Common and well-documented HLA alleles: 2012 update to the CWD catalogue.

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.

Distinct distribution of killer-cell immunoglobulin-like receptor genes in the Mugil and Ilaita areas of Papua New Guinea.

The frequency of the killer-cell immunoglobulin-like receptor (KIR) genes and transmembrane alleles of KIR2DL4 were studied in coastal (Mugil community) and inland (Ilaita community) communities in Papua New Guinea. Linkage disequilibria between KIR genes and between alleles of KIR2DL4 and the KIR genes were similar to those found in other populations suggesting conservation of the usual gene order in Papua New Guinean haplotypes. Significant differences in the frequency of KIR genes were found between the two populations despite being separated by only 300 km. Examples of individuals who lacked the KIR2DL4 gene and others whose KIR2DL4 allele appeared to have 11 adenines in the polyadenine tract in exon 6 were identified. A relatively low frequency of the KIR A haplotype was found in both populations and particularly in the inland community. The KIR gene frequencies were consistent with the inland Ilaita community being closely related to Australian Aborigines and southern Indians, whereas the KIR gene frequencies of the coastal Mugil community appeared to have been influenced either by recent or ancient admixture from populations with a higher frequency of the KIR A haplotype.

Genetic variations in loci relevant to natural killer cell function are affected by ethnicity but are generally not correlated with susceptibility to HIV-1.

Polymorphisms in cell surface receptors of natural killer cells and their ligands on target cells can affect susceptibility to viral infections including human immunodeficiency virus (HIV)-1. We found that the carriage of the human leukocyte antigen (HLA)-G minus 14-bp polymorphism, LILRB1 single nucleotide polymorphism rs1061680, and activating and inhibitory killer immunoglobulin-like receptors (KIRs) were different when data were compared between Caucasian, African Americans and Asian populations. However, carriage was similar when HIV-1 patients were compared with control donors, with the exception of the African American cohort.

Lack of KIR2DL4 gene in a fertile Caucasian woman.

Killer cell immunoglobulin-like receptor (KIR2DL4) gene is present in virtually all humans. It encodes a receptor present on uterine and decidual natural killer (NK) cells and some peripheral blood NK cells. Its only known ligand is human leukocyte antigen-G molecule expressed on extravillous trophoblasts invading the decidua. Therefore, KIR2DL4 has been regarded as a molecule important for successful pregnancy. However, a multiparous woman from Africa, lacking KIR2DL4 gene, was described suggesting that this gene is not absolutely required for successful human reproduction. Here, we describe a Polish woman who delivered a child and who is not only lacking KIR2DL4 gene, but also possessing a KIR genotype virtually identical to that of the African woman mentioned above. Their genotypes are compared with few other KIR2DL4-negative genotypes and haplotypes described so far.

The novel HLA-B*15:180 allele appears to be a recombinant B*08/B*15 allele.

Human leukocyte antigen B-*15:180 is a B*08/B*15 recombinant allele similar to B*15:29 with substitutions positions at 97, 292, 538, 539.

Incidence of humoral sensitization in HLA partially mismatched hematopoietic stem cell transplantation.

As part of the 15th International Histocompatibility and Immunogenetics Workshop (IHIWS), seven centers participated in a collaborative project to determine whether any significant humoral sensitization occurred post-transplant among recipients of HLA partially mismatched hematopoietic cell transplants (HCTs). A total of 140 donor/recipient pairs were enrolled with a total of 367 pre-and post-transplant sera analyzed. The majority of the samples (69.1%) were obtained within 30-90 days post-HCT. HLA-specific antibodies were defined using single antigen bead assays on a Luminex platform with a positive cutoff value of 1000 normalized median fluorescence intensity (MFI). There was an overall incidence of post-HCT sensitization toward donor HLA mismatches of 5.7%; however, all cases were among recipients of one HLA haplotype-mismatched grafts under nonmyeloablative, pre-transplant conditioning. Among the one haplotype-mismatched recipients, 15.7% (8/51) developed donor HLA-specific antibodies and 29.4% also had antibodies directed toward third party HLA antigens. Among the donor-specific antibodies, 9.8% were directed toward HLA class I antigens; 7.8% were against class II antigens; and 2.0% had both class I and II specificity. The relative strength of post-transplant antibodies was low with no significant difference in the mean maximum MFI values between third party and donor-specific antibodies. Because only a small number (10.2%) of the post-transplant samples were obtained 180 days or more post-HCT, longer term study is needed to evaluate any clinical relevance of these low-to-moderate levels of donor-specific antibody in one haplotype-mismatched recipients, as well as to determine whether any other antibodies occur at later times.

Analysis of killer immunoglobulin-like receptor genes in ankylosing spondylitis.

