Donor killer immunoglobulin-like receptor (KIR) genotype-patient cognate KIR ligand combination and antithymocyte globulin preadministration are critical factors in outcome of HLA-C-KIR ligand-mismatched T cell-replete unrelated bone marrow transplantation.

We previously reported the potent adverse effects of killer immunoglobulin-like receptor (KIR) ligand mismatch (KIR-L-MM) on the outcome of T cell-replete unrelated hematopoietic stem cell transplantation (UR-HSCT) through the Japan Marrow Donor Program. Other UR-HSCT studies have yielded inconsistent results. To address this discrepancy, we evaluated candidate factors contributing to the effects of KIR-L-MM on transplantation outcomes in retrospectively selected hematologic malignancy cases with uniform graft-versus-host disease (GVHD) prophylaxis (n = 1489). KIR-L-MM in the graft-versus-host direction (KIR-L-MM-G) was associated with a higher incidence of acute GVHD (aGVHD; P < .002) and a lower overall survival (OS; P < .0001) only without the preadministration of antithymocyte globulin (ATG). Furthermore, in KIR-L-MM-G, the donor KIR2DS2 gene with the patient cognate C1 ligand was associated with a higher incidence of aGVHD (P = .012). Multivariate analysis by Cox proportional hazard models suggested that donor 2DS2 and ATG preadministration were critical factors in grade III-IV aGVHD (hazard ratio = 1.96; 95% confidence interval = 1.01-3.80; P = .045, and hazard ratio = 0.56; 95% confidence interval = 0.31-0.99; P = .047, respectively). These results indicate that the adverse effects of KIR-L-MM-G depend on combination of donor-activating KIR genotype-patient cognate KIR ligand type and no ATG preadministration, thereby suggesting the importance of these factors in UR-HSCT and in leukemia treatment using natural killer (NK) cell alloreactivity.

Detection of Hpdel among Thais, a deleted allele of the haptoglobin gene that causes congenital haptoglobin deficiency.

BACKGROUND: Congenital haptoglobin deficiency is a risk factor for anaphylactic nonhemolytic transfusion reactions in Japan. The deleted allele of the haptoglobin gene, Hp(del), which causes congenital haptoglobin deficiency, has also been observed in other Northeast Asian populations, such as Korean and Chinese persons. It has not been reported in several African and European-African populations, however, or investigated in other countries. STUDY DESIGN AND METHODS: To investigate the distribution of congenital haptoglobin deficiency in Southeast Asian countries, blood samples collected from 200 randomly selected healthy Thai volunteers were analyzed for serum haptoglobin and the haptoglobin gene. Plasma haptoglobin concentration was measured to identify haptoglobin deficiency. Haptoglobin phenotyping was performed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. The presence of the Hp(del) allele was determined with genomic DNA by an Hp(del)-specific polymerase chain reaction (PCR) method. RESULTS: There were no haptoglobin-deficient subjects detected among the 200 Thais. Their haptoglobin phenotypes were as follows: Hp 1-1 in 10, Hp 2-1 in 81, and Hp 2-2 in 109. Six individuals heterozygous for Hp(del) were detected. The frequency of the Hp(del) allele was calculated to be 0.015. The prevalence of haptoglobin deficiency caused by Hp(del) homozygosity was estimated to be approximately 1 in 4000. CONCLUSION: Congenital haptoglobin deficiency caused by Hp(del) homozygosity is presumed to be present in Thailand as a risk factor for anaphylactic transfusion reactions with a frequency similar to that in Japan. The causative deleted allele of the haptoglobin gene, Hp(del), is distributed among Southeast Asian populations as well as among Northeast Asian populations.

High-risk HLA allele mismatch combinations responsible for severe acute graft-versus-host disease and implication for its molecular mechanism.

In allogenic hematopoietic stem-cell transplantation, an effect of HLA locus mismatch in allele level on clinical outcome has been clarified. However, the effect of each HLA allele mismatch combination is little known, and its molecular mechanism to induce acute graft-versus-host disease (aGVHD) remains to be elucidated. A total of 5210 donor-patient pairs who underwent transplantation through Japan Marrow Donor Program were analyzed. All HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 alleles were retrospectively typed in all pairs. The impacts of the HLA allele mismatch combinations and amino acid substitution positions in 6 HLA loci on severe aGVHD were analyzed. A total of 15 significant high-risk HLA allele mismatch combinations and 1 HLA-DRB1-DQB1 linked mismatch combinations (high-risk mismatch) for severe aGVHD were identified, and the number of high-risk mismatches was highly associated with the occurrence of severe aGVHD regardless of the presence of mismatch combinations other than high-risk mismatch. Furthermore, 6 specific amino acid substitution positions in HLA class I were identified as those responsible for severe aGVHD. These findings provide evidence to elucidate the mechanism of aGVHD on the basis of HLA molecule. Furthermore, the identification of high-risk mismatch, that is, nonpermissive mismatch, would be beneficial for the selection of a suitable donor.

