HLA Bw4 Research Articles

PB1741 KIR‐LIGAND MISMATCH IS NOT PREDICTIVE FOR LEUKEMIA‐FREE SURVIVAL IN PATIENTS WITH FAVORABLE RISK AML AFTER AUTOLOGOUS STEM CELL TRANSPLANTATION.

Background:Evidence suggests that Natural Killer (NK) Cells may play a role in the prevention of relapse of Acute Myeloid Leukemia after allogeneic stem cell transplantation (alloSCT). In a specific condition being HLA mismatched transplants, NK cells may get activated because subsets of donor NK cells miss inhibitory HLA alleles on the patient's cells. This is called. KIR‐ligand mismatch, were subsets of donor NK cells get activated when the patient's HLA alleles are not fitting to one or two of the inhibitory KIRs expressed on the donor cells (Ruggeri, Science 2002). The prerequisite for effective KIR‐ligand mismatch seems to be extensive AGVHD prevention by CD34‐selection of the stem cell graft or intense immunosuppressive conditioning including ATG. In HLA‐matched sibling alloSCT the same beneficial effect of KIR‐ligand mismatch was observed when intense T cell depletion of the graft had been applied (Hsu et al., Blood 2005). It was shown that early post T‐cell depleted alloSCT normally hyporesponsive NK cells expressing KIR for non‐self HLA temporarily displayed full functional capacity (Hsu, Blood 2009).Aims:We hypothesized that a beneficial effect of KIR‐ligand mismatch effect may be present in autologous SCT (autoHCT) in AML, where GVHD obviously is impossible. This would imply that developing NK cells harboring one or two inhibitory KIR receptors (iKIRs) for which no fitting HLA‐allele is present might temporarily get activated in those patients that do not bear all three relevant groups of HLA‐alleles (Bw4, C1 and C2) for which inhibitory KIRs are almost always there (presence of KIR2DL2/3 (fitting C1), KIR2DL1 (fitting C2) and KIR3DL1 (fitting Bw4) is 100%, 92.7% and 92.1% respectively, Hsu, Blood 2005). Approximately 30% of the population bears all three ligands for these three iKIRs and are not susceptible for KIR‐ligand mismatch.Methods:We typed all autologous transplanted AML patients in our center with genetic favorable risk factors (according to ELN 2017) and in first complete remission for HLA Bw4, C1 and C2. 2‐year leukemia‐free survival (LFS) of those patients not susceptible for KIR‐ligand mismatch was compared with LFS of those being susceptible. It was expected that 33% of the auto‐transplanted patients would relapse (Vellenga, Blood 2011). Assuming a meaningful higher 2‐year LFS of 87% for patients missing at least ligand (i.e. 15% higher than observed for the whole population), 2‐year LFS in those not missing a KIR ligand would have been 32% (3x 72 = 216; 216 – (2x 87) = 216 – 174 = 32). To demonstrate this difference 9 patients per group are needed. However, because of the unequal distribution of the two groups (“missing no KIR ligand” vs. “missing at least 1i KIR ligand”) to ensure that the “not missing a KIR ligand group” contains 9 patients at least 3x9=27 patients are needed.Results:We identified 39 favorable risk auto‐transplanted AML patients. Twelve had all HLA ligands for all three iKIRs present, and 27 lacked at least one ligand. 2‐year LFS was 83% and 83% respectively. For those 11 that missed HLA C1 ligands (for which all individuals have a ligand (i.e. KIR2DL2/3) 2‐year LFS was 81%.Summary/Conclusion:2‐year LFS is very high after autoHCT in first complete remission in favorable risk AML and KIR‐ligand mismatch is not relevant.image

Role of natural killer cell activity in HIV infections, aging, and breast cancer survival