OBJECTIVES: To assess the possible association of killer immunoglobulin-like receptor (KIR) genes, specifically KIR3DL1, KIR3DS1 and KIR3DL2, with ankylosing spondylitis (AS). METHODS: 14 KIR genes were genotyped in 200 UK patients with AS and 405 healthy controls using multiplex polymerase chain reaction. Sequence-specific oligonucleotide probes were used to subtype 368 cases with AS and 366 controls for 12 KIR3DL2 alleles. Differences in KIR genotypes and KIR3DL2 allele frequencies were assessed using the chi(2) test. RESULTS: KIR3DL1 and KIR3DS1 gene frequencies were very similar in cases with AS and controls (odds ratio = 1.5, 95% confidence interval 0.8 to 3.0, and odds ratio = 1.02, 95% confidence interval 0.2 to 5.3, respectively). KIR3DL2 allele frequencies were not significantly different between cases with AS and controls. CONCLUSIONS: Neither the KIR gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to AS.

Frequencies of killer immunoglobulin-like receptor genotypes influence susceptibility to spontaneous abortion.

Natural killer (NK) cells are the most abundant lymphocyte population in the decidua. These cells express killer immunoglobulin-like receptors (KIRs), which upon recognition of HLA class I molecules on trophoblasts may either stimulate NK cells (activating KIRs) or inhibit them (inhibitory KIRs) to produce soluble factors necessary for the maintenance of pregnancy. KIR genes exhibit extensive haplotype polymorphism; individuals differ in both the number and kind (activating vs. inhibitory) of KIR genes. This polymorphism affects NK cell reactivity and susceptibility to diseases, including gynecological disorders. Therefore we KIR-genotyped 149 spontaneously aborting women and 117 control multiparae (at least 2 healthy-born children). Several genotypes (i.e. combinations of various KIR genes) were differently distributed among the patients and control subjects. Differences were observed in the numbers and the ratios of activating to inhibitory KIRs between patients and healthy women: (i) genotypes containing 6 activating KIR genes were less frequent and those containing 6 inhibitory KIR genes were more frequent in patients than in control subjects, and (ii) an excess of inhibitory KIRs (activating-to-inhibitory KIR gene ratios of 0.33 to 0.83) was associated with miscarriage, whereas ratios close to equilibrium (0.86-1.25) seemed to be protective. In addition, the results suggest for the first time that sporadic and recurrent spontaneous abortions as well as miscarriage in the presence or absence of autoantibodies may have different KIR genotypic backgrounds.

Alleles and intron polymorphism of KIR3DL1 shown by combination of allele group-specific primers and sequencing.

Allelic polymorphism of killer immunoglobulin-like receptor (KIR) genes may affect receptor expression as well as function. We used a combination of allele group-specific primers and DNA sequencing techniques to verify our KIR genotyping primers in polymerase chain reaction and identified three KIR3DL1 alleles. By sequencing some introns of 3DL1 in 18 genomic DNA samples, we found that a 4-bp insert in intron 1 of 3DL1*002 exists in multiple alleles, but that a 2-bp deletion in intron 7 is unique to 3DL1*01501 and that a 19-bp insert in intron 1 seems specific to the CEPH family 1416. Our data suggest that extensive KIR gene polymorphisms are ubiquitous as well as quite complex.

A novel HLA-B allele, B*4093, sharing sequences with B*4006 and B*0702 in an Asian Indian donor.

A new HLA-B allele (B*4093) in a North Indian Hindu donor differing from B*4006 by four nucleotide substitutions in codon 41.1, codon 44.3, codon 45.1 and codon 50.3 has been identified. This novel allele was part of the A*0211-B*4093-Cw*1502-DRB1*15-DQB1*06 HLA haplotype.

Ethylnitrosourea-induced transplacental carcinogenesis in the mouse: tumor response, DNA binding, and adduct formation.

We have confirmed previous results which suggest that transplacental exposure of fetal mice to carcinogens does not cause an increase in tumor incidence as they mature unless treatment occurs after midorganogenesis. In C3HeB/FeJ mice we found a negligible increase in tumor incidence and multiplicity following transplacental exposure to the direct-acting carcinogen ethylnitrosourea (ENU) on gestation day 10, but significant increases in lung and liver tumor incidence following exposure on days 13 or 15 or in adults. To explore the possibility that this observed difference is due to differences in the biodistribution of the carcinogen or its interaction with cellular macromolecules, the level of covalent binding between ENU and fetal and maternal DNA following an i.p. injection of a dose of 50 mg/kg of tritium-labeled ENU was measured 30 min after its injection into pregnant females on days 10, 13, and 15 of gestation. The DNA from fetal and maternal lung, liver, and brain was isolated and the amount of covalent binding estimated from the dpm/mg DNA recovered. Samples of DNA were hydrolyzed and chromatographed to determine that the bound tritium was associated with ENU-DNA adducts and not as a product of DNA synthesis. The level of binding of ENU to fetal DNA was equivalent at all gestation days studied but was significantly less than maternal tissues. Binding to the DNA of maternal liver was 4-fold greater than to fetal DNA while maternal lung and brain DNA were bound at intermediate levels. We conclude that the lack of carcinogenic response to ENU documented here, in fetal mice exposed early in gestation (day 10), is not due to differences in ENU binding to fetal DNA during development.

