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The emergence of SARS-CoV-2 in 2019 led to a global pandemic with a significant impact on healthcare systems. Healthcare workers were particularly vulnerable due to frequent contact with COVID-19 patients. Despite vaccination, they remained at higher risk as the vaccines provided limited protection against infection with viral variants, like Delta or Omicron BA.1 and BA.5. Three years after the onset of the pandemic, we evaluated SARS-CoV-2 infection frequencies among healthcare workers with varying levels of patient contact: high-risk (frequent COVID-19 patient contact), intermediate-risk (non-COVID-19 patient contact), and low-risk (no patient contact). We assessed their cellular and humoral immune responses based on their vaccination status and number of prior infections. SARS-CoV-2-specific antibodies were measured by immunoglobulin ELISA, and neutralizing antibody titers were determined against the viral variants D614G, Delta, and Omicron BA.1 and BA.5. Cellular immune responses were analyzed using an interferon-γ ELISpot. Notably, three years into the pandemic, healthcare workers in daily contact with COVID-19 patients did not have higher infection rates compared to healthcare workers with non-COVID-19 patient contact or no patient contact. Immune responses were similar across all groups, highlighting the effectiveness of vaccination and current hygiene standards in preventing virus transmission from patients to staff.

Abstract Automated medical image analysis systems often require large amounts of training data with high quality labels, which are difficult and time consuming to generate. This paper introduces Radiology Object in COntext version 2 (ROCOv2), a multimodal dataset consisting of radiological images and associated medical concepts and captions extracted from the PMC Open Access subset. It is an updated version of the ROCO dataset published in 2018, and adds 35,705 new images added to PMC since 2018. It further provides manually curated concepts for imaging modalities with additional anatomical and directional concepts for X-rays. The dataset consists of 79,789 images and has been used, with minor modifications, in the concept detection and caption prediction tasks of ImageCLEFmedical Caption 2023. The dataset is suitable for training image annotation models based on image-caption pairs, or for multi-label image classification using Unified Medical Language System (UMLS) concepts provided with each image. In addition, it can serve for pre-training of medical domain models, and evaluation of deep learning models for multi-task learning.

While SARS-CoV-2 has transitioned to an endemic phase, infections caused by newly emerged variants continue to result in severe, and sometimes fatal, outcomes or lead to long-term COVID-19 symptoms. Vulnerable populations, such as PLWH, face an elevated risk of severe illness. Emerging variants of SARS-CoV-2, including numerous Omicron subvariants, are increasingly associated with breakthrough infections. Adapting mRNA vaccines to these new variants may offer improved protection against Omicron for vulnerable individuals. In this study, we examined humoral and cellular immune responses before and after administering adapted booster vaccinations to PLWH, alongside a control group of healthy individuals. Four weeks following booster vaccination, both groups exhibited a significant increase in neutralizing antibodies and cellular immune responses. Notably, there was no significant difference in humoral immune response between PLWH and the healthy controls. Immune responses declined rapidly in both groups three months post vaccination. However, PLWH still showed significantly increased neutralizing antibody titers even after three months. These findings demonstrate the efficacy of the adapted vaccination regimen. The results suggest that regular booster immunizations may be necessary to sustain protective immunity.

Abstract 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.

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.

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.

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.

Abstract Background CT scans are used in routine clinical practice for the diagnosis and treatment surveillance of non‐small cell lung cancer (NSCLC). However, more sensitive methods are desirable. Liquid biopsy analyses of RNA and DNA can offer more sensitive diagnostic approaches. Cell‐free RNA (cfRNA) has been described in several malignancies, but its clinical utility has not previously been explored. Methods We evaluated the clinical utility of cfRNA for early detection and surveillance of tumor disease in a proof‐of‐concept study. Using real‐time‐droplet digital polymerase chain reaction we characterized a candidate transcript (MORF4L2) in plasma samples from 41 advanced stage, 38 early stage NSCLC and 39 healthy samples. We compared its diagnostic performance with tumor markers and evaluated its utility for disease monitoring. Results MORF4L2 cfRNA was more abundant in patients than in healthy donors (p

IntroductionTriple 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 methodsTo 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.ResultssHLA-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 responseConclusionThe 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.

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.