Abstract Natural killer cell activity participates in the elimination of tumors and virus infections. Data presented here include their role in a virus infection (HIV), metastatic breast cancer, and aging per se. We have analyzed two gene segments MHC HLA class I ligands and NK receptors Each have single amino acid differences. Together these alleles can identify long term survivors. Homozygosity of HLA–Bw4 supertype in HIV progression has been described. Since HLA-KIR genetic interactions of HLA-Bw4 with KIR (3DS1) only included Bw4, with the exception of B*27 and B*44 that use different mechanism of protection, on the clinical outcome of HIV infection, NK–ligand interactions (NKG2A-HLA-E with HLA class I leader peptides) explain the long term non-progressors (LTNP) to AIDS. HLA-B alleles have Methionine (Met) or Threonine (Thr) at second position (P2) of their leader peptide. HLA-Bw4 alleles, with exception of B*38, encode leader peptide with Thr at P2 and HLA-Bw6 genes that encode Met at P2 are B*07, B*08 and B*14. All others have Thr at P2. Comparisons of the Thr in LTNP with progressors, up to 15 years. with non-infected Caucasian controls were significant. Also, aged Mexicans demonstrated significant increase of homozygosity of Thr at P2 and Mexican patients with metastatic breast cancer were studied 10 years after treatment with surgery and chemotherapy, The 39 survivors demonstrated significant increase of homozygosity of Thr at P2 compared to controls without cancer and the 37 non survivors. These findings need to be studied in different malignancies and different ethnicities as well as in mouse models. Our findings support the immunogenetic theory of cancer and aging.

Three-dimensional structural modelling and calculation of electrostatic potentials of HLA Bw4 and Bw6 epitopes to explain the molecular basis for alloantibody binding: toward predicting HLA antigenicity and immunogenicity.

We have previously shown that qualitative assessment of surface electrostatic potential of HLA class I molecules helps explain serological patterns of alloantibody binding. We have now used a novel computational approach to quantitate differences in surface electrostatic potential of HLA B-cell epitopes and applied this to explain HLA Bw4 and Bw6 antigenicity. Protein structure models of HLA class I alleles expressing either the Bw4 or Bw6 epitope (defined by sequence motifs at positions 77 to 83) were generated using comparative structure prediction. The electrostatic potential in 3-dimensional space encompassing the Bw4/Bw6 epitope was computed by solving the Poisson-Boltzmann equation and quantitatively compared in a pairwise, all-versus-all fashion to produce distance matrices that cluster epitopes with similar electrostatics properties. Quantitative comparison of surface electrostatic potential at the carboxyl terminal of the α1-helix of HLA class I alleles, corresponding to amino acid sequence motif 77 to 83, produced clustering of HLA molecules in 3 principal groups according to Bw4 or Bw6 epitope expression. Remarkably, quantitative differences in electrostatic potential reflected known patterns of serological reactivity better than Bw4/Bw6 amino acid sequence motifs. Quantitative assessment of epitope electrostatic potential allowed the impact of known amino acid substitutions (HLA-B*07:02 R79G, R82L, G83R) that are critical for antibody binding to be predicted. We describe a novel approach for quantitating differences in HLA B-cell epitope electrostatic potential. Proof of principle is provided that this approach enables better assessment of HLA epitope antigenicity than amino acid sequence data alone, and it may allow prediction of HLA immunogenicity.

Natural Killer KIR3DS1 Is Closely Associated with HCV Viral Clearance and Sustained Virological Response in HIV/HCV Patients

AimTo evaluate the influence of the presence of the killer cell immunoglobulin-like receptor (KIR) 3DS1 on HCV treatment response in HIV/HCV genotype 1 co-infected patientsMethodsHIV/HCV co-infected patients were included. KIR3DS1, their specific HLA-B ligands and IL28B gene were genotyped. Reductions of plasma HCV RNA levels between baseline and week 1, week 2 and week 4 were analyzed for IL28B genotype and KIR3DS1 (HLA Bw4 or Bw6). Rapid and sustained virological response (RVR and SVR) rates were also analyzed.ResultsSixty HIV/HCV genotype 1 co-infected patients were included. Patients with KIR3DS1 and Bw4 had higher rates of HCV viral decline than those who were not carriers of KIR3DS1 (week1: p = 0.01; week2: p = 0.038; week 4: p = 0.03). Patients carrying KIR3DS1/Bw4 had higher rates of RVR and SVR than those who did not carry KIR3DS1 (RVR: 46.15% versus 17.02%, p = 0.012; SVR: 63.6% versus 13 26.5%, p = 0.031). With respect to patients carrying the IL28B-CC genotype, those with KIR3DS1/Bw4 had greater rates of HCV viral clearance (week1: p<0.001; week2: p = 0.01; week 4: p = 0.02), RVR (p = 0.015) and SVR (p = 0.029) than those not carrying KIR3DS1.ConclusionOur results show that the KIR3DS1 genotype has a positive effect on HCV viral clearance during the first weeks of Peg-IFN/RBV treatment in HCV/HCV co-infected patients bearing genotype 1, and higher RVR and SVR rates.

