Frequency of HLA-DP-specific antibodies and a possible new cross-reacting group.

Clinical studies have demonstrated that HLA-DP-specific antibodies can be detrimental to a transplanted kidney. The number of patients affected is proportional to the frequency of DP antibodies. We determined the frequency of HLA-DP-specific antibodies en toto and in the absence of cross-reactive DR antibodies. Of 650 waitlisted renal patients, 271 (42%) were reactive with HLA-DP antigens in solid-phase immunoassays. Of these 271 sera, 58 (21%) were negative for reactivity with cross-reactive DR antigens, and 16 (5.9%) had no class II antibody other than DP. Eliminating sera containing DR cross-reactive antibodies reduced the frequency but not the overall strength of DP antibodies. Although most DP antibodies were not expected to yield a positive cytotoxicity crossmatch, 2 DP-specific antibodies yielded cytotoxic crossmatch tests with titers of >512. The occurrence of HLA-DP-specific antibody differed significantly between previously transplanted (62%) and nontransplanted (38%) patients, but no difference was observed among patients categorized by race or sex. One serum demonstrated strong cross-reactivity between DP and DRB1*01:03 in the absence of DR1 or DR11 reactivity. Sequence alignments were performed and a possible new cross-reactivity between DRB1*01:03 and DP2, DP9, DP10, DP13, DP16, and DP17 was defined. Two additional sera confirmed this cross-reactivity.

In a study for acne vulgaris, sequence-based HLA typing showed a novel DPB1 allele, DPB1*2402.

In this paper, we characterize the novel human leucocyte antigen (HLA)-DPB1*2402 allele that we found in a patient suffering from acne vulgaris. In comparison to the closest related allele DPB1*0401, HLA-DPB1*2402 has a single nucleotide exchange at position 115 (202), T replaces G. In consequence, codon 39 (68) TAC encodes for tyrosine in the novel allele instead of aspartic acid 39 (68) GAC in DPB1*0401.

HLA-DPB1, -DRB1, and -DQB1 polymorphism defined in Ewenki ethnic minority of China Inner Mongolia Autonomous Region.

In the present study, DNA typing for human leucocyte antigen (HLA)-DPB1, -DRB1, and -DQB1 was performed using polymerase chain reaction-sequence-based-typing (PCR-SBT) method on 94 randomly selected, healthy, unrelated individuals from the Ewenki ethnic population in Inner Mongolia Autonomous Region of China. A total of 64 alleles: 25 in DRB1, 19 in DQB1 and 20 in DPB1, were found. Among the 25 detected DRB1 alleles, DRB1*090102, DRB1*030101, DRB1*040101, DRB1*070101, and DRB1*120101/1206 were commonly observed, with frequencies of 16.0%, 13.3%, 10.1%, 7.4%, and 7.4%, respectively. The most predominant DQB1 allele was DQB1*030101/0309 with the frequency of 27.7%, followed by DQB1*0201/0202 (19.7%), DQB1*030302 (12.8%), DQB1*060101/060103 (6.4%), and DQB1*050201 (5.9%). Of the 20 detected DPB1 alleles, DPB1*020102 was the most frequent allele with the frequency of 25.5%. DPB1*0402 (21.3%), DPB1*0401 (20.2%), DPB1*0501 (10.6%) and DPB1*4101 (3.7%) were also very frequent alleles. The most frequent two-locus haplotypes observed in the Ewenki were: DRB1*030101-DQB1*0201/0202(10.7%), DRB1*090102-DQB1*03032(9.8%), DRB1*070101-DQB1*0201/0202 (5.5%), DRB1*070101-DQB1*030302 (5.2%) and DRB1*120101/1206-DQB1*030101/0309 (4.6%). The distribution of the HLA class II alleles and haplotypes frequencies as well as the dendrogram showed that the Ewenki population belongs to the northern group of Chinese.

Rapid detection of human HLA transgenes in pigs by fluorescence in situ hybridization (FISH) for adjuvant study of human xenotransplantation.

