Academic Appointments

  • Emeritus Faculty - University Medical Line, Pathology

Honors & Awards

  • Paul I. Terasaki Clinical Science Award, American Society for Histocompatibility & Immunogenetics (9/30/15)

Professional Education

  • AB, San Francisco State University, Biology (1965)
  • MA, San Francisco State University, Embryology (1973)
  • PhD, University of California, Los Angeles, Microbiology & Immunology (1995)

2023-24 Courses

All Publications

  • Class II HLA Serotype Associations with End-Stage Renal Disease due to Membranous Nephropathy among Caucasians in the United States Charu, V., Tyan, D., Troxell, M., Kambham, N. NATURE PUBLISHING GROUP. 2020: 1569–71
  • Class II HLA Serotype Associations with End-Stage Renal Disease due to Membranous Nephropathy among Caucasians in the United States Charu, V., Tyan, D., Troxell, M., Kambham, N. NATURE PUBLISHING GROUP. 2020: 1569–71
  • DSA-FXM: Accelerated donor specific flow crossmatch discriminating class I and II antibody specifically and only to donor HLA for determining true incompatibility. Transplantation Chen, G., Lin, L., Tyan, D. B. 2019


    BACKGROUND: Worldwide, a final crossmatch is the gold standard for determining compatibility between patient and donor prior to solid organ transplantation and preventing hyperacute rejection. In the absence of autoantibodies, an incompatible crossmatch in a sensitized patient is attributed to mismatched donor human leukocyte antigens (HLA). However, current physical crossmatch methods cannot distinguish reactivity to HLA from other clinically irrelevant cell surface targets nor the class of HLA if it is the target. Result interpretation is difficult or impossible when autoantibodies, alloantibodies, and/or therapeutic antibodies coexist.METHODS: Herein we describe a unique donor specific flow crossmatch (DSA-FXM) that distinguishes HLA class I and/or II DSA bound to HLA antigens on the donor cell surface in their native conformation that is not impacted by rituximab, ATG (after absorption), or auto-antibodies. It is HLA specific.RESULTS: We compared results of single antigen antibody testing, auto- and allo-FXM using traditional FXM and our DSA-FXM method from 94 patients (enriched for auto+/allo+ pairs; n=64) against 110 donors (338 tests) and show that, in our cohort, positive traditional FXM results are not directed to donor HLA 60.25% of the time, and negative traditional FXM results are missing HLA DSA 36.2% of the time based on the DSA-FXM.CONCLUSIONS: We demonstrate that the DSA-FXM is able to define categorically distinct and clinically important HLA antibody profiles in half the time required for the standard FXM, potentially shortening cold ischemia time and providing clinicians with unambiguous essential information regarding HLA compatibility when time is critical.

    View details for DOI 10.1097/TP.0000000000002900

    View details for PubMedID 31385929

  • Mapping and definition of HLA class I and II serologic epitopes using an unbiased reverse engineering strategy. Human immunology Yamamoto, F., Wang, L., Chang, C., Tyan, D. B. 2019


    Current models describing HLA epitopes are both theoretical and empirical. Each has limitations yielding discordant results and increasingly complex modeling. The models make a priori assumptions that epitopes must be present only on the mature protein, solvent accessible, on the 'top' (peptide binding surface) of the molecule, restricted to the same class as the antibody, and in the same position on the target allele if reactive to more than one locus. Results obtained counter to these assumptions are routinely discounted. For the 17th International Histocompatibility and Immunogenetics Workshop, we developed a reverse engineering algorithm to define epitopes without these assumptions on a cohort of 332 primary transplant pairs. Complete NGS typing of the transcribed (including leader) genomic DNA for 11 HLA loci of donor and recipient and DSA assignment by single antigen beads was performed. Our results show that, when grouped by 16 class I and II allele specific DSA, uniform clusters and 172 specific amino acid target epitopes are recognized by recipients despite originating from disparate HLA pairs. Data also show that these targets can be in the leader, alpha 3, transmembrane and cytoplasmic domains, thus calling into question current assumptions regarding immunogenic epitopes. Comparisons of amino acid epitopes defined by the Terasaki and Duquesnoy groups (TerEp and EpRegistry) are given.

    View details for DOI 10.1016/j.humimm.2019.04.004

    View details for PubMedID 31122739

  • A reverse-engineering strategy utilizing the integration of single antigen beads and NGS HLA genotypes to detect potential antibody inducing epitopes. Human immunology Chang, C., Yamamoto, F., Tyan, D. B. 2019


    Central to the idea of antibody recognition is some degree of foreignness of the target antigen compared to the antibody producer. Epitopes are distinct regions on an antigen to which antibody can be elicited and bound. However, for HLA antigens, there is no consensus definition of what represents the minimal functional immunogenic unit of dissimilarity. To assess this in an unbiased way, we developed a reverse engineering software strategy based on donor specific antibodies defined by single antigen beads and full length genomic high resolution HLA typing by NGS of recipients and donors (332 transplant pairs). Starting with the ATG of Exon 1 and moving stepwise one amino acid at a time for each of the following triplets, the algorithm compared every possible amino acid triplet of the recipient and donor for 11 loci (A, B, C, DRB1, DRB3, DRB4, DRB5, DQA1, DQB1, DPA1, DPB1). Results were agnostic with respect to HLA class, not restricted to just the mature protein, and not influenced by existing maps (e.g., IMGT, or epitope models). We also developed web-based functions in the 17th IHIWS database to collect the unbiased triplets so that we could group the transplant pairs with the same donor specific antibodies and find shared triplets within the groups as potential core or essential epitopes that trigger the antibody formation. Profiling the pairs where the same DSA was identified led to identification of discrete amino acid triplets shared among the pairs irrespective of HLA match. The potential epitopes were mapped onto the 3D protein structure for reference.

    View details for PubMedID 30978444

  • Sensitization in Heart Transplantation: Emerging Knowledge A Scientific Statement From the American Heart Association CIRCULATION Colvin, M. M., Cook, J. L., Chang, P. P., Hsu, D. T., Kiernan, M. S., Kobashigawa, J. A., Lindenfeld, J., Masri, S., Miller, D. V., Rodriguez, E., Tyan, D. B., Zeevi, A., Amer Heart Assoc, Heart Failure Transplantation Comm, Council Clinical Cardiology, Council Cardiovasc Dis Young, Council Cardiovasc Stroke Nursing, Council Cardiovasc Surg Anesthesia 2019; 139 (12): E553–E578


    Sensitization, defined as the presence of circulating antibodies, presents challenges for heart transplant recipients and physicians. When present, sensitization can limit a transplantation candidate's access to organs, prolong wait time, and, in some cases, exclude the candidate from heart transplantation altogether. The management of sensitization is not yet standardized, and current therapies have not yielded consistent results. Although current strategies involve antibody suppression and removal with intravenous immunoglobulin, plasmapheresis, and antibody therapy, newer strategies with more specific targets are being investigated.