Killer Ig-like receptor (KIR) compatibility plays a role in the prevalence of acute GVHD in unrelated hematopoietic cell transplants for AML.

Killer Ig-like receptor (KIR) is a major cluster of the natural killer cell receptors and may play a role in the outcome of hematopoietic cell transplants. A total of 65 AML cases transplanted with T-replete hematopoietic cells from unrelated donors were retrospectively KIR-genotyped by a multiplex PCR method of our own design. The KIR gene frequency and genotype patterns in these 130 samples were consistent with the data in the literature. Based upon overall inhibitory and activating KIR genes in both donors and patients, we developed an algorithm to calculate a compatibility score for each transplant case as plus, zero or minus. Significantly higher incidence (18/20, 90%) of acute (a) GVHD (grade II-IV) was found in the transplant cases with plus scores than that (25/45, 56%) in the cases with zero or minus scores (P < 0.01). When the scores are sorted in the opposite way, fewer cases (13/26, 50%) of aGVHD were found in the transplants with minus scores than that (30/39, 77%) in the transplants with zero or plus scores (P < 0.05). The difference of aGVHD prevalence between the plus score and minus score groups is highly significant (P < 0.01). KIR genotype compatibility calculated by this algorithm may predict aGVHD incidence and be helpful in choosing donors.

Inhibition of neutrophil shape change by an inhibitor of chemotaxis.

Human mononuclear cells exposed to staphylococcal peptidoglycan in serum-free culture rapidly produce an inhibitor of neutrophil chemotaxis which we have previously described. We found that this inhibitor of chemotaxis has its most potent effect on the inhibition of neutrophil shape change from a spherical to a polarized configuration. In order to quantify this shape change inhibition, we developed an assay using flow cytometric techniques. Neutrophils exposed to a chemoattractant simultaneously change their shape and decrease their forward angle light scattering intensity (delta FLS) with a correlation coefficient of 0.886 (p less than 0.001). In 51 experiments, neutrophils pretreated with the inhibitor of chemotaxis decreased their FLS by only 6.8 +/- 1.3 channels, while neutrophils pretreated with medium or control culture supernatants decreased theirs by 26.4 +/- 1.9 and 20.5 +/- 3.0 channels respectively (p less than 0.001). The factor which causes inhibition of shape change was indistinguishable from the inhibitor of chemotaxis by physical properties and chromatography. We conclude that this inhibitor of chemotaxis may act by inhibiting a physiologic step at or before shape change.

Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat.

Myoepithelial and secretory cells from the mammary gland of the lactating rat have been isolated, purified, and characterized. Mammary tissue was dissociated with collagenase into basket-like networks of myoepithelial cells and single secretory cells. Because of their larger size, the myoepithelial cell networks could be separated from other mammary and blood cells by differential centrifugation. Isolated secretory cells were purified by isopycnic centrifugation in 25% bovine serum albumin. The purified myoepithelial and secretory cells were viable, as shown by the incorporation of 32P into distinct macromolecules that were separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both myoepithelial and secretory cells retained their characteristic morphology after isolation and purification, as shown by light, transmission, and scanning electron microscopies. The isolated myoepithelial cells were unique and, thus, distinguishable from other mammary cells in a number of respects; they 1) contracted in response to the addition of oxytocin, 2) bound [3H]oxytocin specifically, 3) accounted for the content of alkaline phosphatase and [Na+ + K+]ATPase in mammary tissue, and 4) reacted specifically with antiserum prepared against purified myoepithelial cells. The purified secretory cells were unique in possessing glucose-6-phosphate dehydrogenase activity. The different cell markers not only gave independent estimates of the purity of the cell fractions, but they also may be helpful in identifying mammary cells in stages of differentiation and neoplastic transformation.

Isolation of B lymphocytes for HLA-DR typing using monoclonal antibodies.

