Donor KIR genotype based outcome prediction after allogeneic stem cell transplantation: no land in sight.

Optimizing natural killer (NK) cell alloreactivity could further improve outcome after allogeneic hematopoietic cell transplantation (alloHCT). The donor's Killer-cell Immunoglobulin-like Receptor (KIR) genotype may provide important information in this regard. In the past decade, different models have been proposed aiming at maximizing NK cell activation by activating KIR-ligand interactions or minimizing inhibitory KIR-ligand interactions. Alternative classifications intended predicting outcome after alloHCT by donor KIR-haplotypes. In the present study, we aimed at validating proposed models and exploring more classification approaches. To this end, we analyzed samples stored at the Collaborative Biobank from HLA-compatible unrelated stem cell donors who had donated for patients with acute myeloid leukemia (AML) or myelodysplastic neoplasm (MDS) and whose outcome data had been reported to EBMT or CIBMTR. The donor KIR genotype was determined by high resolution amplicon-based next generation sequencing. We analyzed data from 5,017 transplants. The median patient age at alloHCT was 56 years. Patients were transplanted for AML between 2013 and 2018. Donor-recipient pairs were matched for HLA-A, -B, -C, -DRB1, and -DQB1 (79%) or had single HLA mismatches. Myeloablative conditioning was given to 56% of patients. Fifty-two percent of patients received anti-thymocyte-globulin-based graft-versus-host disease prophylaxis, 32% calcineurin-inhibitor-based prophylaxis, and 7% post-transplant cyclophosphamide-based prophylaxis. We tested several previously reported classifications in multivariable regression analyses but could not confirm outcome associations. Exploratory analyses in 1,939 patients (39%) who were transplanted from donors with homozygous centromeric (cen) or telomeric (tel) A or B motifs, showed that the donor cen B/B-tel A/A diplotype was associated with a trend to better event-free survival (HR 0.84, p=.08) and reduced risk of non-relapse mortality (NRM) (HR 0.65, p=.01). When we further dissected the contribution of B subtypes, we found that only the cen B01/B01-telA/A diplotype was associated with a reduced risk of relapse (HR 0.40, p=.04) while all subtype combinations contributed to a reduced risk of NRM. This exploratory finding has to be validated in an independent data set. In summary, the existing body of evidence is not (yet) consistent enough to recommend use of donor KIR genotype information for donor selection in routine clinical practice.

Body odours as putative chemosignals in the father-child relationship: New insights on paternal olfactory kin recognition and preference from infancy to adolescence.

Children's body odours are effective chemical cues in the parent-child relationship. Mothers can recognize the odour of their child and prefer this odour over that of unfamiliar children. This effect is mediated by genetic similarity and developmental stage and is therefore suited to promote parental care at pre-pubertal stage, while facilitating incest avoidance at (post-)pubertal stage. The present study tested whether similar mechanisms apply to fathers. Therefore n = 56 fathers evaluated body odour samples of their own and of unfamiliar children in varying genetic and developmental stages. Genetic status was determined by human leucocyte antigen (HLA) profiling, developmental status by standardized assessment of pubertal status and steroid hormone concentration (estradiol, testosterone). Similar to mothers, fathers identified their own child's body odour above chance and preferred that odour. The paternal preference did not relate to HLA similarity but decreased with increasing age of the child. The decline was associated with higher pubertal stages in daughters only, which supports the hypothesis of odour-mediated incest prevention in opposite-sex parent-child dyads.

Providing hematopoietic stem cell products from unrelated donors to the world: DKMS donor centers and DKMS Registry.

Allogeneic hematopoietic stem cell (HSC) transplantation is a curative therapy for many severe blood diseases. As many patients have no suitable family donor, large unrelated donor registries and donor centers have been established in many countries, along with an international system for the provision of unrelated donor HSC products. As an essential part of this system, DKMS operates donor centers in 7 countries with a total of 12.2 million donors and over 114,000 donations so far, and a multinational donor registry. In 2022, DKMS donors contributed 57.5% of all cross-border donations worldwide. In this review, we describe the international system for the provision of unrelated donor HSC products as well as tasks and responsibilities of donor registries and donor centers. We also discuss relevant aspects of DKMS donor centers, namely donor file composition, matching and donation probabilities and actual donations, and the unique multinational approach of the DKMS Registry.

