Toshio Yabe

Activation of human Vα24NKT cells by α-glycosylceramide in a CD1d-restricted and Vα24TCR-mediated manner

Vα14NK(natural killer) T cells play an important role in controlling tumors or in preventing autoimmunity in the murine system. Vα24NKT cells, the human counterpart of Vα14NKT cells, may contribute to controlling the progression of autoimmune diseases in humans. These findings show the possibility that ligand(s) for these NKT cells can control the above-mentioned pathological conditions. Specific glycolipids such as α-galactosylceramide (α-GalCer) and α-glucosylceramide (α-GlcCer) have been identified as ligand(s) recognized by murine Vα14NKT cells in a CD1d-restricted manner, but it remains unclear whether these glycolipids are ligand(s) for Vα24NKT cells in humans. To determine whether α-glycosylceramide is presented by CD1d molecules in humans, we initially established a Vα24NKT cell line specific for α-glycosylceramide using dendritic cell (DC) like cells from normal peripheral blood mononuclear cells (PBMC) in an autologous mixed leukocyte reaction (auto-MLR) system, and characterized the Vα24NKT cell line. The Vα24NKT cells were CD3+CD4−CD8−Vα24+Vβ11+NKRP1A+ and specifically proliferated in response to α-glycosylceramide in CD1d-restricted and Vα24TCR-mediated manner. The phenotypic and functional similarities between murine Vα14NKT cells and human Vα24NKT cells suggest that Vα24NKT cells may play an important role in controlling tumors or in preventing autoimmunity as observed with Vα14NKT cells.

CD8 + NKR-P1A + T cells preferentially accumulate in human liver

A unique subset of T cells that co‐express NKR‐P1, which is a lectin type of NK receptor and is thought to have a major role in triggering NK activity, has been identified. In mice, NK1.1 (mouse NKR‐P1C)+ T cells, called NKT cells, preferentially accumulate in the liver and bone marrow. They predominantly use invariant Vα14 chain TCR and phenotypically are CD4+CD8− or CD4−CD8− T cells. In this study, we analyzed, phenotypically and functionally, the NKR‐P1A (analogue of murine NKR‐P1C)+ T cells resident in the human liver. Here, we show that in complete contrast to the NKT cells in the mouse liver, the majority of NKR‐P1A+ T cells in the human liver are CD8+ and their TCR repertoire is not skewed to Vα24 TCR, the homologue of murine Vα14 TCR. Almost all of the NKR‐P1A+ T cells in the human liver expressed CD69, suggesting that they were activated. Furthermore, the NKR‐P1A+ T cells in the human liver exhibited strong cytotoxicity against a variety of tumor cell lines including K562, Molt4 and some colonic adenocarcinoma cell lines.

Biological significance of HLA locus matching in unrelated donor bone marrow transplantation

We hypothesized that the compatibility of each HLA loci between donor and patient induced divergent transplant-related immunologic responses, which attributed to the individualized manifestation of clinical outcomes. Here, we analyzed 7898 Japanese pairs transplanted with T-cell-replete marrow from an unrelated donor with complete HLA allele typing data. Multivariable competing risk regression analyses were conducted to evaluate the relative risk (RR) of clinical outcomes after transplantation. A significant RR of HLA allele mismatch compared with match was seen with HLA-A, -B, -C, and -DPB1 for grade III-IV acute graft-versus-host disease (GVHD), and HLA-C for chronic GVHD. Of note, only HLA-C and HLA-DPB1 mismatch reduced leukemia relapse, and this graft-versus-leukemia effect of HLA-DPB1 was independent of chronic GVHD. HLA-DRB1 and HLA-DQB1 double (DRB1_DQB1) mismatch was revealed to be a significant RR for acute GVHD and mortality, whereas single mismatch was not. Thus, the number of HLA-A, -B, -C, -DPB1, and DRB1_DQB1 mismatches showed a clear-cut risk difference for acute GVHD, whereas the number of mismatches for HLA-A, -B, -C, and DRB1_DQB1 showed the same for mortality. In conclusion, we determined the biological response to HLA locus mismatch in transplant-related immunologic events, and provide a rationale for use of a personalized algorithm for unrelated donor selection.