OBJECTIVES: We have recently generated several lines of transgenic pigs for HLA-DP and -DQ to elucidate the role of HLA-II antigens in the modulation of cell-mediated rejection of xenotransplantation. Using fluorescence in situ hybridization (FISH) analysis, the aim of this study was to determine integration sites and to test zygosity of these transgenes in the piglets after cross mating. METHODS: Blood lymphocytes of transgenic pigs for HLA-DP and -DQ were collected and cultured. Chromosome spreads were prepared by standard methodology. Gene constructs of HLA-DP A1+B1, -DQ A1 & B1 were labeled with fluorescein isothiocyanate or Texas Red by nick-translation. Hybridization was based on a standard FISH protocol. RESULTS: FISH analysis revealed that the HLA-DP probe hybridized to porcine chromosome 6, while both HLA-DQ A1 and B1 probes hybridized to porcine chromosome 11 at the same site. There was no cross-hybridization of HLA transgenes to the swine leukocyte antigen complex. Mosaic integration of HLA-DQ transgenes in the genome of F0, but full penetrance in F1 after selective breeding was observed. Both HLA-DP and HLA-DQ lines were determined to be heterozygous at the integration site. CONCLUSION: By FISH, we have detected specific integration sites of the HLA-DP and -DQ transgenes in pig genome and determined mosaic levels and zygosity types of these transgenes. We conclude that FISH is both sensitive and labor-efficient in confirming and differentiating transgenic pigs for multiple rejection-regulatory genes by visualizing individual integration sites in chromosomes or interphase nuclei.

Loss of B7.2 (CD86) and intracellular adhesion molecule 1 (CD54) expression is associated with decreased tumor-infiltrating T lymphocytes in diffuse B-cell large-cell lymphoma.

Tumor-infiltrating CD8+ T-lymphocytes (T-TILs) are thought to be relevant to immunosurveillance of several tumor types including B-cell non-Hodgkin's lymphoma. B- and T-lymphocyte interactions via cellular adhesion molecules (CAMs), recognition molecules (HLAs), and costimulatory molecules (CSMs) are necessary for optimal antigen-specific T-cell activation to occur and may be important in generating effective host T-TIL responses. We previously found that low T-TIL response (CD8+ T cells < 6%) correlates with statistically shorter relapse-free survival in patients with diffuse large-cell lymphoma (DLCL). We now extend our observations in 71 DLCL patients by analyzing malignant B-cell expression of the following molecules important in T-cell activation: (a) recognition molecules [MHC I (MAS and MCA) and MHC II (HLA-DR, -DP, -DQ)]; (b) CAMs [leukocyte function antigen 1 (CD11a and CD18) and intracellular adhesion molecule 1 (CD54)]; and (c) CSMs [B7.1 (CD80) and B7.2 (CD86)]. Eighteen patients (25%) had low a T-TIL response, and 53 patients (75%) had a high T-TIL response. Overall, expression of the MHC class H molecules HLA-DR and HLA-DQ was most conserved. The loss of B7.2 (P = 0.04), intracellular adhesion molecule 1 (P = 0.0004), MAS (P = 0.02), and HLA-DR (P = 0.0004) expression was significantly associated with decreased T-TIL response. In 100% of patients with low T-TIL responses, at least one HLA, CAM, or CSM was undetectable on the malignant B cells by immunohistochemical staining (mean number of molecules lost = 2.67). In contrast, 49% of patients with high T-TIL responses had no losses in HLA, CAM, or CSM expression (mean number of molecules lost = 0.89). The mean number of absent molecules (HLA, CAM, or CSM) was significantly associated with T-TIL response (P = 0.0001). We conclude that loss of HLA, CAM, or CSM expression on malignant B cells is associated with a poor host T-cell immune response. In addition, because patients with low T-TIL response had lost expression of multiple cellular adhesion, recognition, and costimulatory molecules, our results suggest that a combination of immunorestorative therapies may be required to generate effective antitumor T-cell responses in B-cell DLCL.