Effects of HLA allele and killer immunoglobulin-like receptor ligand matching on clinical outcome in leukemia patients undergoing transplantation with T-cell-replete marrow from an unrelated donor.

The responsible human leukocyte antigen (HLA) locus and the role of killer immunoglobulin-like receptor (KIR) ligand matching on transplantation outcome were simultaneously identified by multivariate analysis in 1790 patients with leukemia who underwent transplantation with T-cell-replete marrow from an unrelated donor (UR-BMT) through the Japan Marrow Donor Program. The graft-versus-leukemia (GVL) effect depended on leukemia cell type. HLA-C mismatch reduced the relapse rate in acute lymphoblastic leukemia (ALL) (hazard ratio [HR] = 0.47; P = .003), and HLA-DPB1 mismatch reduced it in chronic myeloid leukemia (CML) (HR = 0.35; P < .001). In contrast, KIR2DL ligand mismatch in the graft-versus-host (GVH) direction (KIR-L-MM-G) increased in ALL (HR = 2.55; P = .017). An increased rejection rate was observed in KIR2DL ligand mismatch in the host-versus-graft direction (HR = 4.39; P = .012). Acute GVH disease (GVHD) was increased not only in the mismatch of HLA-A, -B, -C, and -DPB1, but also in KIR-L-MM-G. As a whole, the mismatch of HLA-A, -B, and -DQB1 locus and KIR-L-MM-G resulted in increased mortality. In conclusion, not only the mismatch of HLA-C and -DPB1, but also KIR-L-MM-G affected leukemia relapse, which should be considered based on leukemia cell type. Furthermore, KIR-L-MM induced adverse effects on acute GVHD (aGVHD) and rejection, and brought no survival benefits to patients with T-cell-replete UR-BMT.

Genomewide association analysis of human narcolepsy and a new resistance gene.

Human narcolepsy is a hypersomnia that is affected by multiple genetic and environmental factors. One genetic factor strongly associated with narcolepsy is the HLA-DRB1*1501-DQB1*0602 haplotype in the human leukocyte antigen region on chromosome 6, whereas the other genetic factors are not clear. To discover additional candidate regions for susceptibility or resistance to human narcolepsy, we performed a genomewide association study, using 23,244 microsatellite markers. Two rounds of screening with the use of pooled DNAs yielded 96 microsatellite markers (including 16 markers on chromosome 6) with significantly different estimated frequencies in case and control pools. Markers not located on chromosome 6 were evaluated by the individual typing of 95 cases and 95 controls; 30 markers still showed significant associations. A strong association was displayed by a marker on chromosome 21 (21q22.3). The surrounding region was subjected to high-density association mapping with 14 additional microsatellite markers and 74 SNPs. One microsatellite marker (D21S0012m) and two SNPs (rs13048981 and rs13046884) showed strong associations (P < .0005; odds ratios 0.19-0.33). These polymorphisms were in a strong linkage disequilibrium, and no other polymorphism in the region showed a stronger association with narcolepsy. The region contains three predicted genes--NLC1-A, NLC1-B, and NLC1-C--tentatively named "narcolepsy candidate-region 1 genes," and NLC1-A and NLC1-C were expressed in human hypothalamus. Reporter-gene assays showed that the marker D21S0012m in the promoter region and the SNP rs13046884 in the intron of NLC1-A significantly affected expression levels. Therefore, NLC1-A is considered to be a new resistance gene for human narcolepsy.

Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis.

Leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.

Genetic origins of the Ainu inferred from combined DNA analyses of maternal and paternal lineages.