Haplotype reconstruction for genetically complex regions with ambiguous genotype calls: Illustration by the KIR gene region.

Advances in DNA sequencing technologies have enabled genotyping of complex genetic regions exhibiting copy number variation and high allelic diversity, yet it is impossible to derive exact genotypes in all cases, often resulting in ambiguous genotype calls, that is, partially missing data. An example of such a gene region is the killer-cell immunoglobulin-like receptor (KIR) genes. These genes are of special interest in the context of allogeneic hematopoietic stem cell transplantation. For such complex gene regions, current haplotype reconstruction methods are not feasible as they cannot cope with the complexity of the data. We present an expectation-maximization (EM)-algorithm to estimate haplotype frequencies (HTFs) which deals with the missing data components, and takes into account linkage disequilibrium (LD) between genes. To cope with the exponential increase in the number of haplotypes as genes are added, we add three components to a standard EM-algorithm implementation. First, reconstruction is performed iteratively, adding one gene at a time. Second, after each step, haplotypes with frequencies below a threshold are collapsed in a rare haplotype group. Third, the HTF of the rare haplotype group is profiled in subsequent iterations to improve estimates. A simulation study evaluates the effect of combining information of multiple genes on the estimates of these frequencies. We show that estimated HTFs are approximately unbiased. Our simulation study shows that the EM-algorithm is able to combine information from multiple genes when LD is high, whereas increased ambiguity levels increase bias. Linear regression models based on this EM, show that a large number of haplotypes can be problematic for unbiased effect size estimation and that models need to be sparse. In a real data analysis of KIR genotypes, we compare HTFs to those obtained in an independent study. Our new EM-algorithm-based method is the first to account for the full genetic architecture of complex gene regions, such as the KIR gene region. This algorithm can handle the numerous observed ambiguities, and allows for the collapsing of haplotypes to perform implicit dimension reduction. Combining information from multiple genes improves haplotype reconstruction.

High population frequencies of MICA copy number variations originate from independent recombination events.

MICA is a stress-induced ligand of the NKG2D receptor that stimulates NK and T cell responses and was identified as a key determinant of anti-tumor immunity. The MICA gene is located inside the MHC complex and is in strong linkage disequilibrium with HLA-B. While an HLA-B*48-linked MICA deletion-haplotype was previously described in Asian populations, little is known about other MICA copy number variations. Here, we report the genotyping of more than two million individuals revealing high frequencies of MICA duplications (1%) and MICA deletions (0.4%). Their prevalence differs between ethnic groups and can rise to 2.8% (Croatia) and 9.2% (Mexico), respectively. Targeted sequencing of more than 70 samples indicates that these copy number variations originate from independent nonallelic homologous recombination events between segmental duplications upstream of MICA and MICB. Overall, our data warrant further investigation of disease associations and consideration of MICA copy number data in oncological study protocols.

HLA-DP on Epithelial Cells Enables Tissue Damage by NKp44+ Natural Killer Cells in Ulcerative Colitis.