    View details for DOI 10.1161/CIR.0000000000000598

    View details for Web of Science ID 000469318300002

    View details for PubMedID 30776902

  • Human leukocyte antigens antibodies after lung transplantation: Primary results of the HALT study AMERICAN JOURNAL OF TRANSPLANTATION Hachem, R. R., Kamoun, M., Budev, M. M., Askar, M., Ahya, V. N., Lee, J. C., Levine, D. J., Pollack, M. S., Dhillon, G. S., Weill, D., Schechtman, K. B., Leard, L. E., Golden, J. A., Baxter-Lowe, L., Mohanakumar, T., Tyan, D. B., Yusen, R. D. 2018; 18 (9): 2285–94


    Donor-specific antibodies (DSA) to mismatched human leukocyte antigens (HLA) are associated with worse outcomes after lung transplantation. To determine the incidence and characteristics of DSA early after lung transplantation, we conducted a prospective multicenter observational study that used standardized treatment and testing protocols. Among 119 transplant recipients, 43 (36%) developed DSA: 6 (14%) developed DSA only to class I HLA, 23 (53%) developed DSA only to class II HLA, and 14 (33%) developed DSA to both class I and class II HLA. The median DSA mean fluorescence intensity (MFI) was 3197. We identified a significant association between the Lung Allocation Score and the development of DSA (HR = 1.02, 95% CI: 1.001-1.03, P = .047) and a significant association between DSA with an MFI ≥ 3000 and acute cellular rejection (ACR) grade ≥ A2 (HR = 2.11, 95% CI: 1.04-4.27, P = .039). However, we did not detect an association between DSA and survival. We conclude that DSA occur frequently early after lung transplantation, and most target class II HLA. DSA with an MFI ≥ 3000 have a significant association with ACR. Extended follow-up is necessary to determine the impact of DSA on other important outcomes.

    View details for PubMedID 29687961

  • Complement-activating donor-specific anti-HLA antibodies and solid organ transplant survival: A systematic review and meta-analysis PLOS MEDICINE Bouquegneau, A., Loheac, C., Aubert, O., Bouatou, Y., Viglietti, D., Empana, J., Ulloa, C., Murad, M., Legendre, C., Glotz, D., Jackson, A. M., Zeevi, A., Schaub, S., Taupin, J., Reed, E. F., Friedewald, J. J., Tyan, D. B., Suesal, C., Shapiro, R., Woodle, E., Hidalgo, L. G., O'Leary, J., Montgomery, R. A., Kobashigawa, J., Jouven, X., Jabre, P., Lefaucheur, C., Loupy, A. 2018; 15 (5): e1002572


    Anti-human leukocyte antigen donor-specific antibodies (anti-HLA DSAs) are recognized as a major barrier to patients' access to organ transplantation and the major cause of graft failure. The capacity of circulating anti-HLA DSAs to activate complement has been suggested as a potential biomarker for optimizing graft allocation and improving the rate of successful transplantations.To address the clinical relevance of complement-activating anti-HLA DSAs across all solid organ transplant patients, we performed a meta-analysis of their association with transplant outcome through a systematic review, from inception to January 31, 2018. The primary outcome was allograft loss, and the secondary outcome was allograft rejection. A comprehensive search strategy was conducted through several databases (Medline, Embase, Cochrane, and Scopus). A total of 5,861 eligible citations were identified. A total of 37 studies were included in the meta-analysis. Studies reported on 7,936 patients, including kidney (n = 5,991), liver (n = 1,459), heart (n = 370), and lung recipients (n = 116). Solid organ transplant recipients with circulating complement-activating anti-HLA DSAs experienced an increased risk of allograft loss (pooled HR 3.09; 95% CI 2.55-3.74, P = 0.001; I2 = 29.3%), and allograft rejection (pooled HR 3.75; 95% CI: 2.05-6.87, P = 0.001; I2 = 69.8%) compared to patients without complement-activating anti-HLA DSAs. The association between circulating complement-activating anti-HLA DSAs and allograft failure was consistent across all subgroups and sensitivity analyses. Limitations of the study are the observational and retrospective design of almost all included studies, the higher proportion of kidney recipients compared to other solid organ transplant recipients, and the inclusion of fewer studies investigating allograft rejection.In this study, we found that circulating complement-activating anti-HLA DSAs had a significant deleterious impact on solid organ transplant survival and risk of rejection. The detection of complement-activating anti-HLA DSAs may add value at an individual patient level for noninvasive biomarker-guided risk stratification.National Clinical Trial protocol ID: NCT03438058.

    View details for PubMedID 29799874

  • The management of antibodies in heart transplantation: An ISHLT consensus document JOURNAL OF HEART AND LUNG TRANSPLANTATION Kobashigawa, J., Colvin, M., Potena, L., Dragun, D., Crespo-Leiro, M. G., Delgado, J. F., Olymbios, M., Parameshwar, J., Patel, J., Reed, E., Reinsmoen, N., Rodriguez, E., Ross, H., Starling, R. C., Tyan, D., Urschel, S., Zuckermann, A. 2018; 37 (5): 537–47


    Despite the successes from refined peri-operative management techniques and immunosuppressive therapies, antibodies remain a serious cause of morbidity and mortality for patients both before and after heart transplantation. Patients awaiting transplant who possess antibodies against human leukocyte antigen are disadvantaged by having to wait longer to receive an organ from a suitably matched donor. The number of pre-sensitized patients has been increasing, a trend that is likely due to the increased use of mechanical circulatory support devices. Even patients who are not pre-sensitized can go on to produce donor-specific antibodies after transplant, which are associated with worse outcomes. The difficulty in managing antibodies is uncertainty over which antibodies are of clinical relevance, which patients to treat, and which treatments are most effective and safe. There is a distinct lack of data from prospective trials. An international consensus conference was organized and attended by 103 participants from 75 centers to debate contentious issues, determine the best practices, and formulate ideas for future research on antibodies. Prominent experts presented state-of-the-art talks on antibodies, which were followed by group discussions, and then, finally, a reconvened session to establish consensus where possible. Herein we address the discussion, consensus points, and research ideas.

    View details for PubMedID 29452978

  • Application, technical issues, and interpretation of C1q for graft outcome. Current opinion in organ transplantation Tyan, D. B. 2017; 22 (5): 505-510


    Significant interest and controversy surround the use of C1q for determining risk of antibody-mediated rejection (AMR) and graft loss. Alternate models for predicting outcomes have been proposed. This review focuses on the correlation of currently utilized assays for outcome, together with the technical and theoretical limitations, to distill current thinking.Results demonstrate that C1q status is significantly correlated with AMR and graft loss. There is general consensus that C1q is more clinically relevant for graft outcome than neat IgG MFI. IgG titers, subclass, and other complement assays have now been studied to determine if they are more relevant. Only IgG3 and possibly C3d fixation have shown added value to C1q for outcome correlation. Direct parallel titer comparisons of C1q and IgG are lacking and the correlation is unknown.Overall, results confirm the correlation with C1q+ donor-specific antibody (DSA) for AMR and graft loss. The association is stronger posttransplant. C1q+ de novo antibody appears to be especially detrimental portending graft loss in about 1-2.5 years post detection. Recommendations to biopsy and treat at time of de novo C1q+ antibody detection have been suggested by several groups.

