High-Throughput Next-Generation Sequencing of the Kidd Blood Group: Unexpected Antigen Expression Properties of Four Alleles and Detection of Novel Variants.

Background: The blood supply for patients with foreign ethnic backgrounds can be challenging, as they often have blood group and HPA patterns that differ from the variants prevalent in the German population. In addition, hemoglobinopathies requiring regular blood transfusion may be more common in such populations. High-throughput genotyping tests can facilitate the identification of the most compatible blood products, thereby reducing the risk of transfusion reactions. The present study reports the results of a molecular study for the Kidd (JK) blood group. Allele frequencies and antigen prevalence data are presented for >8,000 individuals of various origins. Material and Methods: More than 8,000 blood donors were genotyped for 22 blood group systems and 5 HPA genes using an amplicon-based next-generation sequencing (NGS) approach. As part of the test system, we focused on the JK system in more detail. Double-ARMS PCR analysis was performed for the haplotype phasing of the JK1/JK2 and two more common synonymous polymorphisms. We performed transcript analysis to detect potential alternative splice products. For a subset of samples, a comparison between serotype and red cell genotype was conducted. Allele frequencies were determined for geographically different panels of individuals. Results: We successfully genotyped the JK blood group for 99.6% of the samples. Haplotype phasing revealed 96 different alleles. For several alleles that carry one of the synonymous SNVs c.588A>G and c.810G>A, we could not confirm the reported JK phenotypes. We found a higher frequency of JK:1 alleles for all populations except Iraqis. JK*01W.01 alleles were more common in the Asian groups and sub-Saharan Africans. A variant of the allele JK*02N.01 was present exclusively in Southeast Asians. Conclusion: Genotyping for JK antigens with a targeted NGS assay can easily be performed in routine. The interpretation that c.588A>G leads to a weak phenotype and c.810G>A to a null phenotype is questionable. IDs as well as the descriptions of alleles carrying these SNVs should be revised in the ISBT JK table.

Prognostic markers for the clinical course in the blood of patients with SARS-CoV-2 infection.

BACKGROUND: The presentation of peptides and the subsequent immune response depend on the MHC characteristics and influence the specificity of the immune response. Several studies have found an association between HLA variants and differential COVID-19 outcomes and have shown that HLA genotypes are associated with differential immune responses against SARS-CoV-2, particularly in severely ill patients. Information, whether HLA haplotypes are associated with the severity or length of the disease in moderately diseased individuals is absent. METHODS: Next-generation sequencing-based HLA typing was performed in 303 female and 231 male non-hospitalized North Rhine Westphalian patients infected with SARS-CoV2 during the first and second wave. For HLA-Class I, we obtained results from 528 patients, and for HLA-Class II from 531. In those patients, who became ill between March 2020 and January 2021, the 22 most common HLA-Class I (HLA-A, -B, -C) or HLA-Class II (HLA -DRB1/3/4, -DQA1, -DQB1) haplotypes were determined. The identified HLA haplotypes as well as the presence of a CCR5Δ32 mutation and number of O and A blood group alleles were associated to disease severity and duration of the disease. RESULTS: The influence of the HLA haplotypes on disease severity and duration was more pronounced than the influence of age, sex, or ABO blood group. These associations were sex dependent. The presence of mutated CCR5 resulted in a longer recovery period in males. CONCLUSION: The existence of certain HLA haplotypes is associated with more severe disease.

Interleukin-7 and soluble Interleukin-7 receptor levels in type 1 diabetes - Impact of IL7RA polymorphisms, HLA risk genotypes and clinical features.

High soluble IL-7 receptor (sIL-7R) serum levels and associated single nucleotide polymorphisms in the IL7RA gene were found in autoimmune diseases including type 1 diabetes. Further determinants on sIL-7R and IL-7 availability as well as changes during type 1 diabetes disease course remain elusive. Here we performed multiparameter analysis to identify influential genetic and disease-associated factors on sIL-7R and IL-7 serum levels during type 1 diabetes disease course (239 children) and in healthy controls (101 children). We found higher sIL-7R serum concentrations at type 1 diabetes onset and decreasing levels during therapy whereas IL-7 was only higher in long term patients as compared to controls. Multiple linear regression analyses revealed several factors, including IL7RA SNP rs6897932 and HLA risk haplotypes, influencing sIL-7R levels but not IL-7, which was solely associated with the sIL-7R. This study revealed unexpected complexity in the regulation of the sIL-7R but not for IL-7.

Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling.

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants. Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19. Results: We observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions. Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity.

Next Generation Sequencing Identifies the HLA-DQA1*03:03 Allele in the Type 1 Diabetes Risk-Associated HLA-DQ8 Serotype.

The highest genetic type 1 diabetes risk is conferred by HLA class II haplotypes defined by alleles at the HLA-DR and -DQ loci. The combination of HLA-DQA1*03:01 and DQB1*03:02 alleles (summarized as 'HLA-DQ8') is reported to be among the two most prevalent HLA class II haplotypes in Caucasian type 1 diabetes patients. This classification is based on conventional genotyping of exon 2 of the DQ gene locus and excludes exon 3. In this study, HLA genotyping on the type 1 diabetes susceptibility loci HLA-DRB1, DQA1 and DQB1 was performed using a high-resolution next generation sequencing method. In addition to the routinely examined exon 2, exon 3 was also sequenced. Samples from 229 children with type 1 diabetes were included and compared to a cohort of 9,786 controls. In addition to previously described HLA-DQ haplotypes in type 1 diabetes patients, we found that as well as HLA-DQA1*03:01,HLA-DQA1*03:03 also contributed to HLA-DQ8. HLA-DQA1*03:03 differs from HLA-DQA1*03:01 by one nucleotide substitution in exon 3 at position 160, leading to a single amino acid replacement. DRB1*04:05 was exclusively associated with DQA1*03:03 whereas the DRB1*04:01 haplotype comprised either DQA1*03:01 or DQA1*03:03. Significantly increased type 1 diabetes risk was confirmed for all these haplotypes with only minor differences between DQA1*03:01 and DQA1*03:03 alleles. This study identified the HLA-DQA1*03:03 allele as an addition to the already known type 1 diabetes risk haplotypes, and can contribute to more precise HLA genotyping approaches.

Interleukin-7-dependent nonclassical monocytes and CD40 expression are affected in children with type 1 diabetes.

The important role of IL-7 in the generation of self-reactive T-cells in autoimmune diseases is well established. Recent studies on autoimmunity-associated genetic polymorphisms indicated that differential IL-7 receptor (IL-7R) expression of monocytes may play a role in the underlying pathogenesis. The relevance of IL-7-mediated monocyte functions in type 1 diabetes remains elusive. In the present study, we characterized monocyte phenotype and IL-7-mediated effects in children with type 1 diabetes and healthy controls with multicolor flow cytometry and t-distributed Stochastic Neighbor-Embedded (t-SNE)-analyses. IL-7R expression of monocytes rapidly increased in vitro and was boosted through LPS. In the presence of IL-7, we detected lower monocyte IL-7R expression in type 1 diabetes patients as compared to healthy controls. This difference was most evident for the subset of nonclassical monocytes, which increased after IL-7 stimulation. t-SNE analyses revealed IL-7-dependent differences in monocyte subset distribution and expression of activation and maturation markers (i.e., HLA-DR, CD80, CD86, CD40). Notably, monocyte CD40 expression increased considerably by IL-7 and CD40/IL-7R co-expression differed between patients and controls. This study shows the unique effects of IL-7 on monocyte phenotype and functions. Lower IL-7R expression on IL-7-induced CD40high monocytes and impaired IL-7 response characterize monocytes from patients with type 1 diabetes.

Association of HLA genotypes, AB0 blood type and chemokine receptor 5 mutant CD195 with the clinical course of COVID-19.

