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.

Remission induction versus immediate allogeneic haematopoietic stem cell transplantation for patients with relapsed or poor responsive acute myeloid leukaemia (ASAP): a randomised, open-label, phase 3, non-inferiority trial.

BACKGROUND: Whether high-dose cytarabine-based salvage chemotherapy, administered to induce complete remission in patients with poor responsive or relapsed acute myeloid leukaemia scheduled for allogeneic haematopoietic stem-cell transplantation (HSCT) after intensive conditioning confers a survival advantage, is unclear. METHODS: To test salvage chemotherapy before allogeneic HSCT, patients aged between 18 and 75 years with non-favourable-risk acute myeloid leukaemia not in complete remission after first induction or untreated first relapse were randomly assigned 1:1 to remission induction with high-dose cytarabine (3 g/m2 intravenously, 1 g/m2 intravenously for patients >60 years or with a substantial comorbidity) twice daily on days 1-3 plus mitoxantrone (10 mg/m2 intravenously) on days 3-5 or immediate allogeneic HSCT for the disease control group. Block randomisation with variable block lengths was used and patients were stratified by age, acute myeloid leukaemia risk, and disease status. The study was open label. The primary endpoint was treatment success, defined as complete remission on day 56 after allogeneic HSCT, with the aim to show non-inferiority for disease control compared with remission induction with a non-inferiority-margin of 5% and one-sided type 1 error of 2·5%. The primary endpoint was analysed in both the intention-to-treat (ITT) population and in the per-protocol population. The trial is completed and was registered at ClinicalTrials.gov, NCT02461537. FINDINGS: 281 patients were enrolled between Sept 17, 2015, and Jan 12, 2022. Of 140 patients randomly assigned to disease control, 135 (96%) proceeded to allogeneic HSCT, 97 (69%) after watchful waiting only. Of 141 patients randomly assigned to remission induction, 134 (95%) received salvage chemotherapy and 128 (91%) patients subsequently proceeded to allogeneic HSCT. In the ITT population, treatment success was observed in 116 (83%) of 140 patients in the disease control group versus 112 (79%) of 141 patients with remission induction (test for non-inferiority, p=0·036). Among per-protocol treated patients, treatment success was observed in 116 (84%) of 138 patients with disease control versus 109 (81%) of 134 patients in the remission induction group (test for non-inferiority, p=0·047). The difference in treatment success between disease control and remission induction was estimated as 3·4% (95% CI -5·8 to 12·6) for the ITT population and 2·7% (-6·3 to 11·8) for the per-protocol population. Fewer patients with disease control compared with remission induction had non-haematological adverse events grade 3 or worse (30 [21%] of 140 patients vs 86 [61%] of 141 patients, χ2 test p<0·0001). Between randomisation and the start of conditioning, with disease control two patients died from progressive acute myeloid leukaemia and zero from treatment-related complications, and with remission induction two patients died from progressive acute myeloid leukaemia and two from treatment-related complications. Between randomisation and allogeneic HSCT, patients with disease control spent a median of 27 days less in hospital than those with remission induction, ie, the median time in hospital was 15 days (range 7-64) versus 42 days (27-121, U test p<0·0001), respectively. INTERPRETATION: Non-inferiority of disease control could not be shown at the 2·5% significance level. The rate of treatment success was also not statistically better for patients with remission induction. Watchful waiting and immediate transplantation could be an alternative for fit patients with poor response or relapsed acute myeloid leukaemia who have a stem cell donor available. More randomised controlled intention-to-transplant trials are needed to define the optimal treatment before transplantation for patients with active acute myeloid leukaemia. FUNDING: DKMS and the Gert and Susanna Mayer Stiftung Foundation.

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.

ICH Q14-inspired novel approach to establish an SFC-based purity method for carbamazepine.

The proposed ICH Q14 guideline "Analytical procedure development" describes science and risk-based approaches for development and maintenance of analytical procedures suitable for the assessment of the quality of drug substances and drug products. As a case study, the systematic development and validation of a supercritical fluid chromatography (SFC)-based purity method for carbamazepine is presented. Systematic analytical quality by design (AQbD) principles were applied using the software package Fusion QbD to the method development approach. The relationship between chromatographic parameters and the responses of interest were examined to improve the reliability of the method by understanding, reducing, and controlling sources of variability. Method performance qualification in terms of method robustness was finally carried out with the parameters that were classified as critical after method development and a validation study met previously set acceptance criteria. The developed SFC purity method for carbamazepine demonstrated readiness as a viable alternative to the official HPLC method published in the Ph.Eur. with improved peak resolution, improved peak symmetry, and faster analysis times (3 min vs. 80 min for the official method). Its inherent reliability illustrates the superiority of AQbD in method development and application for drug quality assurance.

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.

5 years DKMS Chile: approach, results and impact of the first unrelated stem cell donor center in Chile.

