Fluoxetine and Sertraline Potently Neutralize the Replication of Distinct SARS-CoV-2 Variants.

The pandemic caused by SARS-CoV-2 is still a major health problem. Newly emerging variants and long-COVID-19 represent a challenge for the global health system. In particular, individuals in developing countries with insufficient health care need easily accessible, affordable and effective treatments of COVID-19. Previous studies have demonstrated the efficacy of functional inhibitors of acid sphingomyelinase against infections with various viruses, including early variants of SARS-CoV-2. This work investigated whether the acid sphingomyelinase inhibitors fluoxetine and sertraline, usually used as antidepressant molecules in clinical practice, can inhibit the replication of the former and recently emerged SARS-CoV-2 variants in vitro. Fluoxetine and sertraline potently inhibited the infection with pseudotyped virus-like particles and SARS-CoV-2 variants D614G, alpha, delta, omicron BA.1 and omicron BA.5. These results highlight fluoxetine and sertraline as priority candidates for large-scale phase 3 clinical trials at different stages of SARS-CoV-2 infections, either alone or in combination with other medications.

Lymphocyte Function at Baseline Could Be a New Predictor of Tumor Burden following Six Cycles of Radium-223 Therapy in Patients with Metastasized, Castration-Resistant Prostate Cancer.

Previous data indicate that one cycle of treatment with radium-223 (223Ra) did not significantly impair lymphocyte function in patients with metastasized, castration-resistant prostate cancer. The aim of the current study was to assess in 21 patients whether six cycles of this therapy had an effect on lymphocyte proliferation and interferon-γ and interleukin (IL)-10 ELISpot results. Lymphocyte proliferation after stimulation with microbial antigens and the production of interferon-γ continuously decreased after six cycles of radionuclide therapy, reaching statistical significance (p < 0.05) at months 1, 2, 4, and/or 6 after therapy. One month after the last cycle of therapy, 67% of patients showed a decrease in tumor burden. The tumor burden correlated negatively with IL-10 secretion at baseline, e.g., after stimulation with tetanus antigen (p < 0.0001, r = -0.82). As determined by receiver operating characteristic (ROC) curve analysis, tetanus-specific IL-10 spots at baseline had the highest predictive value (p = 0.005) for tumor burden at month 6, with an area under the curve (AUC) of 0.90 (sensitivity 100%, specificity 78%). In conclusion, we observed an additive effect of treatment with 223Ra on immune function and found that IL-10 secretion at baseline predicted tumor burden at month 6 after treatment.

T Cell Responses against Orthopoxviruses in HIV-Positive Patients.

A global outbreak of predominantly sexually transmitted mpox infections, outside endemic regions, was reported in May 2022. Thereafter, risk groups were vaccinated against smallpox, a structurally related orthopoxvirus. In the current study, we analyzed T cell responses against peptides derived from orthopoxviruses in 33 HIV-positive patients after two vaccinations against smallpox and in 10 patients after mpox infection. We established an ELISpot assay, detecting either the secretion of the pro-inflammatory cytokine interferon (IFN)-γ or interleukin (IL)-2. After vaccination, 21 out of 33 patients (64%) showed specific IFN-γ secretion and 18 (55%) specific IL-2 secretion, defined as >3-fold higher specific value than negative control and at least 4 spots above the negative control. After mpox infection, all patients showed specific IFN-γ secretion and 7 out of 10 (70%) IL-2 secretion. In vaccinated patients, IFN-γ responses were significantly lower than in patients with mpox infection (median response 4.5 vs. 21.0 spots, p < 0.001). The same trend was observed for IL-2 responses. After mpox infection, IL-2 ELISpot results positively correlated with CD8+ T cells (p < 0.05). Thus, T cell responses were detectable in two thirds of HIV-positive patients after vaccination and were even more abundant and vigorous after mpox infection.

Impact of the COVID-19 outbreak on blood supply in two large university hospitals.