BACKGROUND & AIMS: Ulcerative colitis (UC) is characterized by severe inflammation and destruction of the intestinal epithelium, and is associated with specific risk single nucleotide polymorphisms in HLA class II. Given the recently discovered interactions between subsets of HLA-DP molecules and the activating natural killer (NK) cell receptor NKp44, genetic associations of UC and HLA-DP haplotypes and their functional implications were investigated. METHODS: HLA-DP haplotype and UC risk association analyses were performed (UC: n = 13,927; control: n = 26,764). Expression levels of HLA-DP on intestinal epithelial cells (IECs) in individuals with and without UC were quantified. Human intestinal 3-dimensional (3D) organoid cocultures with human NK cells were used to determine functional consequences of interactions between HLA-DP and NKp44. RESULTS: These studies identified HLA-DPA1∗01:03-DPB1∗04:01 (HLA-DP401) as a risk haplotype and HLA-DPA1∗01:03-DPB1∗03:01 (HLA-DP301) as a protective haplotype for UC in European populations. HLA-DP expression was significantly higher on IECs of individuals with UC compared with controls. IECs in human intestinal 3D organoids derived from HLA-DP401pos individuals showed significantly stronger binding of NKp44 compared with HLA-DP301pos IECs. HLA-DP401pos IECs in organoids triggered increased degranulation and tumor necrosis factor production by NKp44+ NK cells in cocultures, resulting in enhanced epithelial cell death compared with HLA-DP301pos organoids. Blocking of HLA-DP401-NKp44 interactions (anti-NKp44) abrogated NK cell activity in cocultures. CONCLUSIONS: We identified an UC risk HLA-DP haplotype that engages NKp44 and activates NKp44+ NK cells, mediating damage to intestinal epithelial cells in an HLA-DP haplotype-dependent manner. The molecular interaction between NKp44 and HLA-DP401 in UC can be targeted by therapeutic interventions to reduce NKp44+ NK cell-mediated destruction of the intestinal epithelium in UC.

The genotype list string code syntax for exchanging nomenclature-level genotyping results in clinical and research data management and analysis systems.

The nomenclatures used to describe HLA and killer-cell immunoglobulin-like receptor (KIR) alleles distinguish unique nucleotide and peptide sequences, and patterns of expression, but are insufficient for describing genotyping results, as description of ambiguities and relations across loci require terminology beyond allele names. The genotype list (GL) String grammar describes genotyping results for genetic systems with defined nomenclatures, like HLA and KIR, documenting what is known and unknown about a given genotyping result. However, the accuracy of a GL String is dependent on the reference database version under which it was generated. Here, we describe the GL string code (GLSC) system, which associates each GL String with meta-data describing the specific reference context in which the GL String was created, and in which it should be interpreted. GLSC is a defined syntax for exchanging GL Strings in the context of a specific gene-family namespace, allele-name code-system, and pertinent reference database version. GLSC allows HLA and KIR genotyping data to be transmitted, parsed and interpreted in the appropriate context, in an unambiguous manner, on modern data-systems, including Health Level 7 Fast Healthcare Interoperability Resource systems. Technical specification for GLSC can be found at https://glstring.org.

Genotype List String 1.1: Extending the Genotype List String grammar for describing HLA and Killer-cell Immunoglobulin-like Receptor genotypes.

The Genotype List (GL) String grammar for reporting HLA and Killer-cell Immunoglobulin-like Receptor (KIR) genotypes in a text string was described in 2013. Since this initial description, GL Strings have been used to describe HLA and KIR genotypes for more than 40 million subjects, allowing these data to be recorded, stored and transmitted in an easily parsed, text-based format. After a decade of working with HLA and KIR data in GL String format, with advances in HLA and KIR genotyping technologies that have fostered the generation of full-gene sequence data, the need for an extension of the GL String system has become clear. Here, we introduce the new GL String delimiter "?," which addresses the need to describe ambiguity in assigning a gene sequence to gene paralogs. GL Strings that do not include a "?" delimiter continue to be interpreted as originally described. This extension represents version 1.1 of the GL String grammar.

HLA allele and haplotype frequencies of registered stem cell donors in Chile.

Patients in need of hematopoietic stem cell transplantation often rely on unrelated stem cell donors matched in certain human leukocyte antigen (HLA) genes. Donor search is complicated by the extensive allelic variability of the HLA system. Therefore, large registries of potential donors are maintained in many countries worldwide. Population-specific HLA characteristics determine the registry benefits for patients and also the need for further regional donor recruitment. In this work, we analyzed HLA allele and haplotype frequencies of donors of DKMS Chile, the first Chilean donor registry, with self-assessed "non-Indigenous" (n=92,788) and "Mapuche" (n=1,993) ancestry. We identified HLA alleles that were distinctly more abundant in the Chilean subpopulations than in worldwide reference populations, four of them particularly characteristic for the Mapuche subpopulation, namely B*39:09g, B*35:09, DRB1*04:07g, and DRB1*16:02g. Both population subsamples carried haplotypes of both Native American and European origin at high frequencies, reflecting Chile's complex history of admixture and immigration. Matching probability analysis revealed limited benefits for Chilean patients (both non-Indigenous and Mapuche) from donor registries of non-Chilean donors, thus indicating a need for ongoing significant donor recruitment efforts in Chile.