The Ainu, a minority ethnic group from the northernmost island of Japan, was investigated for DNA polymorphisms both from maternal (mitochondrial DNA) and paternal (Y chromosome) lineages extensively. Other Asian populations inhabiting North, East, and Southeast Asia were also examined for detailed phylogeographic analyses at the mtDNA sequence type as well as Y-haplogroup levels. The maternal and paternal gene pools of the Ainu contained 25 mtDNA sequence types and three Y-haplogroups, respectively. Eleven of the 25 mtDNA sequence types were unique to the Ainu and accounted for over 50% of the population, whereas 14 were widely distributed among other Asian populations. Of the 14 shared types, the most frequently shared type was found in common among the Ainu, Nivkhi in northern Sakhalin, and Koryaks in the Kamchatka Peninsula. Moreover, analysis of genetic distances calculated from the mtDNA data revealed that the Ainu seemed to be related to both the Nivkhi and other Japanese populations (such as mainland Japanese and Okinawans) at the population level. On the paternal side, the vast majority (87.5%) of the Ainu exhibited the Asian-specific YAP+ lineages (Y-haplogroups D-M55* and D-M125), which were distributed only in the Japanese Archipelago in this analysis. On the other hand, the Ainu exhibited no other Y-haplogroups (C-M8, O-M175*, and O-M122*) common in mainland Japanese and Okinawans. It is noteworthy that the rest of the Ainu gene pool was occupied by the paternal lineage (Y-haplogroup C-M217*) from North Asia including Sakhalin. Thus, the present findings suggest that the Ainu retain a certain degree of their own genetic uniqueness, while having higher genetic affinities with other regional populations in Japan and the Nivkhi among Asian populations.

Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity.

Human Valpha24+Vbeta11+ natural killer T (NKT) cells are a distinct CD1d-restricted lymphoid subset specifically and potently activated by alpha-galactosylceramide (alpha-GalCer) (KRN7000) presented by CD1d on antigen-presenting cells. Preclinical models show that activation of Valpha24+Vbeta11+ NKT cells induces effective antitumor immune responses and potentially important secondary immune effects, including activation of conventional T cells and NK cells. We describe the first clinical trial of cancer immune therapy with alpha-GalCer-pulsed CD1d-expressing dendritic cells. The results show that this therapy has substantial, rapid, and highly reproducible specific effects on Valpha24+Vbeta11+ NKT cells and provide the first human in vivo evidence that Valpha24+Vbeta11+ NKT cell stimulation leads to activation of both innate and acquired immunity, resulting in modulation of NK, T-, and B-cell numbers and increased serum interferon-gamma. We present the first clinical evidence that Valpha24+Vbeta11+ NKT cell memory produces faster, more vigorous secondary immune responses by innate and acquired immunity upon restimulation.

Dendritic cell vaccination for patients with chronic myelogenous leukemia.

In this pilot study, we investigated the ability of autologous dendritic cells (DCs) pulsed ex vivo with leukemia-specific peptide to stimulate host antitumor immunity when administrated as a vaccine. Three patients with chronic myelogenous leukemia (CML) received three series of four administration of bcr-abl peptide-pulsed (1) blood DCs injected intravenously, (2) immature monocyte-derived DCs injected intradermally or (3) mature monocyte-derived DCs injected intradermally. Vaccination was well tolerated. No major toxicity occurred in any of the patients. In method (1), one patient developed peptide-specific cellular immune response with no clinical response. In method (2), one patient developed peptide-specific cellular immune response with no clinical response. In method (3), all patients developed peptide-specific cellular immune response with no clinical response. The clinical benefits of bcr-abl peptide-specific vaccination in CML remain to be determined. Further vaccine development is necessary to increase the clinical effect.

The CD1d natural killer T-cell antigen presentation pathway is highly conserved between humans and rhesus macaques.

Natural killer T (NKT) cells play an important role in controlling cancers, infectious diseases and autoimmune diseases. Although the rhesus macaque is a useful primate model for many human diseases such as infectious and autoimmune diseases, little is known about their NKT cells. We analyzed V alpha 24TCR+ T cells from rhesus macaque peripheral blood mononuclear cells stimulated with alpha-galactosylceramide (alpha-GalCer) and interleukin-2. We found that rhesus macaques possess V alpha 24TCR+ T cells, suggesting that recognition of alpha-GalCer is highly conserved between rhesus macaques and humans. The amino acid sequences of the V-J junction for the V alpha 24TCR of rhesus macaque and human NKT cells are highly conserved (93% similarity), and the CD1d alpha1-alpha2 domains of both species are highly homologous (95.6%). These findings indicate that the rhesus macaque is a useful primate model for understanding the contribution of NKT cells to the control of human diseases.

A subject with a novel type I bare lymphocyte syndrome has tapasin deficiency due to deletion of 4 exons by Alu-mediated recombination.