OBJECTIVE: This study aimed to examine the relationship between the decrease in elective procedures and the need for blood donation during the novel coronavirus disease (COVID-19) pandemic at university hospitals. BACKGROUND: The COVID-19 pandemic has immensely impacted transfusion medicine. By cancelling elective surgery, the German government hoped to increase the available resources for patients infected with COVID-19, especially in intensive care units, and prevent the shortage of blood products. METHODS/MATERIALS: Over 26 weeks, from the 3rd of February 2020 to the 2nd of August 2020, during the first phase of the pandemic, we assessed the number of crossmatches, blood group typing, use of donated blood, and case mix indices by retrospectively analysing data from two major university hospitals' information systems in Essen and Hamburg, Germany. Data were pooled, analysed, and compared with that of the same period in the previous year. RESULTS: Following the cessation of elective procedures, the number of requests for crossmatches and blood group typing significantly decreased in 2020 compared to that in 2019. However, the number of blood transfusions required was reduced to a lesser extent. The number of outpatient and inpatient cases significantly decreased, whereas the cases requiring transfusion decreased only. CONCLUSION: During the initial phase of the pandemic, transfusion medicine, especially in large institutions, faced an almost unchanged high demand for donated blood. This should be considered regarding personnel and blood donation allocations. Therefore, we developed a monitoring system to display the availability of blood products in real-time. The quick and easy display of in-stock and expiring blood products can optimise the use of this valuable resource.

HPV-associated head and neck cancer is characterized by distinct profiles of CD8+ T cells and myeloid-derived suppressor cells.

Patients with HPV--localized head and neck cancer (HNC) show inferior outcomes after surgery and radiochemotherapy compared to HPV-associated cancers. The underlying mechanisms remain elusive, but differences in immune status and immune activity may be implicated. In this study, we analyzed immune profiles of CD8+ T cells and myeloid-derived suppressor cells (MDSC) in HPV+ versus HPV- disease.The overall frequency of CD8+ T cells was reduced in HNC versus healthy donors but substantially increased after curative therapy (surgery and/or radiochemotherapy). In HPV+ patients, this increase was associated with significant induction of peripheral blood CD8+/CD45RA-/CD62L- effector memory cells. The frequency of HPV-antigen-specific CD8+ cells was low even in patients with virally associated tumors and dropped to background levels after curative therapy. Pre-therapeutic counts of circulating monocytic MDSC, but not PMN-MDSC, were increased in patients with HPV- disease. This increase was accompanied by reduced fractions of terminally differentiated CD8+ effector cells. HPV- tumors showed reduced infiltrates of CD8+ and CD45RO+ immune cells compared with HPV+ tumors. Importantly, frequencies of tumor tissue-infiltrating PMN-MDSC were increased, while percentages of Granzyme B+ and Ki-67+ CD8 T cells were reduced in patients with HPV- disease.We report differences in frequencies and relative ratios of MDSC and effector T cells in HPV- HNC compared with more immunogenic HPV-associated disease. Our data provide new insight into the immunological profiles of these two tumor entities and may be utilized for more tailored immunotherapeutic approaches in the future.

Optimizing platelet transfusion through a personalized deep learning risk assessment system for demand management.

ABSTRACT: Platelet demand management (PDM) is a resource-consuming task for physicians and transfusion managers of large hospitals. Inpatient numbers and institutional standards play significant roles in PDM. However, reliance on these factors alone commonly results in platelet shortages. Using data from multiple sources, we developed, validated, tested, and implemented a patient-specific approach to support PDM that uses a deep learning-based risk score to forecast platelet transfusions for each hospitalized patient in the next 24 hours. The models were developed using retrospective electronic health record data of 34 809 patients treated between 2017 and 2022. Static and time-dependent features included demographics, diagnoses, procedures, blood counts, past transfusions, hematotoxic medications, and hospitalization duration. Using an expanding window approach, we created a training and live-prediction pipeline with a 30-day input and 24-hour forecast. Hyperparameter tuning determined the best validation area under the precision-recall curve (AUC-PR) score for long short-term memory deep learning models, which were then tested on independent data sets from the same hospital. The model tailored for hematology and oncology patients exhibited the best performance (AUC-PR, 0.84; area under the receiver operating characteristic curve [ROC-AUC], 0.98), followed by a multispecialty model covering all other patients (AUC-PR, 0.73). The model specific to cardiothoracic surgery had the lowest performance (AUC-PR, 0.42), likely because of unexpected intrasurgery bleedings. To our knowledge, this is the first deep learning-based platelet transfusion predictor enabling individualized 24-hour risk assessments at high AUC-PR. Implemented as a decision-support system, deep-learning forecasts might improve patient care by detecting platelet demand earlier and preventing critical transfusion shortages.

Predicting Individual Patient Platelet Demand in a Large Tertiary Care Hospital Using Machine Learning.