BACKGROUND: COVID-19, the pandemic disease caused by infection with SARS-CoV-2, may take highly variable clinical courses, ranging from symptom-free and pauci-symptomatic to fatal disease. The goal of the current study was to assess the association of COVID-19 clinical courses controlled by patients' adaptive immune responses without progression to severe disease with patients' Human Leukocyte Antigen (HLA) genetics, AB0 blood group antigens, and the presence or absence of near-loss-of-function delta 32 deletion mutant of the C-C chemokine receptor type 5 (CCR5). PATIENT AND METHODS: An exploratory observational study including 157 adult COVID-19 convalescent patients was performed with a median follow-up of 250 days. The impact of different HLA genotypes, AB0 blood group antigens, and the CCR5 mutant CD195 were investigated for their role in the clinical course of COVID-19. In addition, this study addressed levels of severity and morbidity of COVID-19. The association of the immunogenetic background parameters were further related to patients' humoral antiviral immune response patterns by longitudinal observation. RESULTS: Univariate HLA analyses identified putatively protective HLA alleles (HLA class II DRB1*01:01 and HLA class I B*35:01, with a trend for DRB1*03:01). They were associated with reduced durations of disease instead decreased (rather than increased) total anti-S IgG levels. They had a higher virus neutralizing capacity compared to non-carriers. Conversely, analyses also identified HLA alleles (HLA class II DQB1*03:02 und HLA class I B*15:01) not associated with such benefit in the patient cohort of this study. Hierarchical testing by Cox regression analyses confirmed the significance of the protective effect of the HLA alleles identified (when assessed in composite) in terms of disease duration, whereas AB0 blood group antigen heterozygosity was found to be significantly associated with disease severity (rather than duration) in our cohort. A suggestive association of a heterozygous CCR5 delta 32 mutation status with prolonged disease duration was implied by univariate analyses but could not be confirmed by hierarchical multivariate testing. CONCLUSION: The current study shows that the presence of HLA class II DRB1*01:01 and HLA class I B*35:01 is of even stronger association with reduced disease duration in mild and moderate COVID-19 than age or any other potential risk factor assessed. Prospective studies in larger patient populations also including novel SARS-CoV-2 variants will be required to assess the impact of HLA genetics on the capacity of mounting protective vaccination responses in the future.

High-Throughput Screening of Blood Donors for Twelve Human Platelet Antigen Systems Using Next-Generation Sequencing Reveals Detection of Rare Polymorphisms and Two Novel Protein-Changing Variants.

BACKGROUND: Exposure to non-matching human platelet alloantigens (HPA) may result in alloimmunization. Antibodies to HPA can be responsible for post-transfusion purpura, refractoriness to donor platelets, and fetal and neonatal alloimmune thrombocytopenia. For the supply of compatible apheresis platelet concentrates, the HPA genotypes are determined in a routine manner. METHODS: Here, we describe a novel method for genotyping twelve different HPA systems simultaneously, including HPA-1 to HPA-5, HPA-9w, HPA-10w, HPA-16w, HPA-19w, HPA-27w, and the novel HPA-34w by means of amplicon-based next-generation sequencing (NGS). Blood donor samples of 757 individuals with a migration background and 547 of Western European ancestry were genotyped in a mass-screening setup. An in-house software was developed for fast and automatic analysis. TaqMan assay and Sanger sequencing results served for validation of the NGS workflow. Finally, blood donors were divided in several groups based on their country of origin and the allele frequencies were compared. RESULTS: For 1,299 of 1,304 samples (99.6%) NGS was successfully performed. The concordance with TaqMan assay and Sanger sequencing results was 99.8%. Allele-calling dropouts that were observed for two samples with the TaqMan assay caused by rare single nucleotide polymorphisms were resolved by NGS. Additionally, twenty rare and two novel variants in the coding regions of the genes ITGB3, GPB1A, ITGBA2, and CD109 were detected. The determined allele frequencies were similar to those published in the gnomAD database. CONCLUSIONS: No significant differences were observed in the distribution of allele frequencies of HPA-1 through HPA-5 and HPA-15 throughout the analyzed groups except for a lower allele frequency for the HPA-1b allele in the group of donors with Southern Asian ancestry. In contrast, other nucleotide variants that have not yet been phenotypically characterized occurred three times more often in blood donors with a migration background. High-throughput amplicon-based NGS is a reliable method for screening HPA genotypes in a large sample cohort simultaneously. It is easily upgradeable for genotyping additional targets without changing the setup or the analysis pipeline. Mass-screening methods will help building up blood donor registries to provide matched blood products.

