therefore demonstrated its feasibility and its efficacy: during early fetal development, foreign cells engraft readily and may result in cure or significant correction of a large variety of inherited diseases.

HLA-A29.2 subtype associated with birdshot retinochoroidopathy.

Birdshot retinochoroidopathy is strongly associated with HLA-A29. This antigen can be divided into two subtypes, A29.1 and A29.2, using an immunoprecipitation method succeeded by one-dimensional electrofocusing gel electrophoresis. We reviewed the HLA typings of 58 white French patients who had birdshot retinochoroidopathy. Of these 58 subjects, 54 (93.1%) had HLA-A29 with a relative risk of 157.30. We further analyzed the HLA-A29 subtypings of 33 patients with birdshot retinochoroidopathy. Evaluation of the results showed that HLA-A29.2 subtype was present in all patients (100%). We concluded that the absence of HLA-A29.1 subtype is statistically significant (P less than .01) in this study of HLA-A29 subtyping.

[Influence of HLA compatibility on cytomegalovirus infection in kidney transplantation]. / Influence de la compatibilité HLA sur l'infection à cytomégalovirus en transplantation rénale.

The influence of HLA A, B, DR on the incidence and symptoms of cytomegalovirus (CMV) infection was investigated in 143 patients who, between October 1st, 1987 and December 31st, 1989, received kidneys from cadaveric donors. Systematic virological monitoring was carried out weekly during the first hospitalization and thereafter at each new hospitalization or in the presence of clinical signs suggestive of viral infection. The diagnosis of CMV was based on positive isolation in blood or urine, or seroconversion, or 4-dilution rise in the anti-CMV antibodies titre. HLA grouping of all recipients was made in the same histocompatibility laboratory. Immunosuppression was obtained with a quadruple therapy consisting of corticosteroids (15 mg/kg before transplantation, then 1 mg/kg for 10 days, then gradually tapering off dosage), azathioprine (2 to 3 mg/day), cyclosporin A (2 mg/kg i.v. followed by an oral dose adjusted to the residual levels) and a randomized treatment with either monoclonal anti-CD3 antibody or anti-thymocyte globulins administered during the first 10 days. The incidence of CMV infection was 56 percent (80/143), with 25 percent of primary infection (20/80). The number of DR compatibilities was found to have a significant influence on the incidence of CMV infection, which rose from 22 to 50 and 65 percent respectively in the group of patients with 2.1 or 0 DR compatibility (P less than 0.02). The degree of B + DR compatibility was also associated with the occurrence of CMV infection, the incidence of which rose from 0 to 36, 59, 43.5 and 71 percent respectively in the group of patients with 4, 3, 2, 1, 0 B + DR compatibility (P less than 0.03). The incidence of primary CMV infection increased with the number of DR incompatibilities, rising from 0 to 29 and 52 percent respectively in the group of patients with 0, 1 or 2 DR incompatibilities. The symptoms and severity of CMV infection were significantly influenced by the degree of DR and B + DR compatibility. Despite a very strong association between graft rejection and CMV infection (P less than 0.000001), no influence of HLA, and particularly DR or B + DR compatibility on the incidence and number of graft rejections could be demonstrated. It is concluded that, under the above-described quadruple therapy, the HLA DR and B + DR compatibility exerts a predominant influence on the occurrence and severity of CMV infection, and that this effect is independent of any action on graft rejection.(ABSTRACT TRUNCATED AT 400 WORDS)

Additional complexity within the HLA-D region: sequence analysis of two new DRw13-DQw7 haplotypes.