Introduction: An increasing shortage of donor blood is expected, considering the demographic change in Germany. Due to the short shelf life and varying daily fluctuations in consumption, the storage of platelet concentrates (PCs) becomes challenging. This emphasizes the need for reliable prediction of needed PCs for the blood bank inventories. Therefore, the objective of this study was to evaluate multimodal data from multiple source systems within a hospital to predict the number of platelet transfusions in 3 days on a per-patient level. Methods: Data were collected from 25,190 (42% female and 58% male) patients between 2017 and 2021. For each patient, the number of received PCs, platelet count blood tests, drugs causing thrombocytopenia, acute platelet diseases, procedures, age, gender, and the period of a patient's hospital stay were collected. Two models were trained on samples using a sliding window of 7 days as input and a day 3 target. The model predicts whether a patient will be transfused 3 days in the future. The model was trained with an excessive hyperparameter search using patient-level repeated 5-fold cross-validation to optimize the average macro F2-score. Results: The trained models were tested on 5,022 unique patients. The best-performing model has a specificity of 0.99, a sensitivity of 0.37, an area under the precision-recall curve score of 0.45, an MCC score of 0.43, and an F1-score of 0.43. However, the model does not generalize well for cases when the need for a platelet transfusion is recognized. Conclusion: A patient AI-based platelet forecast could improve logistics management and reduce blood product waste. In this study, we build the first model to predict patient individual platelet demand. To the best of our knowledge, we are the first to introduce this approach. Our model predicts the need for platelet units for 3 days in the future. While sensitivity underperforms, specificity performs reliably. The model may be of clinical use as a pretest for potential patients needing a platelet transfusion within the next 3 days. As sensitivity needs to be improved, further studies should introduce deep learning and wider patient characterization to the methodological multimodal, multisource data approach. Furthermore, a hospital-wide consumption of PCs could be derived from individual predictions.

In Patients Treated by Selective Internal Radiotherapy, Cellular In Vitro Immune Function Is Predictive of Survival.

In patients with liver malignancies, the cellular immune function was impaired in vitro after selective internal radiotherapy (SIRT). Because immunosuppression varied substantially, in the current study, we investigated in 25 SIRT patients followed up for ten years whether the lymphocyte function was correlated with survival. Peripheral blood mononuclear cells were stimulated with four microbial antigens (tuberculin, tetanus toxoid, Candida albicans and CMV) before therapy and at four time points thereafter, and lymphocyte proliferation was determined by H3-thymidine uptake. The median sum of the responses to these four antigens decreased from 39,464 counts per minute (CPM) increment (range 1080-204,512) before therapy to a minimum of 700 CPM increment on day 7 after therapy (0-93,187, p < 0.0001). At all five time points, the median survival in patients with weaker responses was 2- to 3.5-fold shorter (p < 0.05). On day 7, the median survival in patients with responses below and above the cutoff of a 2 CPM increment was 185 and 523 days, respectively (χ2 = 9.4, p = 0.002). In conclusion, lymphocyte function could be a new predictor of treatment outcome after SIRT.

Elevated sHLA-G plasma levels post chemotherapy combined with ILT-2 rs10416697C allele status of the sHLA-G-related receptor predict poorest disease outcome in early triple-negative breast cancer patients.

Introduction: Triple negative breast cancer (TNBC) shows an aggressive growing and spreading behavior and has limited treatment options, often leading to inferior disease outcome. Therefore, surrogate markers are urgently needed to identify patients at high risk of recurrence and more importantly, to identify additional therapeutic targets enabling further treatment options. Based on the key role of the non-classical human leukocyte antigen G (HLA-G) and its related receptor immunoglobulin-like transcript receptor-2 (ILT-2) in immune evasion mechanisms of tumors, members of this ligand-receptor axis appear to be promising tool for both, defining risk groups and potential therapeutic targets. Materials and methods: To follow this, sHLA-G levels before and after chemotherapy (CT), HLA-G 3' UTR haplotypes, and allele variations rs10416697 at the distal gene promoter region of ILT-2 were defined in healthy female controls and early TNBC patients. The results obtained were associated with clinical status, presence of circulating tumor cell (CTC) subtypes, and disease outcome of patients in terms of progression-free or overall survival. Results: sHLA-G plasma levels were increased in TNBC patients post-CT compared to levels of patients pre-CT or controls. High post-CT sHLA-G levels were associated with the development of distant metastases, the presence of ERCC1 or PIK3CA-CTC subtypes post-CT, and poorer disease outcome in uni- or multivariate analysis. HLA-G 3' UTR genotypes did not influence disease outcome but ILT-2 rs10416697C allele was associated with AURKA-positive CTC and with adverse disease outcome by uni- and multivariate analysis. The prognostic value of the combined risk factors (high sHLA-G levels post-CT and ILT-2 rs10416697C allele carrier status) was an even better independent indicator for disease outcome in TNBC than the lymph nodal status pre-CT. This combination allowed the identification of patients with high risk of early progression/death with positive nodal status pre-CT or with non-pathological complete therapy response. Conclusion: The results of this study highlight for the first time that the combination of high levels of sHLA-G post-CT with ILT-2 rs10416697C allele receptor status is a promising tool for the risk assessment of TNBC patients and support the concept to use HLA-G/ILT-2 ligand-receptor axis as therapeutic targets.