HLA class I expression depends on the formation of a peptide-loading complex composed of class I heavy chain; beta(2)-microglobulin; the transporter associated with antigen processing (TAP); and tapasin, which links TAP to the heavy chain. Defects in TAP result in a class I deficiency called the type I bare lymphocyte syndrome (BLS). In the present study, we examined a subject with a novel type I BLS who does not exhibit apparent TAP abnormalities but who has a tapasin defect. The subject's TAPASIN gene has a 7.4-kilobase deletion between introns 3 and 7; an Alu repeat-mediated unequal homologous recombination may be the cause of the deletion. No tapasin polypeptide was detected in the subject's cells. The cell surface class I expression level in tapasin-deficient cells was markedly reduced but the reduction was not as profound as in TAP-deficient cells. These results suggest that tapasin deficiency is another cause of type I BLS.

Human leukocyte antigen-A specificities and its relation with season of birth in Japanese patients with schizophrenia.

Several studies, including one from Japan, have observed an increase of Human Leukocyte Antigen (HLA)-A24 and A26 in schizophrenia, although others failed to observe the increase. No use of systematic diagnostic criteria and a not-adequately reliable typing technique might have affected the results in the previous studies. We investigated HLA-A specificities in Japanese patients with schizophrenia (DSM-IV), recruited from the same area as in the early Japanese study. A DNA-based technique (polymerase chain reaction-microtiter plate hybridization) was employed. No significant difference was observed in frequencies of any HLA-A specificities between patients and controls, including A24 and A26. No significant association was found between the HLA-A and birth-season in patients. Thus, no evidence was obtained for an association between HLA-A and schizophrenia from the Japanese population.

Anaphylactic transfusion reactions in haptoglobin-deficient patients with IgE and IgG haptoglobin antibodies.

BACKGROUND: Patients with haptoglobin deficiency associated with haptoglobin IgG antibodies, who experienced severe nonhemolytic transfusion reactions (NHTRs), have been identified in Japan. Haptoglobin deficiency therefore might be a risk factor for NHTRs. STUDY DESIGN AND METHODS: A total of 4138 cases of voluntarily reported NHTRs in Japan, including 367 cases of immediate-onset anaphylactic NHTRs, were examined to identify haptoglobin deficiency. Serum haptoglobin IgG and IgE antibodies were determined in haptoglobin-deficient patients to elucidate the mechanism underlying the transfusion reactions. RESULTS: Seven patients with haptoglobin deficiency were identified. Six of them experienced severe and acute NHTRs. Six of them were identified to be homozygous for the Hpdel allele of the haptoglobin gene. Both haptoglobin IgG and IgE antibodies were detected in serum samples of all the patients. The stimulative effects of blood transfusion on the production of hap- toglobin antibodies in the patients and the relation- ship between the presence of the antibodies and the occurrence of the transfusion reactions were observed. CONCLUSION: Anaphylactic NHTRs in these patients with haptoglobin deficiency associated with serum haptoglobin antibodies were suggested to be prevalent in Japan. In addition to IgG antibodies, IgE haptoglobin antibodies detected in the sera of such patients were suggested to play a role in the occurrence of the reactions.

Activation and suppression of renin-angiotensin system in human dendritic cells.

We previously identified the gene expression of renin-angiotensin system in human monocyte-derived dendritic cells (DCs). This study was conducted to examine the mechanisms by which angiotensin II and captopril, the inhibitor of the angiotensin-converting enzyme (ACE), affect human DCs. In DCs, lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-(IL)-1alpha, IL-10, IL-12, and IL-18 was significantly inhibited by captopril. In contrast, angiotensin II treatment resulted in a significant increase in TNF-alpha and IL-6 protein biosynthesis by DCs. In addition, we have studied the global expression of 2400 genes in DCs from two donors. Here, we demonstrated the specific down-regulation of the ACE gene expression in captopril-treated DCs. Our finding indicates the possible activation of NF-kappaB through the up-regulation of expressions of MEFV gene (encoding PYRIN protein) and heterogeneous nuclear ribonucleoprotein R in DCs. This is the first study on the modulation of cytokine and gene expression by angiotensin II and captopril in DCs.

The clinical significance of human leukocyte antigen (HLA) allele compatibility in patients receiving a marrow transplant from serologically HLA-A, HLA-B, and HLA-DR matched unrelated donors.