    View details for DOI 10.1097/MOT.0000000000000454

    View details for PubMedID 28723698

    View details for PubMedCentralID PMC5662152

  • Immunological Effect of Skin Allograft in Burn Treatment: Impact on Future Vascularized Composite Allotransplantation JOURNAL OF BURN CARE & RESEARCH Garza, R. M., Press, B. H., Tyan, D. B., Karanas, Y. L., Lee, G. K. 2017; 38 (3): 169-173


    Skin allografts are the benchmark in temporary burn wound coverage, but allografts are hypothesized to place a high antigenic load on recipients. This project aims to determine the degree of human leukocyte antigen sensitization in burn patients treated with allografts. Serum was obtained from nine adult, nontransfused, and nontransplanted burn patients treated with allografts. Group 1 included patients tested in the acute burn period, while group 2 included different patients tested months to years after injury. A calculated panel reactive antibody (cPRA) percent was assessed for each patient, and data for a control group of 92 adult nontransplanted males were used for comparison. Each patient received allografts from an average 3.55 ± 1.24 different donors. cPRA in group 1 was lower than in group 2 (6 ± 12% vs 42 ± 33%, P = .08). cPRA in the study group was significantly higher than in the control group (26 ± 31% vs 8 ± 17%, P = .0075). Burn patients who receive skin allograft demonstrate increased immunological sensitization compared with unsensitized controls. Detection of human leukocyte antigen antibody is lower in the acute burn period than months to years after injury. Increased sensitization may ultimately limit burn patients' candidacy for vascularized composite allotransplantation or decrease success of these procedures.

    View details for DOI 10.1097/BCR.0000000000000458

    View details for Web of Science ID 000399817800007

  • CPRA for allocation of kidneys in the US: More candidates >= 98% CPRA, lower positive crossmatch rates and improved transplant rates for sensitized patients HUMAN IMMUNOLOGY Baxter-Lowe, L. A., Kucheryavaya, A., Tyan, D., Reinsmoen, N. 2016; 77 (5): 395-402


    In 2009 calculated panel reactive antibody (CPRA) replaced PRA as the metric for HLA sensitization in the US kidney allocation system. During the next four years, registrants with at least one unacceptable antigen increased (34-40%) and registrants with ≥98% PRA/CPRA increased from 7% to 9% of the waitlist. These changes were accompanied by a reduction in kidney offers refused for positive crossmatch: 14,137 (1.7%) in 2009 and 3,310 in 2013 (0.4%). Registrants with ≥98% PRA/CPRA had highest rates of refusal but also showed substantial improvement (20% in 2009 vs 8% in 2013). For registrants with ≥98% PRA/CPRA, 45% of accepted offers in 2009 were not transplanted into the intended recipient compared to 11% in 2013. Transplant rates remained low for these patients (∼50/1000 active patient-years), but rates improved for patients with 80-97% PRA/CPRA (223/1000 active patient-years in 2009 vs 354/1000 in 2013). In 2013, 40% regraft candidates had CPRA ≥98% compared to 4% of primary graft candidates. More females than males were ≥98% CPRA (14% vs 7%) and more females had CPRA above 0 (50% vs 28%). In the CPRA era, listing of unacceptable antigens increased, positive crossmatches were diminished and transplant rates for sensitized patients improved.

    View details for DOI 10.1016/j.humimm.2016.03.003

    View details for Web of Science ID 000375741700004

    View details for PubMedID 27012168

  • Antibody-mediated rejection of the lung: A consensus report of the International Society for Heart and Lung Transplantation. journal of heart and lung transplantation Levine, D. J., Glanville, A. R., Aboyoun, C., Belperio, J., Benden, C., Berry, G. J., Hachem, R., Hayes, D., Neil, D., Reinsmoen, N. L., Snyder, L. D., Sweet, S., Tyan, D., Verleden, G., Westall, G., Yusen, R. D., Zamora, M., Zeevi, A. 2016; 35 (4): 397-406


    Antibody-mediated rejection (AMR) is a recognized cause of allograft dysfunction in lung transplant recipients. Unlike AMR in other solid-organ transplant recipients, there are no standardized diagnostic criteria or an agreed-upon definition. Hence, a working group was created by the International Society for Heart and Lung Transplantation with the aim of determining criteria for pulmonary AMR and establishing a definition. Diagnostic criteria and a working consensus definition were established. Key diagnostic criteria include the presence of antibodies directed toward donor human leukocyte antigens and characteristic lung histology with or without evidence of complement 4d within the graft. Exclusion of other causes of allograft dysfunction increases confidence in the diagnosis but is not essential. Pulmonary AMR may be clinical (allograft dysfunction which can be asymptomatic) or sub-clinical (normal allograft function). This consensus definition will have clinical, therapeutic and research implications.

    View details for DOI 10.1016/j.healun.2016.01.1223

    View details for PubMedID 27044531

  • Very high resolution single pass HLA genotyping using amplicon sequencing on the 454 next generation DNA sequencers: Comparison with Sanger sequencing HUMAN IMMUNOLOGY Yamamoto, F., Hoeglund, B., Fernandez-Vina, M., Tyan, D., Rastrou, M., Williams, T., Moonsamy, P., Goodridge, D., Anderson, M., Erlich, H. A., Holcomb, C. L. 2015; 76 (12): 910-916

    View details for DOI 10.1016/j.humimm.2015.05.002

    View details for Web of Science ID 000366437900006

    View details for PubMedID 26037172

  • Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing. Journal of clinical microbiology Sahoo, M. K., Tan, S. K., Chen, S. F., Kapusinszky, B., Concepcion, K. R., Kjelson, L., Mallempati, K., Farina, H. M., Fernández-Viña, M., Tyan, D., Grimm, P. C., Anderson, M. W., Concepcion, W., Pinsky, B. A. 2015; 53 (10): 3226-3233


    BK virus (BKV) infection and end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep sequencing methodology and bioinformatics pipeline that identifies BKV variants across the genome and at BKV-specific HLA-A2, HLA-B0702, and HLA-B08 restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets and fragmentation libraries were sequenced on the Ion Torrent PGM. An error model and variant calling algorithm were developed to accurately identify rare variants. 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range 2-37, interquartile range 10), with the majority of variants (77%) detected at a frequency of less than 5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

    View details for DOI 10.1128/JCM.01385-15

    View details for PubMedID 26202116

  • HLA desensitization with bortezomib in a highly sensitized pediatric patient PEDIATRIC TRANSPLANTATION May, L. J., Yeh, J., Maeda, K., Tyan, D. B., Chen, S., Kaufman, B. D., Bernstein, D., Rosenthal, D. N., Hollander, S. A. 2014; 18 (8): E280-E282


    The proteasome inhibitor bortezomib has been used with variable success in the treatment of AMR following heart transplant. There is limited experience with this agent as a pretransplant desensitizing therapy. We report a case of successful HLA desensitization with a bortezomib-based protocol prior to successful heart transplantation. A nine-yr-old boy with dilated cardiomyopathy, not initially sensitized to HLA (cPRA of zero), required three days of ECMO, followed by implantation of a Heartmate II LVAD. Within six wk, the patient developed de novo class I IgG and C1q complement-fixing HLA antibodies with a cPRA of 100%. Two doses of IVIG (2 g/kg) failed to reduce antibody levels, although two courses of a novel desensitization protocol consisting of rituximab (375 mg/m(2) ), bortezomib (1.3 mg/m(2)  × 5 doses), and plasmapheresis reduced his cPRA to 0% and 87% by the C1q and IgG assays, respectively. He underwent heart transplantation nearly two months later. The patient is now >one yr post-transplant, is free of both AMR and ACR, and has no detectable donor-specific antibodies by IgG or C1q. Proteasome inhibition with bortezomib and plasmapheresis may be an effective therapy for HLA desensitization pretransplant.