Antibody responses after sequential vaccination with PCV13 and PPSV23 in kidney transplant recipients.

PURPOSE: Vaccination against Streptococcus pneumoniae is recommended in transplant recipients to reduce the morbidity and mortality from invasive pneumococcal disease. Previous studies indicate that transplant recipients can produce specific antibodies after vaccination with the 13-valent pneumococcal conjugate vaccine Prevenar 13 (PCV13) or the pneumococcal polysaccharide vaccine Pneumovax 23 (PPSV23). National guidelines recommend sequential vaccination with PCV13 followed by PPSV23 in kidney transplant patients. However, there are currently no data on the serological response in kidney transplant recipients, who received a sequential vaccination with PCV13 and PPSV23. METHODS: In the current study, we sequentially vaccinated 46 kidney transplant recipients with PCV13 and PPSV23 and determined global and serotype-specific anti-pneumococcal antibody responses in the year following vaccination. RESULTS: Serotype-specific and global anti-pneumococcal antibody concentrations were significantly higher compared to baseline. We observed that serotype-specific antibody responses varied by serotype (between 2.2- and 2.9-fold increase after 12 months). The strongest responses after 12 months were detected against the serotypes 9N (2.9-fold increase) and 14 (2.8-fold increase). Global antibody responses also varied with respect to immunoglobulin class. IgG2 revealed the highest increase (2.7-fold), IgM the lowest (1.7-fold). Sequential vaccination with both vaccines achieved higher antibody levels in comparison with a historical cohort studied at our institute, that was vaccinated with PCV13 alone. During the 12-months follow-up period, none of the patients developed pneumococcal-associated pneumonia or vaccination-related allograft rejection. CONCLUSION: In conclusion, we strongly recommend sequential vaccination over single immunization in kidney transplant recipients.

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury.

BACKGROUND: Human mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia-ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action. METHODS: Nine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples. RESULTS: Intranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression. CONCLUSION: Our data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

Long-Term Follow-Up after Adoptive Transfer of BK-Virus-Specific T Cells in Hematopoietic Stem Cell Transplant Recipients.

The BK virus (BKV) causes severe hemorrhagic cystitis in hematopoietic stem cell transplant (HSCT) recipients. To eliminate reactivated BKV, symptomatic patients can be treated with a reduction of the immunosuppressive therapy, with the antiviral drug cidofovir, or with virus-specific T cells (VSTs). In the current study, we compared the effect of VSTs to other treatment options, following up specific T cells using interferon-gamma ELISpot assay. We observed BKV large T-specific cellular responses in 12 out of 17 HSCT recipients with BKV-related cystitis (71%). In recipients treated with VSTs, 6 out of 7 showed specific T-cell responses, and that number in those without VSTs was 6 out of 10. In comparison, 27 out of 50 healthy controls (54%) responded. In HSCT recipients treated for BKV-related cystitis, absolute CD4+ T-cell numbers and renal function correlated with BKV-specific cellular responses (p = 0.03 and 0.01, respectively). In one patient, BKV-specific cellular immunity could already be detected at baseline, on day 35 after HSCT and prior to VSTs, and remained increased until day 226 after VSTs (78 vs. 7 spots increment). In conclusion, the ELISpot appears to be suitable to sensitively monitor BKV-specific cellular immunity in HSCT recipients, even early after transplantation or in the long term after VSTs.

Qualification of a multidonor mixed lymphocyte reaction assay for the functional characterization of immunomodulatory extracellular vesicles.