Abstract Background Patients with multiple sclerosis receiving ocrelizumab-treatment are in desperate need of a protection against SARS-CoV-2 infection. Methods In this study, Euroimmun semi-quantitative Anti-SARS-CoV-2 IgG for detection of humoral response and ELISpot assays for detection of SARS-CoV-2-specific T-cell-response were used in 10 ocrelizumab-treated patients with multiple sclerosis twice vaccinated with Comirnaty® mRNA vaccine. This data was compared with a control group of 20 age- and sex-matched healthy volunteers, who had all previously received a full SARS-CoV-2 mRNA vaccination with Comirnaty® or Spikevax®. Results While all subjects in the control group had high humoral response to the vaccination, in B-cell-depleted individuals a significantly reduced antibody response to vaccination against SARS-CoV-2 was observed. SARS-CoV-2 specific T-cell-response, however, did not differ significantly between both cohorts. Conclusions T-cell-mediated response to Comirnaty® vaccination is observable despite attenuated humoral response in B-cell-depleted patients. This might enable partial protection against COVID-19. Trial registration Retrospectively registered.

Abstract 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

Background and aimsThere 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.MethodsHLA 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.ResultsConsistent 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.ConclusionThe 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.

Immunocompromised patients are at increased risk for a severe course of COVID-19. Treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection with anti-SARS-CoV-2 monoclonal antibodies (mAbs) has become widely accepted. However, the effects of mAb treatment on the long-term primary cellular response to SARS-CoV-2 are unknown. In the following study, we investigated the long-term cellular immune responses to SARS-CoV-2 Spike S1, Membrane (M) and Nucleocapsid (N) antigens using the ELISpot assay in unvaccinated, mAb-treated immunocompromised high-risk patients. Anti-SARS-CoV-2 mAb untreated though vaccinated COVID-19 immunocompromised patients, vaccinated SARS-CoV-2 immunocompromised patients without COVID-19 and vaccinated healthy control subjects served as control groups. The cellular immune response was determined at a median of 5 months after SARS-CoV-2 infection. Our data suggest that immunocompromised patients develop an endogenous long-term cellular immune response after COVID-19, although at low levels. A better understanding of the cellular immune response will help guide clinical decision making for these vulnerable patient cohorts.

Summary: Background: Immune activation is a significant contributor to HIV pathogenesis and disease progression. In virally-suppressed individuals on ART, low-level immune activation has been linked to several non-infectious comorbid diseases. However, studies have not been systematically performed in sub-Saharan Africa and thus the impact of demographics, ART and regional endemic co-infections on immune activation is not known. We therefore comprehensively evaluated in a large multinational African cohort markers for immune activation and its distribution in various settings. Methods: 2747 specimens from 2240 people living with HIV (PLWH) and 477 without HIV from the observational African Cohort Study (AFRICOS) were analyzed for 13 immune parameters. Samples were collected along with medical history, sociodemographic and comorbidity data at 12 HIV clinics across 5 programs in Uganda, Kenya, Tanzania and Nigeria. Data were analyzed with univariate and multivariate methods such as random forests and principal component analysis. Findings: Immune activation was markedly different between PLWH with detectable viral loads, and individuals without HIV across sites. Among viremic PLWH, we found that all immune parameters were significantly correlated with viral load except for IFN-α. The overall inflammatory profile was distinct between men and women living with HIV, in individuals off ART and with HIV viremia. We observed stronger differences in the immune activation profile with increasing viremia. Using machine learning methods, we found that geographic differences contributed to unique inflammatory profiles. We also found that among PLWH, age and the presence of infectious and/or noninfectious comorbidities showed distinct inflammatory patterns, and biomarkers may be used to predict the presence of some comorbidities. Interpretation: Our findings show that chronic immune activation in HIV-1 infection is influenced by HIV viral load, sex, age, region and ART use. These predictors, as well as associations among some biomarkers and coinfections, influence biomarkers associated with noncommunicable diseases. Funding: This work was supported by the President's Emergency Plan for AIDS Relief via a cooperative agreement between the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., and the U.S. Department of Defense [W81XWH-11-2-0174, W81XWH-18-2-0040]. The investigators have adhered to the policies for protection of human subjects as prescribed in AR 70–25. This article was prepared while Michael A. Eller was employed at Henry M. Jackson Foundation for the Advancement of Military Medicine for the U.S. Military HIV Research Program. The views expressed are those of the authors and should not be construed to represent the positions of the US Army or the Department of Defense. The opinions expressed in this article are the author's own, and do not reflect the view of the National Institutes of Health, the U.S. Department of Health and Human Services, or the U.S. government.

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.

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.