Activating KIR and HLA Bw4 Ligands Are Associated to Decreased Susceptibility to Pemphigus Foliaceus, an Autoimmune Blistering Skin Disease

The KIR genes and their HLA class I ligands have thus far not been investigated in pemphigus foliaceus (PF) and related autoimmune diseases, such as pemphigus vulgaris. We genotyped 233 patients and 204 controls for KIR by PCR-SSP. HLA typing was performed by LABType SSO reagent kits. We estimated the odds ratio, 95% confidence interval and performed logistic regression analyses to test the hypothesis that KIR genes and their known ligands influence susceptibility to PF. We found significant negative association between activating genes and PF. The activating KIR genes may have an overlapping effect in the PF susceptibility and the presence of more than three activating genes was protective (OR = 0.49, p = 0.003). A strong protective association was found for higher ratios activating/inhibitory KIR (OR = 0.44, p = 0.001). KIR3DS1 and HLA-Bw4 were negatively associated to PF either isolated or combined, but higher significance was found for the presence of both together (OR = 0.34, p<10−3) suggesting that the activating function is the major factor to interfere in the PF pathogenesis. HLA-Bw4 (80I and 80T) was decreased in patients. There is evidence that HLA-Bw4(80T) may also be important as KIR3DS1 ligand, being the association of this pair (OR = 0.07, p = 0.001) stronger than KIR3DS1-Bw4(80I) (OR = 0.31, p = 0.002). Higher levels of activating KIR signals appeared protective to PF. The activating KIR genes have been commonly reported to increase the risk for autoimmunity, but particularities of endemic PF, like the well documented influence the environmental exposure in the pathogenesis of this disease, may be the reason why activated NK cells probably protect against pemphigus foliaceus.

Investigation of the HLA class I antigens in patients with primary spontaneous pneumothorax

There is no report investigating the human leukocyte antigen system (HLA) class I alleles and haplotypes in the patients with primary spontaneous pneumothorax (PSP) without any familial history in the literature. We investigated the association of these alleles and haplotypes, and the occurrence of the PSP in Turkish patients. Materials and methods: The study group consisted of 20 patients diagnosed as PSP (without any familial history), and 20 healthy volunteers as control group. All the participants were Turkish male nonsmokers. Their genomic DNAs were extracted from venous samples, and the HLA class I alleles and haplotypes were analysed. Results: The HLA Bw4 allele was significantly increased in the study group (80% vs. 60%, P = 0.05). The frequencies of the HLA Cw7, and B18 alleles were higher in the study group (35% vs. 15%, and 20% vs. 0%, respectively, P > 0.05), and there was a high ratio of the Cw7 homozygotism (30% vs. 10%, P > 0.05). Conclusion: HLA Bw4, B18, and Cw7 alleles may play a genetic role in the development of the nonfamilial PSP in the Turkish population, but further accumulation of the cases are necessary to clarify whether the HLA-typing can confirm the development of a nonfamilial PSP.

Increased proportion of KIR3DS1 homozygotes in HIV-exposed uninfected individuals

Natural killer (NK) cell activity is increased in individuals who remain uninfected despite repeated exposures to HIV. Given that a combined major histocompatibility complex (MHC) class I and killer immunoglobulin-like receptor (KIR) KIR3D genotype has been linked to rate of HIV disease progression, we assessed whether these genotypes played a role in protection from infection. The study genotyped 80 HIV-exposed uninfected (EU) and 304 subjects in HIV primary infection (PI) at the MHC class IB and KIR3DS/L1 loci. KIR3D genotyping was performed by sequence-specific primer polymerase chain reaction using two pairs of specific primers for each locus. The MHC class IB locus was typed by sequence-specific oligonucleotide polymerase chain reaction and sequencing to resolve Bw4 and Bw6 alleles and the amino acid present at position 80. Comparison of the genetic distribution of KIR3D, HLA Bw4 and HLA Bw4-I80 genotypes in EU versus PI subjects reveal an increased proportion of KIR3DS1 homozygotes in EU (11/80, 13.8%) compared to subjects in PI (16/304, 5.3%). Analyses of combined MHC class I and KIR3D expression show no differences between the two populations. Homozygosity for the activating NK receptor KIR3DS1, may contribute to the more active NK cell function observed in EU and their relative resistance to HIV infection.