Correction: Reinauer et al., The Clinical Course of Patients with Preschool Manifestation of Type 1 Diabetes Is Independent of the HLA DR-DQ Genotype. Genes 2017, 8, 146.

The article entitled "The Clinical Course of Patients with Preschool Manifestation of Type 1 Diabetes is Independent of the HLA DR-DQ Genotype" contained a calculation error in Table 2 and the statistical methods used were not completely described.[...].

Derivation and characterization of integration-free iPSC line ISRM-UM51 derived from SIX2-positive renal cells isolated from urine of an African male expressing the CYP2D6 *4/*17 variant which confers intermediate drug metabolizing activity.

SIX2-positive renal cells isolated from urine from a 51year old male of African origin bearing the CYP2D6 *4/*17 variant were reprogrammed by nucleofection of a combination of two episomal-based plasmids omitting pathway (TGFß, MEK and GSK3ß) inhibition. The induced pluripotent stem cells (iPSCs) were characterized by immunocytochemistry, embryoid body formation, DNA-fingerprinting and karyotype analysis. Comparative transcriptome analyses with human embryonic stem cell lines H1 and H9 revealed a Pearson correlation of 0.9243 and 0.9619 respectively.

The Clinical Course of Patients with Preschool Manifestation of Type 1 Diabetes Is Independent of the HLA DR-DQ Genotype.

INTRODUCTION: Major histocompatibility complex class II genes are considered major genetic risk factors for autoimmune diabetes. We analysed Human Leukocyte Antigen (HLA) DR and DQ haplotypes in a cohort with early-onset (age < 5 years), long term type 1 diabetes (T1D) and explored their influence on clinical and laboratory parameters. METHODS: Intermediate resolution HLA-DRB1, DQA1 and DQB1 typing was performed in 233 samples from the German Paediatric Diabetes Biobank and compared with a local control cohort of 19,544 cases. Clinical follow-up data of 195 patients (diabetes duration 14.2 ± 2.9 years) and residual C-peptide levels were compared between three HLA risk groups using multiple linear regression analysis. RESULTS: Genetic variability was low, 44.6% (104/233) of early-onset T1D patients carried the highest-risk genotype HLA-DRB1*03:01-DQA1*05:01-DQB1*02:01/DRB1*04-DQA1*03:01-DQB1*03:02 (HLA-DRB1*04 denoting 04:01/02/04/05), and 231 of 233 individuals carried at least one of six risk haplotypes. Comparing clinical data between the highest (n = 83), moderate (n = 106) and low risk (n = 6) genotypes, we found no difference in age at diagnosis (mean age 2.8 ± 1.1 vs. 2.8 ± 1.2 vs. 3.2 ± 1.5 years), metabolic control, or frequency of associated autoimmune diseases between HLA risk groups (each p > 0.05). Residual C-peptide was detectable in 23.5% and C-peptide levels in the highest-risk group were comparable to levels in moderate to high risk genotypes. CONCLUSION: In this study, we saw no evidence for a different clinical course of early-onset T1D based on the HLA genotype within the first ten years after manifestation.

GEMMs addressing Pax5 loss-of-function in childhood pB-ALL.

Germline mutations in transcription factors, which are implicated in hematopoiesis in general or specifically in B-cell differentiation have recently been described to confer an inherited risk to pB-ALL with often reduced penetrance. Predicting leukemia development, therapy response and long term follow up of mutation carriers is challenging because experience from large patient cohorts and their long term follow up are not available. Genetically Engineered Murine Models (GEMMs) represent a promising approach to create individualized and precise models reproducing the molecular makeup of the human disease. This review focuses on PAX5 loss-of-function and summarizes techniques of murine model generation, available GEMMs, which mimic Pax5 loss-of-function in leukemia development and discusses the challenges and drawbacks of these models. These aspects are discussed in the context of creating a robust model, which serves not only for validation of the relevance of a genomic alteration in pB-ALL but at the same time as a valid preclinical model.