α-Glycosylceramides Enhance the Antitumor Cytotoxicity of Hepatic Lymphocytes Obtained from Cancer Patients by Activating CD3−CD56+ NK Cells In Vitro

α-Glycosylceramides, such as α-galactosylceramide and α-glucosylceramide, induce antitumor immunity in various murine cancer models. In the murine hepatic metastasis model, Vα14 TCR+NK1.1+ T cells, which accumulate preferentially in the liver, are considered to play a key role in the induction of antitumor immunity by α-glycosylceramides. We recently reported that Vα24 TCR+ NKT cells, the human homologues of murine Vα14 TCR+NK1.1+cells, are rarely seen among freshly isolated human hepatic lymphocytes. Therefore, it is important to examine whether α-glycosylceramides also enhance the antitumor cytotoxicity of human hepatic lymphocytes, as they have been shown to do in murine systems, to determine the usefulness of α-glycosylceramides in cancer immunotherapy in humans. Here, we show that α-glycosylceramides greatly enhance the cytotoxicity of human hepatic lymphocytes obtained from cancer patients against the tumor cell lines, K562 and Colo201, in vitro. The direct effector cells of the elicited cytotoxicity were CD3−CD56+ NK cells. Even though Vα24 TCR+NKT cells proliferated remarkably in response to α-glycosylceramides, they did not contribute directly to the cytotoxicity. Our observations strongly suggest the potential usefulness of α-glycosylceramides for immunotherapy of liver cancer in humans based on their ability to activate CD3−CD56+ NK cells in the liver.

Splice acceptor site mutation of the transporter associated with antigen processing-1 gene in human bare lymphocyte syndrome

Expression of histocompatibility leukocyte antigen (HLA) class I molecules on the cell surface depends on the heterodimer of the transporter associated with antigen processing 1 and 2 (TAP1 and TAP2), which transport peptides cleaved by proteasome to the class I molecules. Defects in the TAP2 protein have been reported in two families with HLA class I deficiency, the so-called bare lymphocyte syndrome (BLS) type I. We have, to our knowledge, identified for the first time a splice site mutation in the TAP1 gene of another BLS patient. In addition, class I heavy chains (HCs) did not form the normal complex with tapasin in the endoplasmic reticulum (ER) of the cells of our patient.

Significant Association of KIR2DL3-HLA-C1 Combination with Cerebral Malaria and Implications for Co-evolution of KIR and HLA

Cerebral malaria is a major, life-threatening complication of Plasmodium falciparum malaria, and has very high mortality rate. In murine malaria models, natural killer (NK) cell responses have been shown to play a crucial role in the pathogenesis of cerebral malaria. To investigate the role of NK cells in the developmental process of human cerebral malaria, we conducted a case-control study examining genotypes for killer immunoglobulin-like receptors (KIR) and their human leukocyte antigen (HLA) class I ligands in 477 malaria patients. We found that the combination of KIR2DL3 and its cognate HLA-C1 ligand was significantly associated with the development of cerebral malaria when compared with non-cerebral malaria (odds ratio 3.14, 95% confidence interval 1.52–6.48, P = 0.00079, corrected P = 0.02). In contrast, no other KIR-HLA pairs showed a significant association with cerebral malaria, suggesting that the NK cell repertoire shaped by the KIR2DL3-HLA-C1 interaction shows certain functional responses that facilitate development of cerebral malaria. Furthermore, the frequency of the KIR2DL3-HLA-C1 combination was found to be significantly lower in malaria high-endemic populations. These results suggest that natural selection has reduced the frequency of the KIR2DL3-HLA-C1 combination in malaria high-endemic populations because of the propensity of interaction between KIR2DL3 and C1 to favor development of cerebral malaria. Our findings provide one possible explanation for KIR-HLA co-evolution driven by a microbial pathogen, and its effect on the global distribution of malaria, KIR and HLA.

Variations of human killer cell lectin-like receptors: common occurrence of NKG2-C deletion in the general population.

CD94 and NKG2 are members of the NK cell receptor families, and are encoded in the natural killer gene complex (NKC) on human chromosome 12p12–13, one of the candidate chromosomal regions for rheumatic diseases. To examine a possible association between variations in CD94 and NKG2 genes and genetic susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), we carried out a systematic polymorphism screening of NKG2-A (KLRC1), NKG2-C (KLRC2) and CD94 (KLRD1) genes on a population basis. In NKG2-A, previously considered to be highly conserved, 10 polymorphisms in the noncoding region and introns, as well as one rare variation leading to an amino acid substitution within the transmembrane region, c.238T>A (Cys80Ser), were detected. In NKG2-C, in addition to the previously described two nonsynonymous substitutions, c.5G>A (Ser2Asn) and c.305C>T (Ser102Phe), two polymorphisms were newly detected in the noncoding region. In CD94, only one single nucleotide substitution was identified in the 5′ untranslated region. When the patients and healthy individuals were genotyped for these variations, no significant association was observed. However, although statistically not significant, NKG2-A c.238T>A (Cys80Ser) was observed in three patients with RA, but in none of the healthy individuals and the patients with SLE. Unexpectedly, in the process of polymorphism screening, we identified homozygous deletion of NKG2-C in approximately 4.3% of healthy donors; under the assumption of Hardy–Weinberg equilibrium, the allele frequency of NKG2-C deletion was estimated to be 20.7%. These results demonstrated that, although human NKG2-A, -C and CD94 are generally conserved with respect to amino acid sequences, NKG2-A is polymorphic in the noncoding region, and that the number of genes encoded in the human NKC is variable among individuals, as previously shown for the leukocyte receptor complex (LRC), HLA and Fcγ receptor (FCGR) regions.