World Marrow Donor Association guidelines for the reporting of novel HLA alleles.

The guidelines for the implementation and reporting of HLA nomenclature for the World Marrow Donor Association have served as a reliable standard for communication of HLA data in the hematopoietic cell transplantation process. Wider use of next-generation sequencing made a special provision of the guidelines increasingly pertinent: how to communicate novel HLA alleles. Novel alleles need to be recognized by the WHO Nomenclature Committee for Factors of the HLA system to obtain official allele designations. Until then they have to be handled according to the specific rules. Leaving the actual rules basically unchanged we give some advice on how to communicate novel alleles to best facilitate the search process for cases where novel alleles are identified on donor or patient side.

Large case-control study indicates no association of KIR genotype and risk of developing acute myeloid leukemia.

Immunogenetic association studies may give rise to new hypotheses on the immune surveillance of cancer. We hypothesized that certain combinations of killer immunoglobulin-like receptor (KIR) and HLA genotypes may enhance natural killer (NK) cell immunity against nascent acute myeloid leukemia (AML) and, thereby, lead to a skewed genotype distribution among patients. For this purpose, we analyzed KIR and HLA genotypes of 1767 German patients with AML and compared the results with that of the data of 51 890 German volunteers who had registered with German bone marrow donor file (DKMS). Patient samples were retrieved from the Collaborative Biobank and the biorepository of the Study Alliance Leukemia. All samples were genotyped with high-resolution amplicon-based next-generation sequencing. Because of the large number of controls, this study was very sensitive to detect the impact of KIR genotype. Knowledge on KIRs and their cognate HLA ligands allowed for testing of several hypotheses of NK cell-mediated endogenous leukemia surveillance. We did not find significant differences between the 2 cohorts in regard to the presence or absence of single KIR genes. When grouped based on telomeric or centromeric gene content, the major haplotypes A/A, A/B, and B/B were equally distributed among patients and control subjects. Using information on KIRs and their HLA ligands, we further tested receptor-ligand models and summation models without revealing markedly significant differences between patients and controls, albeit we observed a trend pointing at a minor protective effect of a low number of inhibitory KIR/KIR-ligand pairs. The results suggest that the KIR/KIR-ligand genotype has no effect on the susceptibility for the development of de novo AML.

Post-COVID-19 condition in the German working population: A cross-sectional study of 200,000 registered stem cell donors.

BACKGROUND: The SARS-CoV-2 pandemic has strained health systems worldwide, and infection numbers continue to rise. While previous data have already shown that many patients suffer from symptoms for months after an acute infection, data on risk factors and long-term outcomes are incomplete, particularly for the working population. OBJECTIVES: We aimed to provide information on the prevalence of post-COVID-19 conditions in a subset of the German working-age population (18-61 years old) and to analyze risk factors. METHODS: We conducted an online survey with a health questionnaire among registered potential stem cell donors with or without a self-reported history of polymerase chain reaction (PCR)-confirmed SARS-CoV-2 infection. Logistic regression models were used to examine the risks of severity of acute infection, sex, age, body mass index, diabetes mellitus, and arterial hypertension medication on post-COVID-19 symptoms. RESULTS: A total of 199,377 donors reported evaluable survey questionnaires-12,609 cases had a history of SARS-CoV-2 infection and 186,768 controls had none. Overall, cases reported physical, cognitive, and psychological complaints more frequently compared to controls. Increased rates of complaints persisted throughout 15 months postinfection, for example, 28.4%/19.3% of cases/controls reported fatigue (p <0.0001) and 9.5%/3.6% of cases/controls reported loss of concentration (p <0.0001). No significant differences were observed in the frequency of reported symptoms between 3 and 15 months postinfection. Multivariate analysis revealed a strong influence of the severity of the acute SARS-CoV-2 infection episode and age on the risk for post-COVID-19 conditions. CONCLUSION: We report the prevalence of post-COVID-19 conditions in mainly unvaccinated individuals with SARS-CoV-2 infections between February 2020 and August 2021. The severity of the acute course and age were major risk factors. Vaccinations may reduce the risk of post-COVID-19 conditions by reducing the risk of severe infections.