KIR ligand C2 is associated with increased susceptibility to childhood ALL and confers an elevated risk for late relapse.

A role for HLA class I polymorphism in childhood acute lymphoblastic leukemia (ALL) has been suggested for many years, but unambiguous associations have not been found. Here, we show that the HLA-C-encoded supertypic epitope C2, which constitutes a high-affinity ligand for the inhibitory natural killer (NK) cell receptor KIR2DL1, is significantly increased in ALL patients (n = 320; P = .005). Stratification for ethnicity and disease subtype revealed a strong association of C2 with B-ALL in German cases (P = .0004). The effect was independent of KIR2DS1 and KIR2DL1 allelic polymorphism and copy number. Analysis of clinical outcome revealed a higher incidence of late relapse (> 2.5 years) with increasing number of C2 alleles (P = .014). Our data establish C2 as novel risk factor and homozygosity for C1 as protective for childhood B-ALL supporting a model in which NK cells are involved in immunosurveillance of pediatric B-ALL via interaction of KIR with HLA-C ligands.

HIV-1 seroreversion in HIV-1-infected children: do genetic determinants play a role?

BACKGROUND: HIV-1 seroreversion in infants with vertically transmitted HIV-1 infection who started ART in the first months of life has been reported in only a subset of patients. However, the reason why most infants remain seropositive despite similar treatment response is not understood. Here, we assessed whether HIV-1 seroreversion in maternally infected infants is associated with genetic determinants. METHODS: HIV-1-infected infants with a history of documented HIV-1 seroreversion were identified throughout Germany using a standardized questionnaire. At study entry immune reconstitution and anti-HIV-1 antibody expression were monitored as clinical parameters. To search for genetic determinants high-resolution HLA genotyping was performed. In addition, the coding sequence of the chemokine receptor CCR5 was analyzed by Sanger sequencing regarding potential mutations. RESULTS: Patients showed normal numbers and frequencies of lymphocyte subpopulations. Five out of eight patients still had seronegative HIV-1 antibody status at study entry. HLA genotyping revealed the enrichment of HLA-DQB1*03 and DQB1*06 alleles within the patient cohort. Only one patient was found to carry a 32 bp-deletion within the CCR5 gene. CONCLUSION: Our results indicate that the phenotype of HIV-1 seroreversion in infants might correlate with the presence of HLA class II alleles DQB1*03 and DQB1*06. This finding supports the idea of genetic predisposition determining HIV-1 seroreversion in vertically infected infants effectively treated with ART.

Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-matched related and unrelated hematopoietic stem cell transplantation.

The effect of minor H antigen mismatching on the occurrence of graft-versus-host disease (GvHD) and graft-versus-leukemia (GvL) after HLA-matched hematopoietic stem cell transplantation (HSCT) has mainly been demonstrated in single-center studies. Yet, the International Histocompatibility and Immunogenetics Workshops (IHIW) provide a collaborative platform to execute crucial large studies. In collaboration with 20 laboratories of the IHIW, the roles of 10 autosomal and 10 Y chromosome-encoded minor H antigens were investigated on GvHD and relapse incidence in 639 HLA-identical related donor (IRD) and 210 HLA-matched unrelated donor (MUD) HSCT recipients. Donor and recipient DNA samples were genotyped for the minor H antigens HA-1, HA-2, HA-3, HA-8, HB-1, ACC-1, ACC-2, SP110, PANE1, UGT2B17, and HY. The correlations with the primary outcomes GvHD (acute or chronic GvHD), survival, and relapse were statistically analyzed. The results of these multicenter analyses show that none of the HLA class I-restricted HY antigens were found to be associated with any of the primary outcomes. Interestingly, of the HLA class II-restricted HY antigens analyzed, HLA-DQ5 positive recipients showed a significantly increased GvHD-free survival in female-to-male HSCT compared with male-to-female HSCT (P = .013). Yet, analysis of the overall gender effect, thus independent of the known HY antigens, between the gender groups demonstrated an increased GvHD incidence in the female-to-male transplantations (P < .005) and a decreased GvHD-free survival in the female-to-male transplantations (P < .001). Of all autosomally encoded minor H antigens, only mismatching for the broadly expressed minor H antigen HA-8 increased the GvHD incidence in IRD HSCT (Hazard ratio [HR] = 5.28, P < .005), but not in MUD HSCT. Most striking was the influence of hematopoietic restricted minor H antigens on GvL as mismatching for hematopoietic minor H antigens correlated with lower relapse rates (P = .078), higher relapse-free survival (P = .029), and higher overall survival (P = .032) in recipients with GvHD, but not in those without GvHD. In conclusion, the significant GvHD effect of the broadly expressed minor H antigen HA-8 favors matching for HA-8 in IRD, but not in MUD, patient/donor pairs. The GvHD-GvL association demonstrating a significant lower relapse in hematopoietic minor H antigen mismatched patient/donor pairs underlines their clinical applicability for adoptive immunotherapy, enhancing the GvL effect in a GvHD controllable manner.