We investigated immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among a group of convalescent, potential blood donors in Germany who had PCR-confirmed SARS-CoV-2 infection. Sixty days after onset of symptoms, 13/78 (17%) study participants had borderline or negative results to an ELISA detecting IgG against the S1 protein of SARS-CoV-2. We analyzed participants with PCR-confirmed infection who had strong antibody responses (ratio >3) as positive controls and participants without symptoms of SARS-CoV-2 infection and without household contact with infected patients as negative controls. Using interferon-γ ELISpot, we observed that 78% of PCR-positive volunteers with undetectable antibodies showed T cell immunity against SARS-CoV-2. We observed a similar frequency (80%) of T-cell immunity in convalescent donors with strong antibody responses but did not detect immunity in negative controls. We concluded that, in convalescent patients with undetectable SARS-CoV-2 IgG, immunity may be mediated through T cells.

Calcium phosphate nanoparticles (CaP-NPs) are biodegradable carriers that can be functionalized with biologically active molecules. As such, they are potential candidates for delivery of therapeutic molecules in cancer therapies. In this context, it is important to explore whether CaP-NPs impair the natural or therapy-induced immune cell activity against cancer cells. Therefore, in this study, we have investigated the effects of different CaP-NPs on the anti-tumor activity of natural killer (NK) cells using different ovarian cancer (OC) cell line models. We explored these interactions in coculture systems consisting of NK cells, OC cells, CaP-NPs, and therapeutic Cetuximab antibodies (anti-EGFR, ADCC-inducing antibody). Our experiments revealed that aggregated CaP-NPs can serve as artificial targets, which activate NK cell degranulation and impair ADCC directed against tumor targets. However, when CaP-NPs were properly dissolved by sonication, they did not cause substantial activation. CaP-NPs with SiO2-SH-shell induced some activation of NK cells that was not observed with polyethyleneimine-coated CaP-NPs. Addition of CaP-NPs to NK killing assays did not impair conjugation of NK with OC and subsequent tumor cytolytic NK degranulation. Therapeutic antibody coupled to functionalized CaP-NPs maintained substantial levels of antibody-dependent cellular cytotoxic activity. Our study provides a cell biological basis for the application of functionalized CaP-NPs in immunologic anti-cancer therapies.

Background: Radiology is the current standard for monitoring treatment responses in lung cancer. Limited sensitivity, exposure to ionizing radiations and related sequelae constitute some of its major limitation. Non-invasive and highly sensitive methods for early detection of treatment failures and resistance-associated disease progression would have additional clinical utility. Methods: We analyzed serially collected plasma and paired tumor samples from lung cancer patients (61 with stage IV, 48 with stages I-III disease) and 61 healthy samples by means of next-generation sequencing, radiological imaging and droplet digital polymerase chain reaction (ddPCR) mutation and methylation assays. Results: A 62% variant concordance between tumor-reported and circulating-free DNA (cfDNA) sequencing was observed between baseline liquid and tissue biopsies in stage IV patients. Interestingly, ctDNA sequencing allowed for the identification of resistance-mediating p.T790M mutations in baseline plasma samples for which no such mutation was observed in the corresponding tissue. Serial circulating tumor DNA (ctDNA) mutation analysis by means of ddPCR revealed a general decrease in ctDNA loads between baseline and first reassessment. Additionally, serial ctDNA analyses only recapitulated computed tomography (CT) -monitored tumor dynamics of some, but not all lesions within the same patient. To complement ctDNA variant analysis we devised a ctDNA methylation assay (methcfDNA) based on methylation-sensitive restriction enzymes. cfDNA methylation showed and area under the curve (AUC) of > 0.90 in early and late stage cases. A decrease in methcfDNA between baseline and first reassessment was reflected by a decrease in CT-derive tumor surface area, irrespective of tumor mutational status. Conclusion: Taken together, our data support the use of cfDNA sequencing for unbiased characterization of the molecular tumor architecture, highlights the impact of tumor architectural heterogeneity on ctDNA-based tumor surveillance and the added value of complementary approaches such as cfDNA methylation for early detection and monitoring