Flow cytometry with anti HLA-antibodies: a simple but highly sensitive method for monitoring chimerism and minimal residual disease after HLA-mismatched stem cell transplantation

Transplantation of HLA-mismatched stem cells may allow determination of chimerism status of single cells by differential expression of HLA molecules. Monoclonal antibodies against HLA antigens can be used to determine the HLA type of sub-populations by standard flow cytometry. Blood samples from 23 patients transplanted from HLA-mismatched family donors were monitored using HLA-specific antibodies. Suitable antibodies could be found for all donor recipient pairs by using differences in HLA Bw4 and Bw6 groups or other serological antigens. Pretransplant controls of donor and recipient were used to correct for variable fluorescence intensities of the antibodies and sub-populations. Owing to the high sensitivity, cell populations with a minimum frequency of 0.1% were detectable. Flow-cytometric analysis was confirmed by chimerism analysis of immunomagnetically isolated T cells by standard PCR technique. In addition to chimerism evaluation, HLA antibodies improved the detection of leukemic cells after transplantation with aberrant phenotype. In conclusion, flow cytometry using antibodies against HLA antigens is an interesting tool for determination of chimerism and minimal residual disease after HLA-mismatched transplantation. Information about the chimerism status is given on a single-cell level and allows fast and convenient analysis of sub-populations.

Impact of KIR and HLA Genotypes on Outcome in Nonmyeloablative Hematopoietic Cell Transplantation (HCT) Using HLA Matched Related Donors.

Killer immunoglobulin-like receptor (KIR)-HLA ligand mismatches in the graft-versus host direction (i.e. the recipient lacks an inhibitory HLA ligand for one or more donor KIRs) appears to be associated with improved outcome in patients (pts) receiving HLA-matched or mismatched T cell-depleted HCT. We investigated the impact of genotypic KIR-ligand mismatches in pts undergoing T cell-replete HCT from an HLA-identical or 5/6 antigen-matched related donor following nonmyeloablative conditioning. Donor KIR genotype (KIR2DL1,KIR2DL2/3,KIR3DL1) and HLA-B and C genotypes were analyzed in 92 pts who received an unmanipulated, G-CSF mobilized peripheral blood HCT from a 6/6 (n=88) or 5/6 (n=4) HLA-matched related donor following conditioning with cyclophosphamide (120mg/m2) and fludarabine (125mg/m2). The conditioning regimen included anti-thymocyte globulin (160mg/kg) for pts at increased risk for graft rejection (heavily transfused, 5/6 HLA-matched donors, or non-sibling related donors, n=19). Indications for transplantation included metastatic renal cell cancer (RCC, n= 37), treatment refractory solid tumors (n=26), hematologic malignancies (n=9) and non-malignant hematologic disorders (n=20). Cyclosporine, alone (n=11) or in combination with either mycophenolate mofetil (n=33) or methotrexate (n=48) was used as graft versus host disease (GVHD) prophylaxis. Sixty-eight donors (73%) demonstrated genotypic evidence of at least one KIR for which the pt lacked a corresponding inhibitory HLA C or B ligand (‘missing KIR ligand'). There was a trend towards a higher incidence of grades 3–4 acute GVHD (37% vs. 17%, p=0.08) and a significantly higher incidence of steroid refractory GVHD (16% vs 0%, p=0.02) in pts lacking an HLA ligand for one or more donor KIR. The ‘missing KIR ligand' effect on response rate was further evaluated in a subset of pts with metastatic RCC undergoing HCT. An HLA ligand for one or more donor KIR was absent in 28/38 (76%) RCC pts. A higher objective response rate (PR or CR) was seen in pts with one or more missing KIR-ligands (12/28, 43%) compared to those whose HLA genotype would predict inhibition of all donor KIRs (2/9, 22%, p=0.24). This effect was most pronounced in pts homozygous for HLA Bw6 who lacked HLA Bw4 ligands capable of inhibiting donor KIR3DL1. Compared to pts not homozygous for HLA-Bw6, Bw6 homozygous RCC pts had a higher response rate (24% vs 58%; p=0.047) and significantly prolonged survival (median 419 vs 1496 days; p=0.005) following HCT. A univariate analysis considering patient age, acute and chronic GVHD, and KIR-HLA ligand mismatches (C mismatch, B mismatch, or > 1 mismatch) showed that a missing HLA-Bw4 ligand and the absence of a ligand for > 1 donor KIR were each associated with improved survival in RCC patients; in a multivariate analysis, absence of an HLA Bw4 ligand for donor KIR3DL1 was the only factor that remained significantly associated with improved survival (p=0.015, OR 0.276, CI 0.097–0.781). Conclusion: KIR-HLA ligand mismatches appear to impact multiple transplant outcomes including the incidence and severity of acute GVHD, tumor response and survival following T-cell replete nonmyeloablative transplantation from HLA-matched related donors. Evaluation of a larger cohort of patients to confirm these findings is currently underway.