    View details for DOI 10.1111/petr.12347

    View details for Web of Science ID 000344360500006

  • Antibody-mediated rejection despite inhibition of terminal complement TRANSPLANT INTERNATIONAL Bentall, A., Tyan, D. B., Sequeira, F., Everly, M. J., Gandhi, M. J., Cornell, L. D., Li, H., Henderson, N. A., Raghavaiah, S., Winters, J. L., Dean, P. G., Stegall, M. D. 2014; 27 (12): 1235-1243


    Terminal complement blockade has been shown to decrease the incidence of early acute antibody-mediated rejection (eAMR) in the first month after positive cross-match kidney transplant recipients, yet some patients still develop eAMR. The current study investigated possible mechanisms of eAMR despite eculizumab treatment. Of the 26 patients treated with eculizumab, two developed clinical eAMR and another patient developed histologic signs of eAMR without graft dysfunction ('subclinical eAMR'). Twenty-three did not have histologic injury on early surveillance biopsies. All 26 patients had therapeutic levels of eculizumab and showed complete blockade of complement in hemolytic assays. High levels of donor-specific alloantibody (DSA) including total IgG, IgG3, and C1q+ DSA were present in patients with and without eAMR, and none correlated well with eAMR. In contrast, IgM DSA was present in only four patients after transplantation: the two patients with clinical eAMR, one patient with subclinical AMR, and one patient without eAMR (P = 0.006 correlation with eAMR). Both clinical eAMR episodes were easily treated with plasma exchange which removed IgM more completely and rapidly than IgG, resulting in normalization of function and histology. These data suggest a possible role of antidonor IgM DSA in the pathogenesis of eAMR in patients treated with terminal complement blockade ( Identifier: NCT00670774).

    View details for DOI 10.1111/tri.12396

    View details for Web of Science ID 000344789900009

  • HLA desensitization with bortezomib in a highly sensitized pediatric patient. Pediatric transplantation May, L. J., Yeh, J., Maeda, K., Tyan, D. B., Chen, S., Kaufman, B. D., Bernstein, D., Rosenthal, D. N., Hollander, S. A. 2014; 18 (8): E280-2


    The proteasome inhibitor bortezomib has been used with variable success in the treatment of AMR following heart transplant. There is limited experience with this agent as a pretransplant desensitizing therapy. We report a case of successful HLA desensitization with a bortezomib-based protocol prior to successful heart transplantation. A nine-yr-old boy with dilated cardiomyopathy, not initially sensitized to HLA (cPRA of zero), required three days of ECMO, followed by implantation of a Heartmate II LVAD. Within six wk, the patient developed de novo class I IgG and C1q complement-fixing HLA antibodies with a cPRA of 100%. Two doses of IVIG (2 g/kg) failed to reduce antibody levels, although two courses of a novel desensitization protocol consisting of rituximab (375 mg/m(2) ), bortezomib (1.3 mg/m(2)  × 5 doses), and plasmapheresis reduced his cPRA to 0% and 87% by the C1q and IgG assays, respectively. He underwent heart transplantation nearly two months later. The patient is now >one yr post-transplant, is free of both AMR and ACR, and has no detectable donor-specific antibodies by IgG or C1q. Proteasome inhibition with bortezomib and plasmapheresis may be an effective therapy for HLA desensitization pretransplant.

    View details for DOI 10.1111/petr.12347

    View details for PubMedID 25174602

  • Sensitization from transfusion in patients awaiting primary kidney transplant. Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association Yabu, J. M., Anderson, M. W., Kim, D., Bradbury, B. D., Lou, C. D., Petersen, J., Rossert, J., Chertow, G. M., Tyan, D. B. 2013; 28 (11): 2908-2918


    Sensitization to human leukocyte antigen (HLA) from red blood cell (RBC) transfusion is poorly quantified and is based on outdated, insensitive methods. The objective was to evaluate the effect of transfusion on the breadth, magnitude and specificity of HLA antibody formation using sensitive and specific methods.Transfusion, demographic and clinical data from the US Renal Data System were obtained for patients on dialysis awaiting primary kidney transplant who had ≥2 HLA antibody measurements using the Luminex single-antigen bead assay. One cohort included patients with a transfusion (n = 50) between two antibody measurements matched with up to four nontransfused patients (n = 155) by age, sex, race and vintage (time on dialysis). A second crossover cohort (n = 25) included patients with multiple antibody measurements before and after transfusion. We studied changes in HLA antibody mean fluorescence intensity (MFI) and calculated panel reactive antibody (cPRA).In the matched cohort, 10 of 50 (20%) transfused versus 6 of 155 (4%) nontransfused patients had a ≥10 HLA antibodies increase of >3000 MFI (P = 0.0006); 6 of 50 (12%) transfused patients had a ≥30 antibodies increase (P = 0.0007). In the crossover cohort, the number of HLA antibodies increasing >1000 and >3000 MFI was higher in the transfused versus the control period, P = 0.03 and P = 0.008, respectively. Using a ≥3000 MFI threshold, cPRA significantly increased in both matched (P = 0.01) and crossover (P = 0.002) transfused patients.Among prospective primary kidney transplant recipients, RBC transfusion results in clinically significant increases in HLA antibody strength and breadth, which adversely affect the opportunity for future transplant.

    View details for DOI 10.1093/ndt/gft362

    View details for PubMedID 24009295

  • Consensus Guidelines on the Testing and Clinical Management Issues Associated With HLA and Non-HLA Antibodies in Transplantation TRANSPLANTATION Tait, B. D., Suesal, C., Gebel, H. M., Nickerson, P. W., Zachary, A. A., Claas, F. H., Reed, E. F., Bray, R. A., Campbell, P., Chapman, J. R., Coates, P. T., Colvin, R. B., Cozzi, E., Doxiadis, I. I., Fuggle, S. V., Gill, J., Glotz, D., Lachmann, N., Mohanakumar, T., Suciu-Foca, N., Sumitran-Holgersson, S., Tanabe, K., Taylor, C. J., Tyan, D. B., Webster, A., Zeevi, A., Opelz, G. 2013; 95 (1): 19-47