BACKGROUND AIMS: Extracellular vesicles (EVs), including exosomes and microvesicles, are released by almost all cells and found in all body fluids. Unknown proportions of EVs transmit specific information from their cells of origin to specific target cells and are key mediators in intercellular communication processes. Depending on their origin, EVs can modulate immune responses, either acting as pro- or anti-inflammatory. With the aim to analyze the immunomodulating activities of EV preparations, especially those from mesenchymal stromal cells (MSCs) in vitro, a multi-donor mixed lymphocyte reaction (mdMLR) assay was established and stressed for its reproducibility. METHODS: To this end, human peripheral blood-derived mononuclear cells (PBMCs) of 12 different healthy donors were pooled warranting mutual allogeneic cross-reactivity, even following an optimized freezing and thawing procedure. After thawing, mixed PBMCs were cultured for 5 days in the absence or presence of EVs to be tested. Reflecting allogeneic reactions, in the absence of EVs, pooled PBMCs form characteristic satellite colonies whose appearance can be modulated by EVs. More quantifiable, the strength of the allogenic reaction is reflected by the content of activated CD4 and CD8 T cells being recognized by means of their CD25 and CD54 expression. RESULTS: Of note, connected to the use of primary cells, independent multi-donor PBMC pools differed in their capability to activate their cultured T cells. Thus, throughout the study, only pooled PBMC batches were used whose activated T-cell contents exceeded 25% of the total T-cell population at culture day 5 and whose contents were reproducibly reduced in the presence of immunomodulatory active MSC-EVs. T-cell activation-suppressing effects of the MSC-EV preparations tested were in all cases accompanied by the impact on monocytes. In the presence of immunomodulatory active MSC-EVs, more monocytes were harvested from mdMLR cultures than in their absence. Furthermore, in the absence of immunomodulatory EVs, most monocytes appeared as non-classical (CD14+CD16+) monocytes, whereas immunomodulatory active MSC-EVs promoted the appearance of classical (CD14++CD16-) and intermediate (CD14++CD16+) monocyte subpopulations. CONCLUSIONS: Overall, the obtained results qualify the mdMLR assay as a robust experimental tool for the evaluation of immunomodulatory potentials of given MSC-EV samples. However, further assay development is required to develop and qualify an authority-acceptable potency assay for clinically applicable MSC-EV products.

Independent human mesenchymal stromal cell-derived extracellular vesicle preparations differentially attenuate symptoms in an advanced murine graft-versus-host disease model.

BACKGROUND AIMS: Extracellular vesicles (EVs) harvested from conditioned media of human mesenchymal stromal cells (MSCs) suppress acute inflammation in various disease models and promote regeneration of damaged tissues. After successful treatment of a patient with acute steroid-refractory graft-versus-host disease (GVHD) using EVs prepared from conditioned media of human bone marrow-derived MSCs, this study focused on improving the MSC-EV production for clinical application. METHODS: Independent MSC-EV preparations all produced according to a standardized procedure revealed broad immunomodulatory differences. Only a proportion of the MSC-EV products applied effectively modulated immune responses in a multi-donor mixed lymphocyte reaction (mdMLR) assay. To explore the relevance of such differences in vivo, at first a mouse GVHD model was optimized. RESULTS: The functional testing of selected MSC-EV preparations demonstrated that MSC-EV preparations revealing immunomodulatory capabilities in the mdMLR assay also effectively suppress GVHD symptoms in this model. In contrast, MSC-EV preparations, lacking such in vitro activities, also failed to modulate GVHD symptoms in vivo. Searching for differences of the active and inactive MSC-EV preparations, no concrete proteins or miRNAs were identified that could serve as surrogate markers. CONCLUSIONS: Standardized MSC-EV production strategies may not be sufficient to warrant manufacturing of MSC-EV products with reproducible qualities. Consequently, given this functional heterogeneity, every individual MSC-EV preparation considered for the clinical application should be evaluated for its therapeutic potency before administration to patients. Here, upon comparing immunomodulating capabilities of independent MSC-EV preparations in vivo and in vitro, we found that the mdMLR assay was qualified for such analyses.

Monitoring the SARS-CoV-2 Pandemic: Prevalence of Antibodies in a Large, Repetitive Cross-Sectional Study of Blood Donors in Germany-Results from the SeBluCo Study 2020-2022.