Introduction: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is performed worldwide to treat blood cancer and other life-threatening blood disorders. As successful transplantation requires an HLA-compatible donor, unrelated donor centers and registries have been established worldwide to identify donors for patients without a family match. Ethnic minorities are underrepresented in large donor registries. Matching probabilities are higher when donors and patients share the same ethnic background, making it desirable to increase the diversity of the global donor pool by recruiting donors in new regions. Here, we report the establishment and the first 5 years of operation of the first unrelated stem cell donor center in Chile, a high-income country in South America with a population of over 19 million. Methods: We used online and in-person donor recruitment practices through patient appeals and donor drives in companies, universities, the armed forces, and public services. After confirmatory typing donors were subjected to medical work-up and cleared for donation. Results: We recruited almost 170,000 donors in 5 years. There were 1,488 requests received for confirmatory typing and donor availability checks, of which 333 resulted in medical work-up, leading to 194 stem cell collections. Products were shipped to Chile (48.5%) and abroad. Even when the COVID-19 pandemic challenged our activities, the number of donors recruited and shipped stem cell products remained steady. In Chile there was an almost 8-fold increase in unrelated donor transplantation activity from 16 procedures in 2016-2018 to 124 procedures in 2019-2021, mainly for pediatric patients following the center's establishment. We estimate that 49.6% of Chilean patients would find at least one matched unrelated donor in the global DKMS donor pool. Discussion: Establishing a DKMS donor center in Chile has significantly increased donor availability for Chilean patients and contributed to an increase of unrelated donor stem cell transplant activity.

Small volume bone marrow aspirates with high progenitor cell concentrations maximize cell therapy dose manufacture and substantially reduce donor hemoglobin loss.

BACKGROUND: Bone marrow (BM) transplantation is a life-saving therapy for hematological diseases, and the BM harbors also highly useful (progenitor) cell types for novel cell therapies manufacture. Yet, the BM collection technique is not standardized. METHODS: Benchmarking our collection efficiency to BM collections worldwide (N = 1248), we noted a great variability of total nucleated cell (TNC) yields in BM products (HPC-M) with superior performance of our center, where we have implemented a small volume aspirate policy. Thus, we next prospectively aimed to assess the impact of BM collection technique on HPC-M quality. For each BM collection (N = 20 donors), small volume (3 mL) and large volume (10 mL) BM aspirates were sampled at 3 time points and analyzed for cell composition. RESULTS: Compared to large volume aspirates, small volume aspirates concentrated more TNCs, immune cells, platelets, hematopoietic stem/progenitor cells, mesenchymal stromal cells (MSCs), and endothelial progenitors. Inversely, the hemoglobin concentration was higher in large volume aspirates indicating more hemoglobin loss. Manufacturing and dosing scenarios showed that small volume aspirates save up to 42% BM volume and 44% hemoglobin for HPC-M donors. Moreover, MSC production efficiency can be increased by more than 150%. CONCLUSIONS: We propose to consider small volume BM aspiration as standard technique for BM collection.

Controlling the elution order of insulin and its analogs in sub-/supercritical fluid chromatography using methanesulfonic acid and 18-crown-6 as mobile phase additives.

The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed-phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 µm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18-crown-6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH).

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.

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.

Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides.

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide's fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 µm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.

Prevalence of nitrosamine contaminants in drug samples: Has the crisis been overcome?

Various drug samples (N = 249; drug substances, tablets, capsules, solutions, crèmes, and more) from the European pharmaceutical market were collected since 2019 and analyzed for 16 nitrosamines (NAs). In 2.0% of the cases, NAs were detected. These findings included four active pharmaceutical ingredients already known for potential NA contamination: losartan (N-nitrosodimethylamine [NDMA] and N-nitrosodiethylamine, simultaneously), valsartan (NDMA), metformin (NDMA) and ranitidine (NDMA). The fifth new finding, which has not been reported yet, discovered contamination of a molsidomine tablet sample with N-nitrosomorpholine (NMor). The tablet contained 144% of the toxicological allowable intake for NMor. NMor was included in our screening from the beginning and is currently the focus of regulatory authorities, but was added to the guidelines only last year. Thus, it may not have been the focus of regulatory investigations for too long. Our results indicate that the majority of drug products in the market are nonhazardous in terms of patient safety and drug purity. Unfortunately, the list of individual affected products keeps growing constantly and new NA cases, such as molsidomine or nitrosated drug substances (nitrosamine drug substance-related impurities [NDSRI]), continue to emerge. We therefore expect nitrosamine screenings to remain a high priority.

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.

Reanalysis of unclear CMV status results from buccal swab samples of potential stem cell donors is an efficient donor registry strategy.

Many stem cell donor registries determine the cytomegalovirus (CMV) IgG serostatus at donor recruitment as it is an important marker for donor selection in the context of hematopoietic stem cell transplantation. To make sample collection less uncomfortable for the donor, we have developed a method that allows CMV status determination from buccal swab samples, thus avoiding blood drawing. However, the determination fails in some cases which leads to new donors being listed for donor search without CMV status, thus hindering donor searches. In this work, we evaluated the success rate of repeating CMV status analysis from a new swab. Our results show that about 90% of the samples could be successfully determined. Due to the great importance of the CMV status in donor search, we consider the retesting approach to be highly recommended for stem cell donor registries.

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.

Application of sub-/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide.

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed-phase liquid chromatography meets these needs and makes it a useful option during research and development. In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design-of-Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry-compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed-phase liquid chromatography were resolved by applying the final method.