    The introduction of solid-phase immunoassay (SPI) technology for the detection and characterization of human leukocyte antigen (HLA) antibodies in transplantation while providing greater sensitivity than was obtainable by complement-dependent lymphocytotoxicity (CDC) assays has resulted in a new paradigm with respect to the interpretation of donor-specific antibodies (DSA). Although the SPI assay performed on the Luminex instrument (hereafter referred to as the Luminex assay), in particular, has permitted the detection of antibodies not detectable by CDC, the clinical significance of these antibodies is incompletely understood. Nevertheless, the detection of these antibodies has led to changes in the clinical management of sensitized patients. In addition, SPI testing raises technical issues that require resolution and careful consideration when interpreting antibody results.With this background, The Transplantation Society convened a group of laboratory and clinical experts in the field of transplantation to prepare a consensus report and make recommendations on the use of this new technology based on both published evidence and expert opinion. Three working groups were formed to address (a) the technical issues with respect to the use of this technology, (b) the interpretation of pretransplantation antibody testing in the context of various clinical settings and organ transplant types (kidney, heart, lung, liver, pancreas, intestinal, and islet cells), and (c) the application of antibody testing in the posttransplantation setting. The three groups were established in November 2011 and convened for a "Consensus Conference on Antibodies in Transplantation" in Rome, Italy, in May 2012. The deliberations of the three groups meeting independently and then together are the bases for this report.A comprehensive list of recommendations was prepared by each group. A summary of the key recommendations follows. Technical Group: (a) SPI must be used for the detection of pretransplantation HLA antibodies in solid organ transplant recipients and, in particular, the use of the single-antigen bead assay to detect antibodies to HLA loci, such as Cw, DQA, DPA, and DPB, which are not readily detected by other methods. (b) The use of SPI for antibody detection should be supplemented with cell-based assays to examine the correlations between the two types of assays and to establish the likelihood of a positive crossmatch (XM). (c) There must be an awareness of the technical factors that can influence the results and their clinical interpretation when using the Luminex bead technology, such as variation in antigen density and the presence of denatured antigen on the beads. Pretransplantation Group: (a) Risk categories should be established based on the antibody and the XM results obtained. (b) DSA detected by CDC and a positive XM should be avoided due to their strong association with antibody-mediated rejection and graft loss. (c) A renal transplantation can be performed in the absence of a prospective XM if single-antigen bead screening for antibodies to all class I and II HLA loci is negative. This decision, however, needs to be taken in agreement with local clinical programs and the relevant regulatory bodies. (d) The presence of DSA HLA antibodies should be avoided in heart and lung transplantation and considered a risk factor for liver, intestinal, and islet cell transplantation. Posttransplantation Group: (a) High-risk patients (i.e., desensitized or DSA positive/XM negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. (b) Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. (c) Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3 to 12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results.A comprehensive list of recommendations is provided covering the technical and pretransplantation and posttransplantation monitoring of HLA antibodies in solid organ transplantation. The recommendations are intended to provide state-of-the-art guidance in the use and clinical application of recently developed methods for HLA antibody detection when used in conjunction with traditional methods.

    View details for DOI 10.1097/TP.0b013e31827a19cc

    View details for Web of Science ID 000313058700007

    View details for PubMedID 23238534

  • C1q Assay for the Detection of Complement Fixing Antibody to HLA Antigens. Methods in molecular biology (Clifton, N.J.) Chen, G., Tyan, D. B. 2013; 1034: 305-311


    Solid phase Luminex(®) and flow cytometric single antigen bead assays offer exquisite sensitivity and specificity for HLA antibody detection. Unlike the historical complement-dependent cytotoxicity (CDC) method, these assays do not distinguish complement fixing from non-complement fixing antibody, the former of which are considered the most clinically relevant in the peri-transplant period. This chapter describes a novel solid phase C1q binding assay to distinguish HLA antibodies that can bind the first component of complement (C1q). These antibodies have the capacity to initiate the complement cascade irrespective of whether that actually occurs. The C1q assay detects many more complement fixing antibodies than are observed by the less sensitive and less specific CDC assay.

    View details for DOI 10.1007/978-1-62703-493-7_16

    View details for PubMedID 23775744

  • New approaches for detecting complement-fixing antibodies CURRENT OPINION IN ORGAN TRANSPLANTATION Tyan, D. B. 2012; 17 (4): 409-415


    Complement-fixing human leukocyte antigen (HLA) antibodies are a contraindication to solid organ transplant. Newer solid phase assays for HLA antibody definition have created treatment quandaries because many more antibodies are detected by these methods. It is unclear which of the antibodies identified are clinically relevant as all IgG-binding antibodies are detected whether they can fix complement or not.Two methods have been developed to assess complement-fixing capability in the solid phase assays: C4d and C1q. These assays, especially the sensitive C1q method, have been reported to more closely correlate with renal and cardiac graft dysfunction, rejection, and graft failure than antibodies detected only by the traditional IgG method. Additionally, the C1q method can be used to predict and monitor desensitization status pre and posttransplant in patients being treated with intravenous immunoglobulin.The availability of these complement-fixing assays provides new tools for making treatment decisions by discriminating antibodies with known clinical relevance and to assess the clinical relevance of binding antibodies that cannot fix complement. The assays also provide a means of assessing when to transplant a patient undergoing desensitization or when to discontinue augmented immunosuppression for resolving antibody-mediated rejection.

    View details for DOI 10.1097/MOT.0b013e328355fb9b

    View details for Web of Science ID 000306483100013

    View details for PubMedID 22710388

  • Complement-fixing donor-specific antibodies identified by a novel C1q assay are associated with allograft loss PEDIATRIC TRANSPLANTATION Sutherland, S. M., Chen, G., Sequeira, F. A., Lou, C. D., Alexander, S. R., Tyan, D. B. 2012; 16 (1): 12-17


    Long-term outcomes following renal transplantation remain disappointing. Recently, interest has focused on the antibody-mediated component of allograft injury and the deleterious effects of DSA. We applied a novel C1q solid-phase assay in parallel with the standard IgG SAB assay to identify DSA with the potential to activate complement by binding C1q. Among 193 consecutive renal transplants at our center, 19.2% developed de novo DSA following transplantation. Of the patients with DSA, 43% had antibodies that bound C1q in vitro [C1q+ DSA]. Patients with C1q+ DSA were more likely to develop allograft loss than patients with DSA that did not bind C1q (46.7% vs. 15%; p = 0.04); patients with C1q+ DSA were nearly six times more likely to lose their transplant than those with C1q- DSA. Additionally, patients with C1q+ DSA who underwent allograft biopsy were more likely to demonstrate C4d deposition (50% vs. 8%; p = 0.03) and meet criteria for acute rejection (60% vs. 17%; p = 0.02) when compared with patients with DSA that did not bind C1q. These data suggest that DSA with the ability to activate complement, as determined by this novel C1q assay, are associated with greater risk of acute rejection and allograft loss.

    View details for DOI 10.1111/j.1399-3046.2011.01599.x

    View details for Web of Science ID 000299154200010

    View details for PubMedID 22093755

  • Complement (C1q) fixing solid-phase screening for HLA antibodies increases the availability of compatible platelet components for refractory patients TRANSFUSION Fontaine, M. J., Kuo, J., Chen, G., Galel, S. A., Miller, E., Sequeira, F., Viele, M., Goodnough, L. T., Tyan, D. B. 2011; 51 (12): 2611-2618


    Immune refractoriness to platelet (PLT) transfusion is primarily due to HLA antibody. Patients at our institution are identified as refractory due to HLA by a Luminex-based immunoglobulin (Ig)G-single-antigen-bead (SAB) assay, but in highly sensitized patients, antigen-negative compatible donors cannot be found due to the high sensitivity of the IgG-SAB method. We developed an assay that detects only HLA antibodies binding the first complement component (C1q). We hypothesized that the C1q-SAB method might be more relevant than the IgG-SAB method because the antibodies identified may activate the complement cascade causing PLT destruction.Thirteen highly sensitized refractory patients received 177 PLT units incompatible by the IgG-SAB method. They were retrospectively retested by the C1q-SAB method. Calculated percent reactive antibody (CPRA) and HLA antibody specificities were compared between the two methods and corrected count increment (CCI) values were analyzed. Additionally the impact of ABO compatibility on CCI responses was evaluated.The mean CPRA value was significantly lower by C1q-SAB (60%) than by IgG-SAB (94%; p < 0.05). Patients showed significantly better CCI (10.6 × 10(9) ± 0.8 × 10(9) /L) with C1q-compatible (n = 134) than with C1q-incompatible PLTs (n = 43) (2.5 × 10(9) ± 0.9 × 10(9) /L/m(2) ; p < 0.0001). ABO compatibility did not significantly impact the CCI values (p < 0.0001). Our results show that 75% of PLT units previously considered incompatible were actually compatible.For highly refractory patients to PLT transfusion, the C1q-based SAB binding assay may be a better method for identifying clinically relevant HLA antibodies and selecting PLT units that will result in acceptable CCI.