Host KIR/HLA-C Genotypes Determine HIV-Mediated Changes of the NK Cell Repertoire and Are Associated With Vpu Sequence Variations Impacting Downmodulation of HLA-C.

NK cells play a pivotal role in viral immunity, utilizing a large array of activating and inhibitory receptors to identify and eliminate virus-infected cells. Killer-cell immunoglobulin-like receptors (KIRs) represent a highly polymorphic receptor family, regulating NK cell activity and determining the ability to recognize target cells. Human leukocyte antigen (HLA) class I molecules serve as the primary ligand for KIRs. Herein, HLA-C stands out as being the dominant ligand for the majority of KIRs. Accumulating evidence indicated that interactions between HLA-C and its inhibitory KIR2DL receptors (KIR2DL1/L2/L3) can drive HIV-1-mediated immune evasion and thus may contribute to the intrinsic control of HIV-1 infection. Of particular interest in this context is the recent observation that HIV-1 is able to adapt to host HLA-C genotypes through Vpu-mediated downmodulation of HLA-C. However, our understanding of the complex interplay between KIR/HLA immunogenetics, NK cell-mediated immune pressure and HIV-1 immune escape is still limited. Therefore, we investigated the impact of specific KIR/HLA-C combinations on the NK cell receptor repertoire and HIV-1 Vpu protein sequence variations of 122 viremic, untreated HIV-1+ individuals. Compared to 60 HIV-1- controls, HIV-1 infection was associated with significant changes within the NK cell receptor repertoire, including reduced percentages of NK cells expressing NKG2A, CD8, and KIR2DS4. In contrast, the NKG2C+ and KIR3DL2+ NK cell sub-populations from HIV-1+ individuals was enlarged compared to HIV-1- controls. Stratification along KIR/HLA-C genotypes revealed a genotype-dependent expansion of KIR2DL1+ NK cells that was ultimately associated with increased binding affinities between KIR2DL1 and HLA-C allotypes. Lastly, our data hinted to a preferential selection of Vpu sequence variants that were associated with HLA-C downmodulation in individuals with high KIR2DL/HLA-C binding affinities. Altogether, our study provides evidence that HIV-1-associated changes in the KIR repertoire of NK cells are to some extent predetermined by host KIR2DL/HLA-C genotypes. Furthermore, analysis of Vpu sequence polymorphisms indicates that differential KIR2DL/HLA-C binding affinities may serve as an additional mechanism how host genetics impact immune evasion by HIV-1.

Engagement of TRAIL triggers degranulation and IFNγ production in human natural killer cells.

NK cells utilize a large array of receptors to screen their surroundings for aberrant or virus-infected cells. Given the vast diversity of receptors expressed on NK cells we seek to identify receptors involved in the recognition of HIV-1-infected cells. By combining an unbiased large-scale screening approach with a functional assay, we identify TRAIL to be associated with NK cell degranulation against HIV-1-infected target cells. Further investigating the underlying mechanisms, we demonstrate that TRAIL is able to elicit multiple effector functions in human NK cells independent of receptor-mediated induction of apoptosis. Direct engagement of TRAIL not only results in degranulation but also IFNγ production. Moreover, TRAIL-mediated NK cell activation is not limited to its cognate death receptors but also decoy receptor I, adding a new perspective to the perceived regulatory role of decoy receptors in TRAIL-mediated cytotoxicity. Based on these findings, we propose that TRAIL not only contributes to the anti-HIV-1 activity of NK cells but also possesses a multifunctional role beyond receptor-mediated induction of apoptosis, acting as a regulator for the induction of different effector functions.

Graphical user interface for the haplotype frequency estimation software Hapl-o-Mat.