SARS-CoV-2 serosurveillance is important to adapt infection control measures and estimate the degree of underreporting. Blood donor samples can be used as a proxy for the healthy adult population. In a repeated cross-sectional study from April 2020 to April 2021, September 2021, and April/May 2022, 13 blood establishments collected 134,510 anonymised specimens from blood donors in 28 study regions across Germany. These were tested for antibodies against the SARS-CoV-2 spike protein and nucleocapsid, including neutralising capacity. Seroprevalence was adjusted for test performance and sampling and weighted for demographic differences between the sample and the general population. Seroprevalence estimates were compared to notified COVID-19 cases. The overall adjusted SARS-CoV-2 seroprevalence remained below 2% until December 2020 and increased to 18.1% in April 2021, 89.4% in September 2021, and to 100% in April/May 2022. Neutralising capacity was found in 74% of all positive specimens until April 2021 and in 98% in April/May 2022. Our serosurveillance allowed for repeated estimations of underreporting from the early stage of the pandemic onwards. Underreporting ranged between factors 5.1 and 1.1 in the first two waves of the pandemic and remained well below 2 afterwards, indicating an adequate test strategy and notification system in Germany.

Peripheral HLA-G/ILT-2 immune checkpoint axis in acute and convalescent COVID-19 patients.

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) can elicits pro-viral activities by down-modulating immune responses. We analysed soluble forms of HLA-G, IL-6 and IL-10 as well as on immune effector cell expression of HLA-G and its cognate ILT-2 receptor in peripheral blood obtained from hospitalised and convalescent COVID-19 patients. Compared with convalescents (N = 202), circulating soluble HLA-G levels (total and vesicular-bound molecules) were significantly increased in hospitalised patients (N = 93) irrespective of the disease severity. During COVID-19, IL-6 and IL-10 levels were also elevated. Regarding the immune checkpoint expression of HLA-G/ILT-2 on peripheral immune effector cells, the frequencies of membrane-bound HLA-G on CD3+ and CD14+ cells were almost identical in patients during and post COVID-19, while the frequency of ILT-2 receptor on CD3+ and CD14+ cells was increased during acute infection. A multi-parametric correlation analysis of soluble HLA-G forms with IL-6, IL-10, activation markers CD25 and CD154, HLA-G, and ILT-2 expression on immune cells revealed a strong positive correlation of soluble HLA-G forms with membrane-bound HLA-G molecules on CD3+/CD14+ cells only in convalescents. During COVID-19, only vesicular-bound HLA-G were positively correlated with the activation marker CD25 on T cells. Thus, our data suggest that the elevated levels of soluble HLA-G in COVID-19 are due to increased expression in organ tissues other than circulating immune effector cells. The concomitant increased expression of soluble HLA-G and ILT-2 receptor frequencies supports the concept that the immune checkpoint HLA-G/ILT-2 plays a role in the immune-pathogenesis of COVID-19.

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.

Programmed death receptor ligand-2 (PD-L2) bearing extracellular vesicles as a new biomarker to identify early triple-negative breast cancer patients at high risk for relapse.

PURPOSE: Based on the tumor-promoting features of extracellular vesicles (EV) and PD-L1/2-bearing EV subpopulations (PD-L1/2EV), we evaluated their potential as surrogate markers for disease progression or eligibility criteria for PD-1 immune checkpoint inhibition (ICI) approaches in early triple-negative breast cancer (TNBC). METHODS: After enrichment of EV from plasma samples of 56 patients before and 50 after chemotherapy (CT), we determined levels of EV particle number and PD-L1/2EV by nanoparticle tracking analysis or ELISA and associated the results with clinical status/outcome and the presence of distinct circulating tumor cells (CTC) subpopulations. RESULTS: Compared to healthy controls, patients had a tenfold higher EV concentration and significantly elevated PD L2EV but not PD L1EV levels. The most important clinical implications were found for PD-L2EV. High PD-L2EV levels were associated with a significantly reduced 3-year progression-free and overall survival (PFS and OS). A loss of PD-L2EV after CT was significantly more prominent in patients achieving pathological complete response (pCR). Increased pre-CT PD-L2EV levels were found in patients having NOTCH1-positive or ERBB3-positive CTC. The presence of ERBB3-positive CTC combined with high pre-CT PD-L2EV resulted in a shorter PFS. CONCLUSION: This study highlights PD L2EV as a promising biomarker for risk assessment of TNBC patients and represents the basic for additional studies introducing PD-L2EV as an eligibility criterion for PD-1 ICI approaches.