Response differences between human CD4(+) and CD8(+) T-cells during CD28 costimulation: implications for immune cell-based therapies and studies related to the expansion of double-positive T-cells during aging.

Since CD28 costimulation is critical for T-cell activation, there is great interest in CD28 as a target for immuntherapeutic approaches. We show that stimulation of human CD4(+) and CD8(+) T-cells differs in their responsiveness to stimulation with anti-CD3/CD28-coated beads, as surrogate antigen-presenting cells. While the CD4(+) subset responded with sustained proliferation, CD8(+) T-cells grew for a limited period only and failed to produce IL-2 beyond the first few days in culture. This decrease is accompanied with an increased rate of apoptosis in CD8(+) T-cells despite Bcl-x(L) expression. The CD8(+) but not the CD4(+) subset developed a reversible double-positive phenotype during CD28 costimulation. This finding may have some bearing on the appearance of double-positive T-cells in human peripheral blood. This double-positive subset was shown to undergo a statistically significantly increase during aging in humans. Taken together, the above data have important implications for immunotherapy and immune senescence.

HLA class II DRB1, DQB1 and DPB1 genotypic associations with peanut allergy: evidence from a family-based and case-control study.

BACKGROUND: Peanut is one of the most common foods provoking allergic reactions and is the most frequent cause of fatal and near-fatal food-induced anaphylaxis. However, as yet, little is known of the genetic and immunological mechanisms which underly peanut allergy. OBJECTIVE: Based on findings in other allergic diseases, we have investigated whether particular human leucocyte antigen (HLA) class II genetic polymorphisms contribute to the development of peanut allergy. METHODS: All individuals from 37 families each containing one or more peanut allergic individuals, plus nine unrelated patients (161 individuals in total, defined as the study group) were typed for the HLA class II DRB1, DQB1 and DPB1 loci, by PCR-based techniques. Genotype frequencies were compared with those found in 293 unrelated controls. RESULTS: Four class II genotypes (DRB1*08 (13.7% vs 4.8%; Pc = 0.026), DRB1*08/12 tyr 16 (22.4% vs 8.2%; Pc = 0.021), DQB1*04 (12.2% vs 2.7%; Pc = 0.0026) and DPB1*0301 (49.1 vs 22.5%; Pc = 0.00062)) were present at a significantly higher frequency in the study group compared with controls. Three of these genotypes (DRB1*08 (18.0%; Pc = 0.027), DRB1*08/12 tyr16 (24.0%; Pc = 0.029) and DQB1*04 (16.7%; Pc = 0.0029)) were also significantly increased in peanut allergic individuals compared with controls. In addition, two genotypes (DPB1*0101 and 0201) were significantly decreased in frequency in the overall study group, but not specifically in peanut allergic individuals. CONCLUSION: While other genetic factors may be important, results from this study indicate that HLA class II genetic polymorphism may play a role in determining susceptibility to peanut allergy.

Cytokine modulatable signalling through macrophage HLA class II. 1. IFN-gamma upregulates the efficiency of Ca2+ mobilization in response to ligation of macrophage HLA-DP.

The human macrophage line 2MAC, established recently from peripheral blood, expresses a number of lineage-specific markers as well as a broad array of intercellular adhesion molecules, including high levels of HLA class I and class II. We have presented evidence elsewhere that 2MAC can be productively applied to the study of signal transduction through macrophage HLA class II. Namely, we showed that ligation of 2MAC HLA class II, but not HLA class I, by monoclonal antibody (mAb) elicits an increase in free cytoplasmic Ca2+ concentration [Ca2+]i. Moreover, this Ca2+ flux appears to be functionally relevant: ligation of HLA-DR, but not HLA class I, by mAb results in the Ca2+ mobilization-dependent induction of tissue factor, the high-affinity cellular receptor for factor VII/VIIa. Here we show that 2MAC is uniquely valuable for addressing the efficiency of signal transduction through HLA class II. Namely, we show here that prior culture of 2MAC cells with interferon-gamma (IFN-gamma) profoundly upregulates subsequent Ca2+ mobilization in response to ligation of HLA-DP in the absence of increased cell surface HLA-DP expression. Because IFN-gamma has no effect on 2MAC HLA-DP expression, IFN-gamma must upregulate Ca2+ mobilization by increasing the efficiency of signal transduction through HLA class II (HLA-DP), by targeting some other component of the macrophage HLA class II signalling pathway.