Population-specific human leukocyte antigen (HLA) haplotype frequencies are an essential basis of advanced algorithms for donor selection in unrelated hematopoietic stem cell transplantation. In 2016, we introduced Hapl-o-Mat, a versatile tool for haplotype frequency estimation based on an expectation-maximization algorithm (https://github.com/DKMS/hapl-o-Mat). Hapl-o-Mat is specifically tailored to the analysis of HLA genes and able to cope with the heterogeneous genotyping data usually found in donor registries. To make Hapl-o-Mat accessible to a wider range of users, we designed a graphical user interface module that considerably facilitates the interaction with the application (https://github.com/DKMS/hapl-o-Mat_GUI). We further provide a precompiled version of Hapl-o-Mat that can be used on Windows personal computers without dependency on additional software libraries (https://github.com/DKMS/hapl-o-Mat_WinBin). We are confident that these new, user-oriented features will encourage more researchers to apply Hapl-o-Mat to their data, thereby increasing knowledge and public availability of population-specific HLA haplotype frequencies.

Estimating HLA haplotype frequencies from homozygous individuals - A Technical Report.

We estimated HLA haplotype frequencies based on individuals homozygous for 4, 5 or 6 loci. Validation of our approach using a sample of over 3.4 million German individuals was successful. Compared to an expectation-maximization algorithm, the errors were larger. However, our approach allows the unequivocal detection of rare haplotypes.

High-throughput Interpretation of Killer-cell Immunoglobulin-like Receptor Short-read Sequencing Data with PING.

The killer-cell immunoglobulin-like receptor (KIR) complex on chromosome 19 encodes receptors that modulate the activity of natural killer cells, and variation in these genes has been linked to infectious and autoimmune disease, as well as having bearing on pregnancy and transplant outcomes. The medical relevance and high variability of KIR genes makes short-read sequencing an attractive technology for interrogating the region, providing a high-throughput, high-fidelity sequencing method that is cost-effective. However, because this gene complex is characterized by extensive nucleotide polymorphism, structural variation including gene fusions and deletions, and a high level of homology between genes, its interrogation at high resolution has been thwarted by bioinformatic challenges, with most studies limited to examining presence or absence of specific genes. Here, we present the PING (Pushing Immunogenetics to the Next Generation) pipeline, which incorporates empirical data, novel alignment strategies and a custom alignment processing workflow to enable high-throughput KIR sequence analysis from short-read data. PING provides KIR gene copy number classification functionality for all KIR genes through use of a comprehensive alignment reference. The gene copy number determined per individual enables an innovative genotype determination workflow using genotype-matched references. Together, these methods address the challenges imposed by the structural complexity and overall homology of the KIR complex. To determine copy number and genotype determination accuracy, we applied PING to European and African validation cohorts and a synthetic dataset. PING demonstrated exceptional copy number determination performance across all datasets and robust genotype determination performance. Finally, an investigation into discordant genotypes for the synthetic dataset provides insight into misaligned reads, advancing our understanding in interpretation of short-read sequencing data in complex genomic regions. PING promises to support a new era of studies of KIR polymorphism, delivering high-resolution KIR genotypes that are highly accurate, enabling high-quality, high-throughput KIR genotyping for disease and population studies.

Individual HLA-A, -B, -C, and -DRB1 Genotypes Are No Major Factors Which Determine COVID-19 Severity.

HLA molecules are key restrictive elements to present intracellular antigens at the crossroads of an effective T-cell response against SARS-CoV-2. To determine the impact of the HLA genotype on the severity of SARS-CoV-2 courses, we investigated data from 6,919 infected individuals. HLA-A, -B, and -DRB1 allotypes grouped into HLA supertypes by functional or predicted structural similarities of the peptide-binding grooves did not predict COVID-19 severity. Further, we did not observe a heterozygote advantage or a benefit from HLA diplotypes with more divergent physicochemical peptide-binding properties. Finally, numbers of in silico predicted viral T-cell epitopes did not correlate with the severity of SARS-CoV-2 infections. These findings suggest that the HLA genotype is no major factor determining COVID-19 severity. Moreover, our data suggest that the spike glycoprotein alone may allow for abundant T-cell epitopes to mount robust T-cell responses not limited by the HLA genotype.