Evidence for Natural Selection on Leukocyte Immunoglobulin-like Receptors for HLA Class I in Northeast Asians

Human leukocyte antigen (HLA) plays a critical role in innate and adaptive immunity and is a well-known example of genes under natural selection. However, the genetic aspect of receptors recognizing HLA molecules has not yet been fully elucidated. Leukocyte immunoglobulin (Ig)-like receptors (LILRs) are a family of HLA class I-recognizing receptors comprising activating and inhibitory forms. We previously reported that the allele frequency of the 6.7 kb LILRA3 deletion is extremely high (71%) in the Japanese population, and we identified premature termination codon (PTC)-containing alleles. In this study, we observed a wide distribution of the high deletion frequency in Northeast Asians (84% in Korean Chinese, 79% in Man Chinese, 56% in Mongolian, and 76% in Buryat populations). Genotyping of the four HapMap populations revealed that LILRA3 alleles were in strong linkage disequilibrium with LILRB2 alleles in Northeast Asians. In addition, PTC-containing LILRA3 alleles were detected in Northeast Asians but not in non-Northeast Asians. Furthermore, flow-cytometric analysis revealed that the LILRB2 allele frequent in Northeast Asians was significantly associated with low levels of expression. F(ST) and extended-haplotype-homozygosity analysis for the HapMap populations provided evidence of positive selection acting on the LILRA3 and LILRB2 loci. Taken together, our results suggest that both the nonfunctional LILRA3 alleles and the low-expressing LILRB2 alleles identified in our study have increased in Northeast Asians because of natural selection. Our findings, therefore, lead us to speculate that not only HLA class I ligands but also their receptors might be sensitive to the local environment.

The human histocompatibility leukocyte antigen (HLA) haplotype is associated with the onset of postherpetic neuralgia after herpes zoster.

&NA; In some herpes zoster patients, pain persists for more than 3 months or more after healing of vesicular eruptions; this condition is termed postherpetic neuralgia (PHN). We have recently reported the association of the human histocompatibility leukocyte antigens (HLA) haplotype, HLA‐A*3303‐B*4403‐DRB1*1302 with PHN patients; however, it has not been determined whether the haplotype is also associated with herpes zoster that did not develop subsequent PHN. To distinguish whether the haplotype is associated with herpes zoster or the development of PHN, we examined if herpes zoster patients without subsequently PHN are also associated with the HLA haplotype or not. Herpes zoster patients were followed up for more than 6 months, and HLA alleles and haplotypes were compared among the PHN patients (n=52), the herpes zoster patients who did not develop PHN (n=42) and healthy controls (n=125). The frequencies of the risk haplotype in the PHN patients, in the healthy controls and in the herpes zoster patients without subsequent PHN were 16.3, 5.2 and 4.8%, respectively. While the frequency of the risk haplotype was significantly higher in the PHN patients than in the healthy controls (P=0.0006), no difference was observed between the herpes zoster patients without subsequent PHN and the healthy controls. No significant association was found between the duration of symptoms or the site of herpes zoster and the HLA alleles and the haplotype. These results suggest that the HLA‐A*3303‐B*4403‐DRB1*1302 haplotype plays an important role in the development of PHN after herpes zoster, but not in the onset of herpes zoster.

Association of HLA-A*3303-B*4403-DRB1*1302 haplotype, but not of TNFA promoter and NKp30 polymorphism, with postherpetic neuralgia (PHN) in the Japanese population.

Herpes zoster is a common disease caused by reactivation of the varicella zoster virus (VZV). In a small number of herpes zoster patients, pain persists beyond 4 weeks or more after healing of vesicular eruptions; this condition is termed postherpetic neuralgia (PHN). Positive associations of human histocompatibility leukocyte antigens (HLA) class I antigens, A33 and B44, with PHN in the Japanese population have been reported. Our hypothesis is that susceptibility genes to PHN might exist in the HLA region and the study objective is to further examine possible associations of genes in HLA class I, II and III regions, HLA-A, -B, -DRB1, tumor necrosis factor α (TNFA) promoter, and a natural killer cell activating receptor, NKp30 polymorphisms with PHN. Although TNFA or NKp30 in the class III region had been considered as a candidate locus, we found no associations of TNFA promoter or NKp30 polymorphisms with PHN in this study. We demonstrated that HLA-A*3303, -B*4403 and -DRB1*1302 alleles were significantly associated with PHN (P=0.0007 for A*3303, P=0.001 for B*4403 and P=0.001 for DRB1*1302). The frequency of the HLA-A*3303-B*4403-DRB1*1302 haplotype was also significantly higher in the PHN patients than in the healthy controls (P=0.0039). Our results suggest that this haplotype might be related to the pathogenesis of PHN.