Identification of HLA class II antigens as the targets of effector clones which may cause transfusion-associated graft-versus-host disease.

We established T cell clones, which were considered to be the possible cause of transfusion-associated graft-versus-host disease (TA-GVHD), from the peripheral blood lymphocytes (PBLs) of two patients. In both cases, several CD4+ cytotoxic T-cell (CTL) clones were established. In case I, the target antigen of the established CD4+ clones was a DRB1*0403-related antigen serologically typed as HLA DR4, which was one of the patient HLA antigens. In case II, the target of four out of five established CD4+ CTL was a DRB1*1302-related antigen. One CD4+ CTL clone showed cytotoxicity against cells carrying A*2402, B*4403, Cw*1403 and DPB1*0401. A monoclonal antibody (mAb) blocking study showed only anti-DP mAb inhibited the cytotoxicity of this clone. Thus, it might be considered that this clone recognizes HLA-DP with its binding peptides derived from either A*2402, B*4403, Cw*1403 or DRB1*1302. Our findings indicate that CD4+ CTLs may play important roles in the aetiology of TA-GVHD and that the antigens of patients recognized by donor-derived effector cells may not always recognize a single HLA antigen.

A significant percentage of normal T lymphocytes express HLA-DP in the peripheral blood.

HLA-DP expression has been widely investigated on T lymphocytes activated under different conditions. In the present study, a double staining procedure was used in flow cytometric experiments to define DP expression on normal peripheral blood T lymphocytes. In about two-thirds of the case analyzed, DP was expressed on a higher percentage of normal peripheral T lymphocytes than DR was. This was particularly true for 1 of the 16 cases investigated in which the percentage of T lymphocytes expressing DP was 46% and in which DP expression was mainly the prerogative of CD8+ and CD56+ lymphocytes.

Changes in the expression of HLA-class II antigens on peripheral blood monocytes from aged humans.

Increased incidence of infections, cancer, monoclonal gammopathies and rheumatic diseases in aged humans has been described. Histocompatibility antigens are involved in the regulation of immune response and it has been suggested that age-related alterations in the murine immune system may be due to changes in the expression of these antigens on the immunocompetent cells. In this paper we study the expression of HLA-DR/DP and HLA-DQ antigens on monocytes from healthy human elderly donors. Results show that peripheral blood monocytes from elderly subjects express decreased levels of HLA-DR/DP antigens (61.5 + 16.3) when compared to young controls (82.5 + 8.5) and increased levels of HLA-DQ antigens (40.4 + 16.8 and 23.6 + 7.1, respectively). The abnormal levels of expression of HLA-class II molecules could be related to the altered immune functions observed in elderly people.

RFLP-defined HLA-DP polymorphism in four ethnic groups.

Patterns of BglII, MspI, and TaqI DP alpha and DP beta hybridizing restriction fragment length polymorphisms are compared with primed lymphocyte typing--determined specificities in 28 10th International Histocompatibility Workshop core cell lines. Correlation of specific RFLP patterns with most recognized DP types is confirmed, although the RFLPs do not distinguish DPw3 from DPw6. Analysis of DP-region nucleotide sequence data shows that the observed RFLPs are those expected to hybridize to the DP alpha and beta probes and are thus not due to cross-hybridization to other genes. The distribution of RFLPs in Chinese, Micronesian, South Indian, and white Australian populations is described. The most frequent DP specificity in Chinese is DPw5. In the other populations DPw4 is the most common specificity, although DPw5 is also relatively common in Micronesians. Four new DP alpha RFLP patterns and three new DP beta patterns are described. There are also numerous unusual combinations of DP alpha and DP beta alleles particularly in the South Indian population.