HLA-DRB1 is by far the most polymorphic locus within the HLA-D region with now well over 40 alleles. Nearly one fourth of these alleles are subtypes of DRw6, and these are in most cases undetectable by routine typing procedures. In this paper we present the molecular characterization of two new Caucasian DRw13-DQw7 haplotypes by DNA sequencing of the polymorphic first domain exons of DRB1 and DRB3 loci. The first haplotype, DRB1*1301-DRB3*0101-DQB1*0301, has arisen by a recombination between locus DRB1 from a DRw13-DQw6 haplotype and DQA1 from a DR4-DQw7 haplotype, as determined by DNA sequencing, DQ oligotyping, and restriction fragment length polymorphism typing. The second haplotype, DRB1*1305-DQB1*0301, is characterized by the novel DRB1*1305 allele differing from DRB1*1301 by three amino acids. It probably arose by a gene conversion event between a DRw13-DQw6 allele and DRB1*1101. This allele represents a DRw11/DRw13 hybrid DR molecule with a DRw13 serological epitope in the second hypervariable region and a Dw5 cellular epitope in the third hypervariable region. As determined by sequencing of locus DRB3, this allele is associated with DRw52b. Our molecular analysis of the complex HLA-DRw13 group now allows unambiguous DNA typing of all five DRw13 alleles with seven oligonucleotides, a significant improvement in the context of organ transplantation.

Selection of unrelated donors for bone marrow transplantation is improved by HLA class II genotyping with oligonucleotide hybridization.

As the demand for donors for bone marrow transplantation increases, the use of HLA-matched, genetically unrelated donors represents a promising strategy. It is well documented that the clinical outcome of bone marrow transplantation is directly dependent on optimal matching for HLA class I and class II specificities. Molecular studies have revealed the existence of a much larger number of HLA class II alleles than was anticipated, many of which cannot be recognized by routine serological typing. Currently this "hidden" polymorphism represents a major limitation to the generalized use of unrelated donors for bone marrow transplantation. It has recently become possible, however, to identify HLA allelic polymorphism directly at the DNA level by hybridization with sequence-specific oligonucleotide probes ("HLA oligotyping") after amplification of DNA by polymerase chain reaction. In this study, we have investigated whether donor-recipient pairs that are fully matched for HLA by serology are truly HLA-DR, -DQ, and -DP identical and to what extent class II differences influence the primary mixed lymphocyte culture. We show that HLA oligotyping, performed on 50 pairs of HLA class I and II serologically matched individuals, can indeed reveal phenotypically relevant allelic differences at either DRB or DQB loci in 56% of these pairs and can therefore improve HLA class II typing and the choice of bone marrow donors quite significantly. Oligotyping for DRB/DQB/DPB polymorphism also allows prediction of a positive mixed lymphocyte culture, as established in 31 donor/recipient combinations, and even detection of polymorphic differences that were not revealed by this test. This approach is well suited for accurate HLA typing of large pools of bone marrow donors and was successfully applied to select fully matched donors for bone marrow transplantation.

The value of platelet transfusions as preparation for kidney transplantation.

The role of platelet transfusion as a preparative method for kidney transplantation is still a matter of debate. Two groups of 28 male patients transplanted between 1983 and 1988, paired for age, date of transplant, absence of anti-HLA antibody and immunosuppressive therapy have been compared. Group I was given 5 purified platelet transfusions at 1-week intervals before transplantation. Each transfusion contained 7.6 x 10(6) platelets contaminated by less than 1 leukocyte in 10(5) platelets. Group II received from 3 to 5 whole blood transfusions. In all cases it was a first transplant from cadaveric donors and previously untransfused patients before entering the protocol. No patient in group I developed cytotoxic antibodies. Acute tubular necrosis occurred with the same incidence in group I and in group II but was more severe and longer in group I, requiring hemodialysis in 62.5% and only 22% in group II. ATN was significantly associated with graft loss in group I (P less than 0.05). The total number of rejections and the number of patients undergoing rejection were not significantly different in both groups. However, the intensity of rejection was significantly higher in group I with 41% (21/51) of severe or irreversible rejections versus 9/46 (19.5%) in group II (P less than 0.05). The first rejection occurred significantly earlier in group I than in group II since 75% of the first rejection episodes occurred in the first 10 days versus 38% in group II (P less than 0.02) with a mean delay of 12.8 +/- 3.2 and 19.10 +/- 3.3 days, respectively. Although platelet transfusions are devoid of leukocytes the incidence of CMV infection was not significantly different in both groups: 57% in group I and 68% in group II. Purified platelet transfusions did not induce humoral immunization but lack of sensitization does not imply indefinite graft prolongation. Because platelets do not carry class II antigens, purified platelets transfusions represent a useful model to analyze the role of class I antigens alone in the induction of unresponsiveness in organ transplantation.