The impact of HLA-C matching depends on the C1/C2 KIR ligand status in unrelated hematopoietic stem cell transplantation.

It was previously shown that chronic myeloid leukemia (CML) patients transplanted with peripheral blood progenitor cells (PBPC) from HLA-C allele-matched donors had better clinical outcome when lacking the HLA-C-encoded KIR epitope C2. We investigated whether this holds true in other diseases and in HLA-C allele-mismatched patients. Twenty-four myelodysplastic syndrome (MDS), 39 acute myeloid leukemia (AML)/CML, and 34 acute lymphoblastic leukemia/non-Hodgkin lymphoma patients received unrelated unmanipulated PBPC. HLA matching was analyzed retrospectively (including DNA-based direct sequencing of HLA-C). Only in AML/CML, the C2 ligand was associated with impaired overall survival (OS, p < 0.05). We next calculated the impact of donor/recipient HLA-C allele matching within the C1 and C2 groups. Surprisingly, AML/CML and MDS patients with C2 ligands profited from HLA-C allele mismatching (OS, p < 0.01), whereas in the C1 group, allele matching was beneficial (p < 0.05). HLA-C allele mismatching in the C2 KIR ligand group was associated with lower TRM (OR 0.48, p < 0.009) and lower relapse rate (OR 2.7 p < 0.1) when compared to allele-matched C2 patients. Thus, patients could be assigned to a low- and a high-risk group according to their C1/C2 ligand status and the HLA-C allele matching degree. These data suggest that four-digit allele matching of HLA-C has differential effects dependent on the presence of C1 and C2 KIR epitopes in the patient.

Analyses of HLA-C-specific KIR repertoires in donors with group A and B haplotypes suggest a ligand-instructed model of NK cell receptor acquisition.

To determine the influence of KIR and HLA class I polymorphism on human NK cell repertoires, 32 different clonotypes representing all possible combinations of 4 inhibitory KIR and NKG2A were analyzed by multicolor flow cytometry. In donors homozygous for the common group A KIR haplotype, a significant influence of HLA-C ligands was seen: KIR repertoires were dominated by clonotypes expressing a single KIR for the respective cognate ligand, either the C1-specific KIR2DL3 or C2-specific KIR2DL1. In contrast, in donors possessing the polymorphic group B haplotypes, a similar adaptation to cognate HLA-C was lacking. We suggest that this discrepancy is largely the result of a suppressive effect of the group B-specific KIR2DL2 on the frequency of KIR2DL1(+) NK cells. In functional assays, KIR2DL2 not only recognized C1 but also C2 ligands, showing overlapping specificity with KIR2DL1. Moreover, using an NK cell differentiation assay we show sequential acquisition of KIR2DL2 before KIR2DL1 on developing NK cells. Together, these observations are compatible with a ligand-instructed model of NK cell education, in which recognition of HLA class I by an inhibitory receptor (KIR2DL2) suppresses subsequent expression of a second receptor (KIR2DL1) of related specificity. Importantly, the ligand-instructed model fits to the observed KIR repertoires in both broad KIR haplotype groups.