Rituximab-Mediated ADCC Is Augmented by Concomitant Interference with Inhibitory Self-Recognition by Human NK Cells.

The anti-CD20 monoclonal antibody, rituximab is widely used in the treatment of non-Hodgkin lymphomas. However, clinical responses to rituximab are variable. It has been demonstrated that rituximab can lead to tumor cell death by engaging the cellular immune system through antibody dependent cellular cytotoxicity (ADCC). NK cells have been shown to play a critical rule in eliminating rituximab coated B-cells, and the efficiency of killing depends on the interaction between the Fc portion of rituximab and the FcγRIII (CD16) activating receptor on NK cells. NK cell function is regulated by a complex balance of inhibitory and activating signals that enable the cells to survey their surrounding and selectively target and kill targets that do not display a “self” ligand (the “missing self hypothesis”). We hypothesized that interference with inhibitory self-recognition would augment rituximab-induced NK cell-mediated ADCC. Initial studies with the 721.221 B51 (HLA Bw4+) CD20+ cell line and NK92.26.5 cells transduced with human CD16 suggested that interference with KIR3DL1 recognition of Bw4 augmented tumor lysis in the presence of rituximab. To further test this hypothesis we employed human NK cells and autologous EBV transformed B cells from normal volunteers, and blocked the KIR3DL1 inhibitory receptor on NK cells using (Fab′)2 fragments of the DX9 antibody, in conjunction with rituximab exposure. Inhibitory blockade promoted rituximab-mediated cytotoxicity by peripheral blood mononuclear cells in three separate HLABw4+, KIR3DL1+ volunteers. These results suggest that manipulating the balance between inhibitory and activating receptors on NK cells might be applied to improve ADCC and ultimately lead to an improvement in response rates to rituximab and related lymphoma-directed antibodies that mediate ADCC. Supported by R01CA50633.

Effect of Bw4 and Bw6 epitope mismatches on antibody production, acute and chronic rejection, and graft survival in renal allografts.

Highly sensitized patients often have antibodies directed against the HLA Bw4 and Bw6 epitopes. Because of the high frequency of these epitopes, when present, these antibodies result in a high incidence of positive cross-matches. We sought to determine whether antibodies specific for Bw4 or Bw6 affected renal allograft outcome. The effect of mismatches for the HLA class I public epitopes, Bw4 and Bw6, was examined in 72 recipients of one haplotype matched recipients of living, related donor renal allografts selected to control for degree of HLA mismatch. Analysis of the production of HLA-specific antibody was performed for 180 recipients of failed cadaveric allografts by complement-dependent cytotoxicity tests and by an enzyme-linked immunoadsorbent assay (ELISA). No significant difference was observed in the incidence of acute rejection, number of rejection episodes or 1-year allograft survival among Bw4/6 matched versus mismatched recipients of one haplotype matched allografts. Additionally, no significant difference in the development of chronic allograft nephropathy was noted among 56 recipients followed long-term (> or =3 years). In the recipients of failed cadaveric transplants, Bw4/6 mismatching was associated with the frequency and magnitude of production of HLA-specific antibody. However, the panel reactive antibodies correlated with the number of HLA-A and -B mismatches, and there was no additional impact of Bw4/6 mismatching. IgG, HLA-specific antibodies were found to be significantly increased among patients homozygous for Bw4 or Bw6, whether or not there was a Bw4/6 mismatch. Mismatching for Bw4 or Bw6 does not confer any independent, increased risk for humoral sensitization or renal allograft failure.

HLA antigens in Omani patients with vitiligo.

Fifty native Omanis with vitiligo were studied to compare the incidence of HLA ABC and DR antigens with a control population. HLA Bw6 was found in 82% of patients compared with 49% controls (Pc = 0.0009 RR = 4.56) and HLA DR7 occurred in 40% of patients and 9% in controls (Pc = 0.00075 RR = 6.17). HLA DR7 was significantly increased in those patients with acrofacial, compared to focal disease (57% vs. 24% P = 0.038). Sixty-six per cent of the patients in this study had parents who were consanguineous and a positive family history was only found in this group with an incidence of 32%. HLA Bw4 segregated 100% with patients with a positive family history compared with 48% in consanguineous patients without a positive family history (Pc = 0.011 RR = 23). Vitiligo appears to be associated with different HLA antigens in different ethnic groups.