Circulating cell-free messenger RNA enables non-invasive pan-tumour monitoring of melanoma therapy independent of the mutational genotype.

BACKGROUND: Plasma-derived tumour-specific cell-free nucleic acids are increasingly utilized as a minimally invasive, real-time biomarker approach in many solid tumours. Circulating tumour DNA of melanoma-specific mutations is currently the best studied liquid biopsy biomarker for melanoma. However, the combination of hotspot genetic alterations covers only around 80% of all melanoma patients. Therefore, alternative approaches are needed to enable the follow-up of all genotypes, including wild-type. METHODS: We identified KPNA2, DTL, BACE2 and DTYMK messenger RNA (mRNA) upregulated in melanoma versus nevi tissues by unsupervised data mining (N = 175 melanoma, N = 20 normal skin, N = 6 benign nevi) and experimentally confirmed differential mRNA expression in vitro (N = 18 melanoma, N = 8 benign nevi). Circulating cell-free RNA (cfRNA) was analysed in 361 plasma samples (collected before and during therapy) from 100 melanoma patients and 18 healthy donors. Absolute cfRNA copies were quantified on droplet digital PCR. RESULTS: KPNA2, DTL, BACE2 and DTYMK cfRNA demonstrated high diagnostic accuracy between melanoma patients' and healthy donors' plasma (AUC > 86%, p < .0001). cfRNA copies increased proportionally with increasing tumour burden independently of demographic variables and even remained elevated in individuals with radiological absence of disease. Re-analysis of single-cell transcriptomes revealed a pan-tumour origin of cfRNA, including endothelial, cancer-associated fibroblasts, macrophages and B cells beyond melanoma cells as cellular sources. Low baseline cfRNA levels were associated with significantly longer progression-free survival (PFS) (KPNA2 HR = .54, p = .0362; DTL HR = .60, p = .0349) and overall survival (KPNA2 HR = .52, p = .0237; BACE2 HR = .55, p = .0419; DTYMK HR = .43, p = .0393). Lastly, we found that cfRNA copies significantly increased during therapy in non-responders compared to responders regardless of therapy and mutational subtypes and that the increase of KPNA2 (HR = 1.73, p = .0441) and DTYMK (HR = 1.82, p = .018) cfRNA during therapy was predictive of shorter PFS. CONCLUSIONS: In sum, we identified a new panel of cfRNAs for a pan-tumour liquid biopsy approach and demonstrated its utility as a prognostic, therapy-monitoring tool independent of the melanoma mutational genotype.

Immune escape pathways from the HBV core18-27 CD8 T cell response are driven by individual HLA class I alleles.

Background and aims: There is growing interest in T cell-based immune therapies for a functional cure of chronic HBV infection including check-point inhibition, T cell-targeted vaccines or TCR-grafted effector cells. All these approaches depend on recognition of HLA class I-presented viral peptides. The HBV core region 18-27 is an immunodominant target of CD8+ T cells and represents the prime target for T cell-based therapies. Here, a high-resolution analysis of the core18-27 specific CD8+ T cell and the selected escape pathways was performed. Methods: HLA class I typing and viral sequence analyses were performed for 464 patients with chronic HBV infection. HBV-specific CD8+ T-cell responses against the prototype and epitope variants were characterized by flow cytometry. Results: Consistent with promiscuous presentation of the core18-27 epitope, antigen-specific T cells were detected in patients carrying HLA-A*02:01, HLA-B*35:01, HLA-B*35:03 or HLA-B*51:01. Sequence analysis confirmed reproducible selection pressure on the core18-27 epitope in the context of these alleles. Interestingly, the selected immune escape pathways depend on the presenting HLA-class I-molecule. Although cross-reactive T cells were observed, some epitope variants achieved functional escape by impaired TCR-interaction or disturbed antigen processing. Of note, selection of epitope variants was exclusively observed in HBeAg negative HBV infection and here, detection of variants associated with significantly greater magnitude of the CD8 T cell response compared to absence of variants. Conclusion: The core18-27 epitope is highly variable and under heavy selection pressure in the context of different HLA class I-molecules. Some epitope variants showed evidence for impaired antigen processing and reduced presentation. Viruses carrying such escape substitutions will be less susceptible to CD8+ T cell responses and should be considered for T cell-based therapy strategies.