The generation and expansion of functionally competent NK cells in vitro is of great interest for their application in immunotherapy of cancer. Since CD33 constitutes a promising target for immunotherapy of myeloid malignancies, NK cells expressing a CD33-specific chimeric antigen receptor (CAR) were generated. Unexpectedly, we noted that CD33-CAR NK cells could not be efficiently expanded in vitro due to a fratricide-like process in which CD33-CAR NK cells killed other CD33-CAR NK cells that had upregulated CD33 in culture. This upregulation was dependent on the stimulation protocol and encompassed up to 50% of NK cells including CD56dim NK cells that do generally not express CD33 in vivo. RNAseq analysis revealed that upregulation of CD33+ NK cells was accompanied by a unique transcriptional signature combining features of canonical CD56bright (CD117high, CD16low) and CD56dim NK cells (high expression of granzyme B and perforin). CD33+ NK cells exhibited significantly higher mobilization of cytotoxic granula and comparable levels of cytotoxicity against different leukemic target cells compared to the CD33− subset. Moreover, CD33+ NK cells showed superior production of IFNγ and TNFα, whereas CD33− NK cells exerted increased antibody-dependent cellular cytotoxicity (ADCC). In summary, the study delineates a novel functional divergence between NK cell subsets upon in vitro stimulation that is marked by CD33 expression. By choosing suitable stimulation protocols, it is possible to preferentially generate CD33+ NK cells combining efficient target cell killing and cytokine production, or alternatively CD33− NK cells, which produce less cytokines but are more efficient in antibody-dependent applications.

Despite major improvements in diagnostics and therapy in early as well as in locally advanced breast cancer (LABC), metastatic relapse occurs in about 20% of patients, often explained by early micro-metastatic spread into bone marrow by disseminated tumor cells (DTC). Although neoadjuvant chemotherapy (NACT) has been a successful tool to improve overall survival (OS), there is growing evidence that various environmental factors like the non-classical human leukocyte antigen-G (HLA-G) promotes cancer invasiveness and metastatic progression. HLA-G expression is associated with regulatory elements targeting certain single-nucleotide polymorphisms (SNP) in the HLA-G 3’ untranslated region (UTR), which arrange as haplotypes. Here, we systematically evaluated the impact of HLA-G 3’UTR polymorphisms on disease status, on the presence of DTC, on soluble HLA-G levels, and on therapy and disease outcome in non-metastatic LABC patients. Although haplotype frequencies were similar in patients (n = 142) and controls (n = 204), univariate analysis revealed that the UTR-7 haplotype was related to patients with low tumor burden, whereas UTR-4 was associated with tumor sizes >T1. Furthermore, UTR-4 was associated with the presence of DTC, but UTR-3 and UTR-7 were related to absence of DTC. Additionally, increased levels of soluble HLA-G molecules were found in patients carrying UTR-7. Regarding therapy and disease outcome, univariate and multivariate analysis highlighted UTR-1 or UTR-2 as a prognostic parameter indicative for a beneficial course of disease in terms of complete response towards NACT or progression-free survival (PFS). At variance, UTR-4 was an independent risk factor for a reduced OS besides already known parameters. Taken into account the most common HLA-G 3’UTR haplotypes (UTR-1–UTR-7, UTR-18), deduction of the UTR-1/2/4 haplotypes to specific SNPs revealed that the +3003C variant, unique for UTR-4, seemed to favor a detrimental disease outcome, while the +3187G and +3196G variants, unique for UTR-1 or UTR-2, were prognostic parameters for a beneficial course of disease. In conclusion, these data suggest that the HLA-G 3’UTR variants +3003C, +3187G, and +3196G are promising candidates for the prediction of therapy and disease outcome in LABC patients.

The human leukocyte antigen G (HLA-G) is an immune checkpoint molecule with a complex network of interactions with several inhibitory receptors. Although the effect of HLA-G on T cells and NK cells is well studied, the effect of HLA-G on B cells is still largely elusive. B cells are of particular interest in the context of the HLA-G-ILT-2 interaction because the ILT-2 receptor is constitutively expressed on most B cells, whereas it is only present on some subsets of T and NK cells. To characterize the effect of HLA-G5 molecules on B cells, we studied splenic B cells derived from cytomegalovirus (CMV) sero-positive donors after CMV stimulation with antigens in the presence and absence of soluble HLA-G5. In the presence of HLA-G5, increased expression of the ITIM-bearing Ig-like transcript (ILT-2) was observed on B cells, but its expression was not affected by stimulation with CMV antigens. Moreover, it became evident that HLA-G5 exposure resulted in a decreased expression of CD27 and CD38 and, accordingly, in lower proportions of CD19+CD27+CD38+ and higher proportions of CD19+CD27-CD38- B cells. Taken together, our in vitro findings demonstrate that soluble HLA-G5 suppresses markers of B cell activation, suggesting that HLA-G5 has an impact on splenic B cell differentiation and activation. Based on these results, further investigation regarding the role of HLA-G as a prognostic factor and a potential therapeutic agent with respect to B cell function appears reasonable.