    View details for DOI 10.1111/j.1537-2995.2011.03194.x

    View details for PubMedID 21615749

  • Novel C1q assay reveals a clinically relevant subset of human leukocyte antigen antibodies independent of immunoglobulin G strength on single antigen beads HUMAN IMMUNOLOGY Chen, G., Sequeira, F., Tyan, D. B. 2011; 72 (10): 849-858


    It has been known for 40 years that cytotoxic human leukocyte antigen (HLA) antibodies are associated with graft rejection. However, the complement-dependent cytotoxicity assay (CDC) used to define these clinically deleterious antibodies suffers from a lack of sensitivity and specificity. Recently, methods exploiting immunoglobulin G (IgG) antibody binding to HLA single antigen beads (SAB) have overcome sensitivity and specificity drawbacks but introduced a new dilemma: which of the much broader set of antibodies defined by these methods are clinically relevant. To address this, we developed a complement-fixing C1q assay on the HLA SAB that combines sensitivity, specificity, and functional potential into one assay. We compared the CDC, IgG, and C1q assays on 96 sera having 2,118 defined antibodies and determined that CDC detects only 19% of complement-fixing antibodies detected by C1q, whereas C1q detects only 47% of antibodies detected by IgG. In the same patient, there is no predictability by IgG mean fluorescence intensity (MFI) as to which of the antibodies will bind C1q because fixation is independent of MFI values. In 3 clinical studies, C1q(+) antibodies appear to be more highly correlated than those detected by IgG alone for antibody-mediated rejection in hearts as well as for kidney transplant glomerulopathy and graft failure.

    View details for DOI 10.1016/j.humimm.2011.07.001

    View details for Web of Science ID 000295901100011

    View details for PubMedID 21791230

  • A multi-site study using high-resolution HLA genotyping by next generation sequencing TISSUE ANTIGENS HOLCOMB, C. L., Hoeglund, B., ANDERSON, M. W., BLAKE, L. A., Boehme, I., Egholm, M., Ferriola, D., Gabriel, C., Gelber, S. E., Goodridge, D., Hawbecker, S., Klein, R., Ladner, M., Lind, C., Monos, D., Pando, M. J., Proell, J., Sayer, D. C., Schmitz-Agheguian, G., Simen, B. B., THIELE, B., Trachtenberg, E. A., Tyan, D. B., Wassmuth, R., White, S., Erlich, H. A. 2011; 77 (3): 206-217


    The high degree of polymorphism at human leukocyte antigen (HLA) class I and class II loci makes high-resolution HLA typing challenging. Current typing methods, including Sanger sequencing, yield ambiguous typing results because of incomplete genomic coverage and inability to set phase for HLA allele determination. The 454 Life Sciences Genome Sequencer (GS FLX) next generation sequencing system coupled with conexio atf software can provide very high-resolution HLA genotyping. High-throughput genotyping can be achieved by use of primers with multiplex identifier (MID) tags to allow pooling of the amplicons generated from different individuals prior to sequencing. We have conducted a double-blind study in which eight laboratory sites performed amplicon sequencing using GS FLX standard chemistry and genotyped the same 20 samples for HLA-A, -B, -C, DPB1, DQA1, DQB1, DRB1, DRB3, DRB4, and DRB5 (DRB3/4/5) in a single sequencing run. The average sequence read length was 250 base pairs and the average number of sequence reads per amplicon was 672, providing confidence in the allele assignments. Of the 1280 genotypes considered, assignment was possible in 95% of the cases. Failure to assign genotypes was the result of researcher procedural error or the presence of a novel allele rather than a failure of sequencing technology. Concordance with known genotypes, in cases where assignment was possible, ranged from 95.3% to 99.4% for the eight sites, with overall concordance of 97.2%. We conclude that clonal pyrosequencing using the GS FLX platform and CONEXIO ATF software allows reliable identification of HLA genotypes at high resolution.

    View details for DOI 10.1111/j.1399-0039.2010.01606.x

    View details for Web of Science ID 000287092600005

    View details for PubMedID 21299525

  • C1q-Fixing Human Leukocyte Antigen Antibodies Are Specific for Predicting Transplant Glomerulopathy and Late Graft Failure After Kidney Transplantation TRANSPLANTATION Yabu, J. M., Higgins, J. P., Chen, G., Sequeira, F., Busque, S., Tyan, D. B. 2011; 91 (3): 342-347


    Human leukocyte antigen (HLA) antibodies, especially those that fix complement, are associated with antibody-mediated rejection and graft failure. The C1q assay on single antigen beads detects a subset of HLA antibodies that can fix complement and precede C4d deposition. The aim of this study was to determine whether C1q-fixing antibodies distinguish de novo donor-specific antibodies (DSA) that are clinically relevant and harmful.We retrospectively studied 31 of 274 kidney transplant recipients who had pretransplant and concurrent biopsy and serum specimens, 13 with C4d-positive and 18 with C4d-negative staining. We measured IgG and C1q DSA pretransplant and at the time of biopsy using single antigen bead assays. We identified 13 recipients who developed de novo DSA by IgG or C1q and examined associations with C4d deposition, transplant glomerulopathy, and graft failure.Testing for DSA by IgG is more sensitive for C4d deposition (IgG: 100%, 95% confidence interval [CI] 0.60-1; C1q: 75%, 95% CI 0.36-0.96). Testing for DSA by C1q is more specific for transplant glomerulopathy (C1q: 81%, 95% CI 0.57-0.94; IgG: 67%, 95% CI 0.43-0.85) and graft loss (C1q: 79%, 95% CI 0.54-0.93; IgG: 63%, 95% CI 0.39-0.83). Absence of de novo DSA by IgG and C1q has a high negative predictive value for the absence of C4d deposition (IgG: 100%, 95% CI 0.73-1; C1q: 88%, 95% CI 0.62-0.98), transplant glomerulopathy (IgG: 100%, 95% CI 0.73-1; C1q: 100%, 95% CI 0.77-1), and graft failure (IgG: 86%, 95% CI 0.56-0.97; C1q: 88%, 95% CI 0.62-0.98).Monitoring patients with the C1q assay, which detects antibodies that fix complement, offers a minimally invasive means of identifying patients at risk for transplant glomerulopathy and graft loss.