Isolating sufficient B cells for DR typing is often a problem, particularly in patients with low lymphocyte and/or B cell counts. This paper describes a simple method for preparing highly purified B cells using monoclonal antibodies and complement to kill the majority of non-B cells. Mononuclear cells are isolated by standard Ficoll-Hypaque techniques and incubated at 0 degrees C for 10 min with the following monoclonal antibodies: OKT3 (pan T cell), OKT11 (pan T and null cell), and Leu11b (NK cell). Rabbit complement is added and the mixture is incubated at 37 degrees C for 20 min. Dead cells are then removed by Percoll gradient centrifugation. This procedure results in cell preparations containing 90.1 +/- 1.3% B cells as measured by two-colow immunofluorescence with an Epics C flow cytometer. A yield of 52% can routinely be obtained. In contrast, nylon wool adherence results in preparations with only 44 +/- 9.1% B cells with a yield of 31%. B cells prepared by the monoclonal antibody procedure always show stronger and clearer reactions on DR typing trays. These results are consistent with the flow cytometric analyses of the B cell preparations. In summary, this procedure has the following advantages: increased accuracy of DR typing, decreased sample size (only 1.0 X 10(7) mononuclear cells are required), reduced time of cell preparation, and reduction in the number of repeat typings necessitated by poor yields and/or results.

Infectious antecedent of immunoblastic lymphoma. Progressive immunosuppression in a patient with lymphogranuloma venereum.

Angioimmunoblastic lymphadenopathy is a nonmalignant disease of unknown etiology often progressing to immunoblastic lymphoma. Immunologic deficiency is evident in these patients as well as in those with various infections found in association with the acquired immune deficiency syndrome (AIDS). This report describes a previously healthy young woman in whom angioimmunoblastic lymphadenopathy developed in association with lymphogranuloma venereum, with progressive loss of immunologic competence. This deterioration paralleled the evolution of angioimmunoblastic lymphadenopathy into a rapidly fatal immunoblastic lymphoma.

T lymphocyte abnormalities in disseminated histoplasmosis.

Disseminated histoplasmosis is associated with depression of T cell-mediated immunity and in some cases anergy. In this report, two patients with disseminated disease are described. Both had a depression of T cell-mediated immunity as well as other abnormalities of immune response. In one, a patient with relapse, a marked depression in the ratio of T helper to T suppressor cells was noted. Neither patient had any predisposing condition known to be associated with disseminated disease.

Successful transplantation after conversion of a positive crossmatch to negative by dissociation of IgM antibody.

Preliminary crossmatching usually eliminates highly sensitized patients from consideration for renal transplantation. However, if the crossmatch is positive because of the presence of IgM antibody, this activity can be eliminated by treatment with the reducing agent Dithiothreitol (DTT). Successful transplantation may then be possible in patients whose crossmatch is positive due to the presence of IgM antibody. After treatment with DTT, the sera of 25 highly sensitized patients were measured for cytotoxicity against a selected panel of 40 cells. Those whose high %PRA could be attributed to blood transfusions or previous transplants did not change with DTT. Only two patients who had developed high panel reactivity, without a clear cause, had little reactivity remaining after DTT treatment of their sera. To select patients whose crossmatch might be rendered negative by DTT treatment, we developed a "minipanel" screening protocol. Patients whose monthly PRA cells increased greater than 30% from baseline had their serum samples treated with DTT to reduce IgM. The treated sera were tested against a panel of six cells. If there was little or no cytotoxicity, it was assumed that IgM antibody was responsible for the positive crossmatches. All subsequent cadaver donor crossmatches were done with and without DTT treated sera. Five patients (2 living-related; 3 cadaver) with current crossmatches positive before, but negative after, DTT treatment continue to have functioning kidneys 3-15 months after renal transplantation. There were no hyperacute rejections. We conclude that patients with IgM antibody can be successfully transplanted if they have a negative cross-match after reduction of IgM antibody in their serum samples. A "minipanel" helps to identify patients who will benefit from DTT treatment.

Evidence that zero antigen-matched cyclosporine-treated renal transplant recipients have graft survival equal to that of matched recipients. Reevaluation of points.

The value of HLA matching in cadaver renal transplantation (CRT) continues to be debated. It has recently been suggested that increased importance be given to HLA matching for the distribution of cadaver kidneys. Such a policy would add both delay and expense to CRT, which could be justified only by significantly improved results. The results of CRT in 252 cyclosporine treated adult patients transplanted at our institution from November 1984 to April 1989 were reviewed. Kidneys were initially transplanted into crossmatch-negative recipients based on waiting time, regardless of match. From October 1987, a points system, based on United Network for Organ Sharing (UNOS) criteria has been used. Eighty-four pts. with zero antigen match with their donors were compared with 168 pts. sharing 1-6 Ag. Actuarial graft and patient survival were determined by the cumulative life table method and compared using a log rank test. Our results indicated no statistically significant difference in graft survival because of better matching or mismatching. These findings are in keeping with our previously reported long-term results for non-CsA pts. Past predictions of improved graft survival based upon better matching at our institution have not fulfilled expectations, with the exception of 6 Ag matches. In conclusion, increased emphasis on HLA matching with fewer "points" for poorer matches does not appear justifiable.