To improve the clinical outcome of allogeneic hematopoietic stem cell transplantation from an unrelated donor, the identification of human leukocyte antigen (HLA) alleles responsible for immunologic events such as graft-versus-host disease (GVHD), engraftment failure, and graft-versus-leukemia effect is essential. Genomic typing of HLA-A, -B, -C, -DRB1, and -DQB1 was retrospectively performed in 1298 donor-patient pairs in cases where marrow was donated from serologically HLA-A, -B, and -DR compatible donors. Single disparities of the HLA-A, -B, -C, or -DRB1 allele were independent risk factors for acute GVHD, and the synergistic effect of the HLA-C allele mismatch with other HLA allele mismatches on acute GVHD was remarkable. HLA-A and/or HLA-B allele mismatch was found to be a significant factor for the occurrence of chronic GVHD. HLA class I (A, B, and/or C) allele mismatch caused a significantly higher incidence of engraftment failure than HLA match. Significant association of HLA-C allele mismatch with leukemia relapse was not observed. As the result of these events, HLA-A and/or HLA-B allele mismatch reduced overall survival remarkably in both standard-risk and high-risk leukemia cases, whereas the HLA-C mismatch or HLA-class II (DRB1 and/or DQB1) mismatch did not. Furthermore, multiple mismatch of the HLA locus was found to reduce survival in leukemia cases. Thus, the role of the HLA class I allele in unrelated bone marrow transplantation was elucidated. Notably, HLA-C alleles had a different mode from HLA-A or -B alleles for acute GVHD and survival.

High-throughput HBV DNA and HCV RNA detection system using a nucleic acid purification robot and real-time detection PCR: its application to analysis of posttransfusion hepatitis.

BACKGROUND: A high-throughput detection system was developed for HBV DNA and HCV RNA. METHODS: A combination of real-time detection PCR using an automated system (PRISM 7700, PE Biosystems, Foster City, CA) and automatic viral nucleic acid extraction (BioRobot 9604, Qiagen, Hilden, Germany) was used as the high-throughput detection system. An internal control for HBV DNA detection was also developed. RESULTS: Testing of 96 samples for HBV and HCV was completed within 5 hours. The sensitivity of this system almost equals that of the manual method using nested PCR. The addition of an internal control for HBV detection did not affect the sensitivity of the method and confirmed the accuracy of results. It was possible to quantify HBV in HBV+ samples that contain more than 500 genome equivalents per mL. We started using this system from June 1999 for testing stored donor and patient samples to analyze cases of posttransfusion hepatitis and identified three HBV+ donations that were implicated in posttransfusion hepatitis B. CONCLUSION: The high-throughput detection system is a useful tool for HBV DNA and HCV RNA detection because it enables rapid and reliable testing of a large number of samples.

Cutting edge: analysis of human V alpha 24+CD8+ NK T cells activated by alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

Human Valpha24(+) NKT cells constitute a counterpart of mouse Valpha14(+) NKT cells, both of which use an invariant TCR-alpha chain. The human Valpha24(+) NKT cells as well as mouse Valpha14(+) NKT cells are activated by glycolipids in a CD1d-restricted manner and produce many immunomodulatory cytokines, possibly affecting the immune balance. In mice, it has been considered from extensive investigations that Valpha14(+)CD8(+) NKT cells that express invariant TCR do not exist. Here we introduce human Valpha24(+)CD8(+) NKT cells. These cells share important features of Valpha24(+) NKT cells in common, but in contrast to CD4(-)CD8(-) (double-negative) or CD4(+) Valpha24(+) NKT cells, they do not produce IL-4. Our discovery may extend and deepen the research field of Valpha24(+) NKT cells as well as help to understand the mechanism of the immune balance-related diseases.

Generation of Fas-independent CD4+ cytotoxic T-cell clone specific for p190 minor bcr-abl fusion peptide.

In the majority of Ph+ALL patients, p190 bcr-abl fusion protein is generated in the Philadelphia chromosome. The fusion protein may serve as a leukemia antigen because it is not expressed in normal cells and hardly in any other malignancy. From a healthy donor, we have established a p190 bcr-abl fusion peptide-specific CD4+ cytotoxic T-cell clone, activation of which depends on HLA-DRB1*1501. This T-cell clone has a strong cytotoxic activity against autologus MoDCs pulsed with e1a2 peptide and its cytotoxicity is not mediated by Fas/Fas ligand or perforin pathway. Success in establishment of the p190 bcr-abl fusion peptide-specific T-cell clone encourages us to develop a new approach to an effective immunotherapy for Ph+ALL.