    View details for DOI 10.1097/TP.0b013e318203fd26

    View details for Web of Science ID 000286624400014

    View details for PubMedID 21116220

  • Clinical usefulness of a novel C1q assay to detect immunoglobulin G antibodies capable of fixing complement in sensitized pediatric heart transplant patients JOURNAL OF HEART AND LUNG TRANSPLANTATION Chin, C., Chen, G., Sequeria, F., Berry, G., Siehr, S., Bernstein, D., Rosenthal, D., Reinhartz, O., Tyan, D. 2011; 30 (2): 158-163


    Donor-specific antibodies (DSA) against human leukocyte antigens complicate transplantation with the potential for acute antibody-mediated rejection (AMR). Complement-fixing antibodies are required to initiate the complement cascade. Not all DSAs, however, can fix complement.A novel C1q assay was developed to detect the sub-set of immunoglobulin G (IgG) antibodies capable of fixing complement. Sera from 18 pediatric heart transplant patients were analyzed for DSAs using a Luminex platform (Luminex Inc, Austin, TX) and commercially available single-antigen bead assay kits. Biopsy specimens were assessed for AMR using histopathologic criteria and immunohistochemical staining.During the study period, 5 patients had AMR; of these, 2 were C1q virtual crossmatch positive (VXM+) and had persistent C1q DSAs after transplant, and 3 were C1q VXM- but antibody developed immediately after transplant. A positive C1q assay in the immediate post-transplant period had a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 100%, with 100% sensitivity and 100% specificity (Fisher exact p = 0.001). Of 11 patients who were IgG VXM+, 5 had AMR; the IgG VXM had a PPV of 45% and NPV of 100%, with 100% sensitivity and 54% specificity (Fisher exact p = 0.101).The C1q assay can detect a sub-set of antibodies capable of fixing complement and predicts AMR early after transplant. Avoiding only the donor antigens that would be recognized by the C1q assay may accelerate time to transplant by expansion of the donor pool and potentially allows transplantation of previously "incompatible" organs.

    View details for DOI 10.1016/j.healun.2010.08.020

    View details for Web of Science ID 000286545200008

    View details for PubMedID 20951058

  • Complete donor T-cell engraftment 30 days after allogeneic transplantation predicts molecular remission in high-risk chronic lymphocytic leukaemia BRITISH JOURNAL OF HAEMATOLOGY Jones, C. D., Arai, S., Lowsky, R., Tyan, D. B., Zehnder, J. L., Miklos, D. B. 2010; 150 (5): 637-639
  • Review of Heart-Lung Transplantation at Stanford ANNALS OF THORACIC SURGERY Deuse, T., Sista, R., Weill, D., Tyan, D., Haddad, F., Dhillon, G., Robbins, R. C., Reitz, B. A. 2010; 90 (1): 329-337


    Long-term survival after heart-lung transplantation was first achieved in 1981 at Stanford and a total of 217 heart-lung transplantations had been performed by June 2008. This review summarizes Stanford's cumulative experience with heart-lung transplantation, demonstrates the progress that has been made, and discusses past and persistent problems. Diagnostic tools and treatment options for infectious diseases and rejection have changed and patient survival markedly improved over the almost three decades. Eight patients lived longer than 20 years. Further options to treat infections and strategies to control bronchiolitis obliterans syndrome, the main causes of early and long-term mortality, respectively, are required to achieve routine long-term survival.

    View details for DOI 10.1016/j.athoracsur.2010.01.023

    View details for PubMedID 20609821

  • Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes GENOME RESEARCH Norman, P. J., Abi-Rached, L., Gendzekhadze, K., Hammond, J. A., Moesta, A. K., Sharma, D., Graef, T., McQueen, K. L., Guethlein, L. A., Carrington, C. V., Chandanayingyong, D., Chang, Y., Crespi, C., Saruhan-Direskeneli, G., Hameed, K., Kamkamidze, G., Koram, K. A., Layrisse, Z., Matamoros, N., Mila, J., Park, M. H., Pitchappan, R. M., Ramdath, D. D., Shiau, M., Stephens, H. A., Struik, S., Tyan, D., Verity, D. H., Vaughan, R. W., Davis, R. W., Fraser, P. A., Riley, E. M., Ronaghi, M., Parham, P. 2009; 19 (5): 757-769


    Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric "half" was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family.

    View details for DOI 10.1101/gr.085738.108

    View details for Web of Science ID 000265668800009

    View details for PubMedID 19411600

    View details for PubMedCentralID PMC2675964

  • Unusual selection on the KIR3DL1/S1 natural killer cell receptor in Africans NATURE GENETICS Norman, P. J., Abi-Rached, L., Gendzekhadze, K., Korbel, D., Gleimer, M., Rowley, D., Bruno, D., Carrington, C. V., Chandanayingyong, D., Chang, Y., Crespi, C., Saruhan-Direskeneli, G., Fraser, P. A., Hameed, K., Kamkamidze, G., Koram, K. A., Layrisse, Z., Matamoros, N., Mila, J., Park, M. H., Pitchappan, R. M., Ramdath, D. D., Shiau, M., Stephens, H. A., Struik, S., Verity, D. H., Vaughan, R. W., Tyan, D., Davis, R. W., Riley, E. M., Ronaghi, M., Parham, P. 2007; 39 (9): 1092-1099


    Interactions of killer cell immunoglobulin-like receptors (KIRs) with major histocompatibility complex (MHC) class I ligands diversify natural killer cell responses to infection. By analyzing sequence variation in diverse human populations, we show that the KIR3DL1/S1 locus encodes two lineages of polymorphic inhibitory KIR3DL1 allotypes that recognize Bw4 epitopes of protein">HLA-A and HLA-B and one lineage of conserved activating KIR3DS1 allotypes, also implicated in Bw4 recognition. Balancing selection has maintained these three lineages for over 3 million years. Variation was selected at D1 and D2 domain residues that contact HLA class I and at two sites on D0, the domain that enhances the binding of KIR3D to HLA class I. HLA-B variants that gained Bw4 through interallelic microconversion are also products of selection. A worldwide comparison uncovers unusual KIR3DL1/S1 evolution in modern sub-Saharan Africans. Balancing selection is weak and confined to D0, KIR3DS1 is rare and KIR3DL1 allotypes with similar binding sites predominate. Natural killer cells express the dominant KIR3DL1 at a high frequency and with high surface density, providing strong responses to cells perturbed in Bw4 expression.

    View details for DOI 10.1038/ng2111

    View details for Web of Science ID 000249122400018

    View details for PubMedID 17694054

  • Genetic control of human NK cell repertoire JOURNAL OF IMMUNOLOGY Shilling, H. G., Young, N., Guethlein, L. A., Cheng, N. W., Gardiner, C. M., Tyan, D., Parham, P. 2002; 169 (1): 239-247


    Through differential killer cell Ig-like receptor (KIR) and CD94:NKG2 gene expression, human NK cells generate diverse repertoires, each cell having an inhibitory receptor for autologous HLA class I. Using a new method for measuring repertoire difference that integrates multiple flow cytometry parameters, we found individual repertoire stability, but population variability. Correlating repertoire differences with KIR and HLA genotype for 85 sibling pairs reveals the dominant influence of KIR genotype; HLA genotype having a subtle, modulating effect on relative KIR expression frequencies. HLA and/or KIR genotype also influences CD94:NKG2A expression. After HLA-matched stem cell transplantation, KIR repertoires either recapitulated that of the donor or were generally depressed for KIR expression. Human NK cell repertoires are defined by combinations of variable KIR and HLA class I genes and conserved CD94:NKG2 genes.

    View details for Web of Science ID 000176360400031

    View details for PubMedID 12077250

  • Allelic polymorphism synergizes with variable gene content to individualize human KIR genotype JOURNAL OF IMMUNOLOGY Shilling, H. G., Guethlein, L. A., Cheng, N. W., Gardiner, C. M., Rodriguez, R., Tyan, D., Parham, P. 2002; 168 (5): 2307-2315


    Killer Ig-like receptor (KIR) genes are a multigene family on human chromosome 19. KIR genes occur in various combinations on different haplotypes. Additionally, KIR genes are polymorphic. To examine how allelic polymorphism diversifies KIR haplotypes with similar or identical combinations of KIR genes, we devised methods for discriminating alleles of KIR2DL1, -2DL3, -3DL1, and -3DL2. These methods were applied to 143 individuals from 34 families to define 98 independent KIR haplotypes at the allele level. Three novel 3DL2 alleles and a chimeric 3DL1/3DL2 sequence were also identified. Among the A group haplotypes were 22 different combinations of 2DL1, 2DL3, 3DL1, and 3DL2 alleles. Among the B group haplotypes that were unambiguously determined were 15 distinct haplotypes involving 9 different combinations of KIR genes. A and B haplotypes both exhibit strong linkage disequilibrium (LD) between 2DL1 and 2DL3 alleles, and between 3DL1 and 3DL2 alleles. In contrast, there was little LD between the 2DL1/2DL3 and 3DL1/3DL2 pairs that define the two halves of the KIR gene complex. The synergistic combination of allelic polymorphism and variable gene content individualize KIR genotype to an extent where unrelated individuals almost always have different KIR types. This level of diversity likely reflects strong pressure from pathogens on the human NK cell response.

    View details for Web of Science ID 000173990200030

    View details for PubMedID 11859120

  • Human diversity in killer cell inhibitory receptor genes IMMUNITY Uhrberg, M., Valiante, N. M., Shum, B. P., Shilling, H. G., Lienert-Weidenbach, K., Corliss, B., Tyan, D., Lanier, L. L., Parham, P. 1997; 7 (6): 753-763


    The presence and expression of killer inhibitory receptor (KIR) and CD94:NKG2 genes from 68 donors were analyzed using molecular typing techniques. The genes encoding CD94:NKG2 receptors were present in each person, but KIR gene possession varied. Most individuals expressed inhibitory KIR for the three well-defined HLA-B and -C ligands, but noninhibitory KIR genes were more variable. Twenty different KIR phenotypes were defined. Two groups of KIR haplotypes were distinguished and occurred at relatively even frequency. Group A KIR haplotypes consist of six genes: the main inhibitory KIR, one noninhibitory KIR, and a structurally divergent KIR. Allelic polymorphism within five KIR genes was detected. Group B comprises more noninhibitory KIR genes and contains at least one additional gene not represented in group A. The KIR locus therefore appears to be polygenic and polymorphic within the human population.

    View details for Web of Science ID 000071351300003

    View details for PubMedID 9430221

  • Cw*1701 defines a divergent African HLA-C allelic lineage IMMUNOGENETICS WELLS, R. S., Seielstad, M. T., Bunce, M., Tyan, D. B., Bekele, E., Parham, P. 1997; 46 (3): 173-180


    The complete sequence of a new HLA-C allele, Cw*1701, was determined from a South African Zulu individual. Unique features that distinguish Cw*1701 from other HLA-C alleles include multiple point substitutions and an 18 nucleotide insertion in exon 5, which encodes the transmembrane domain. In a phylogenetic analysis of HLA-C sequences, Cw*1701 forms a third, distinct allelic lineage. A comparison of the transmembrane domain of Cw*1701 with other HLA-B and -C alleles reveals that duplications and deletions have been common in the evolution of these loci. A polymerase chain reaction based typing method was used to determine the distribution of this unusual allele in human populations. In contrast to the other two lineages of HLA-C alleles, the Cw*17 lineage is found at high frequencies only in populations of African descent. In addition, the HLA-B/Cw*17 haplotype diversity is higher in Africa.

    View details for Web of Science ID A1997XL60100001

    View details for PubMedID 9211742

  • Heterogeneous phenotypes of expression of the NKB1 natural killer cell class I receptor among individuals of different human histocompatibility leukocyte antigens types appear genetically regulated, but not linked to major histocompatibility complex haplotype JOURNAL OF EXPERIMENTAL MEDICINE Gumperz, J. E., Valiante, N. M., Parham, P., Lanier, L. L., Tyan, D. 1996; 183 (4): 1817-1827


    Natural killer (NK) cells that express the NKB1 receptor are inhibited from killing target cells that possess human histocompatibility leukocyte antigen (HLA) B molecules bearing the Bw4 serological epitope. To investigate whether NKB1 expression is affected by HLA type, peripheral blood lymphocytes of 203 HLA-typed donors were examined. Most donors had a single population of NKB1+ cells, but some had two populations expressing different cell surface levels of NKB1, and others had no detectable NKB1+ cells. Among the donors expressing NKB1, both the relative abundance of NKB1+ NK cells and their level of cell surface expression varied substantially. The percentage of NKB1+ NK cells ranged from 0 to >75% (mean 14.7%), and the mean fluorescence of the positive population varied over three orders of magnitude. For each donor, the small percentage of T cells expressing NKB1 (usually <2%), had a pattern of expression mirroring that of the NK cells. NKB1 expression by NK and T cells remained stable over the 2-yr period that five donors were tested. Patterns of NKB1 expression were not associated with Bw4 or Bw6 serotype of the donor or with the presence of any individual HLA-A or -B antigens. Cells expressing NKB1 are often found in donors who do not possess an appropriate class I ligand, and can be absent in those who express Bw4+ HLA-B antigens. Family studies further suggested that the phenotype of NKB1 expression is inherited but not HLA linked. Whereas identical twins show matching patterns of NKB1 expression, HLA-identical siblings can differ in NKB1 expression, and conversely, HLA-disparate siblings can be similar. Thus NKB1 expression phenotypes are tightly regulated and extremely heterogeneous, but not correlated with HLA type.

    View details for Web of Science ID A1996UH14400054

    View details for PubMedID 8666938



    The ST-16 antigenic specificity of the HLA-B locus is defined as a B39 variant of Mexican-Americans. Nucleotide sequencing of cDNA shows the ST-16 allele (B*3905) differs from B*39011 by a single substitution that substitutes tyrosine for aspartic acid at position 74 of the mature class I heavy chain. The complete coding region sequence for the common caucasoid allele encoding the B38 antigen has been determined. This B*3801 allele differs from B*3802 at two nucleotide substitutions within the Bw4 sequence motif. B*3801 and B*3802 may have been derived independently from B*39011 by conversion events with B alleles donating distinctive Bw4 motifs. A novel allele B*39022 derived from a Colombian Indian differs from the B*39021 allele of Japanese origin at two widely separated silent substitutions. Comparison of sequences for the known B16 alleles suggest that B*39021 and B*39022 were independently derived by recombination from B*39013 and B*39011 respectively.

    View details for Web of Science ID A1995QD28800003

    View details for PubMedID 7725307



    RecA protein-coated probe has been utilized to enrich genomic digests for desired genes in order to facilitate cloning from genomic libraries. Using a previously cloned HLA-B27 gene as the recA-coated enrichment probe, we obtained a mean 108x increase in the ratio of specific to nonspecific plaques in lambda libraries screened for B27 variant alleles of estimated 99% homology to the probe. Class I genes of lesser homology were less enriched: 6.7x for non-B27 genes of estimated greater than 95% homology and 3.7x for other-Class I genes of greater than 80% homology. Loss of genomic DNA during the enrichment procedure can, however, restrict application of this technique whenever starting genomic DNA is very limited. Nevertheless, the impressive reduction in cloning effort and material makes recA enrichment a useful new tool for cloning homologous genes from genomic DNA.

    View details for Web of Science ID A1988P964900029

    View details for PubMedID 2901713