Abstract Background The importance of donor-specific antibodies (DSA) after liver transplantation (LT) for graft and patient survival is an ongoing controversy. So far it has not been elucidated when and in how far DSA are harmful for graft and patient survival. Therefore, we had the aim to investigate the association of DSA with complications after LT. Methods Data of 430 LT recipients were collected and statistically analyzed. Detection of HLA antibodies (Ab) was performed by Luminex assay. Results DSA were detected in 81 patients (18.8%). These were mainly HLA class II Ab (81.5%). HLA class II Ab show a higher MFI (median: 5.300) compared to HLA class I Ab (median: 2.300). There is no association between MFI levels and development of complications after LT. However, cirrhosis occurred significantly more often in DSA positive patients (18%) than in patients without detectable DSA (9%, P = 0.027). All DSA positive patients with cirrhosis of the graft showed HLA class II antibodies (OR: 3.028; 95% CI: 1.51–6.075; P = 0.002). Conclusion Occurrence of HLA class II DSA after LT is associated with graft cirrhosis and may indicate a higher risk to develop graft damage independent on MFI and requires an individualized risk management.

Vulnerable patients such as immunosuppressed or elderly patients are at high risk for a severe course of COVID-19 upon SARS-CoV-2 infection. Immunotherapy with SARS-CoV-2 specific monoclonal antibodies (mAb) or convalescent plasma represents a considerable treatment option to protect these patients from a severe or lethal course of infection. However, monoclonal antibodies are not always available or less effective against emerging SARS-CoV-2 variants. Convalescent plasma is more commonly available and may represent a good treatment alternative in low-income countries. We retrospectively evaluated outcomes in individuals treated with mAbs or convalescent plasma and compared the 30-day overall survival with a patient cohort that received supportive care due to a lack of SARS-CoV-2 specific therapies between March 2020 and April 2021. Our data demonstrate that mAb treatment is highly effective in preventing severe courses of SARS-CoV-2 infection. All patients treated with mAb survived. Treatment with convalescent plasma improved overall survival to 82% compared with 61% in patients without SARS-CoV-2 targeted therapy. Our data indicate that early convalescent plasma treatment may be an option to improve the overall survival of high-risk COVID-19 patients. This is especially true when other antiviral drugs are not available or their efficacy is significantly reduced, which may be the case with emerging SARS-CoV-2 variants.

Programmed cell death protein-1 (PD-1) is an inhibitory co-receptor required for regulating immune responsiveness and maintaining immune homeostasis. As PD-1 can be released as bioactive soluble molecule, we investigated the clinical significance of soluble PD-1 (sPD-1) after allogeneic hematopoietic stem cell transplantation (HSCT) regarding graft-versus-host disease (GvHD), relapse, and overall survival (OS) in a mono-centric cohort of 82 patients. Compared to pre-HSCT and to healthy controls, post-HSCT sPD-1 plasma levels were significantly increased during an observation time of three months. Univariate analysis revealed that low sPD-1 plasma levels at month one, two or three post HSCT were associated with acute GvHD grade III-IV, the onset of moderate/severe chronic GvHD (cGvHD) and inferior OS, DFS, and TRM, respectively. No relationship was detected to relapse rates. sPD-1 plasma levels were significantly increased in ATG-treated patients compared to ATG-untreated patients. Multivariate analysis revealed that a low sPD-1 plasma levels status at one or two month(s) after HSCT is an independent indicator for inferior OS, DFS, or TRM. A low sPD-1 plasma levels status at month three post HSCT is predictive for the onset of moderate/severe cGvHD. Thus, our study pinpoints the soluble inhibitory co-receptor PD-1 as a promising candidate molecule for the prediction of clinical HSCT outcome.

Mycobacterium abscessus complex (MABC) infection has a devastating impact on the course of cystic fibrosis (CF) and non-CF lung disease. Diagnosis of MABC pulmonary disease is challenging, and current diagnostic approaches lack accuracy, especially in CF. In this study, we aimed to establish an MABC-specific interferon-γ release assay to detect host immune responses to MABC and improve diagnostics of MABC infection by the detection of antigen-specific T cells. Four species-specific proteins of MABC were overexpressed in an Escherichia coli expression system. Purified proteins were used to stimulate peripheral blood mononuclear cells of study subjects in an ELISpot assay. Interferon-γ response of 12 subjects with established diagnosis of MABC infection (10 CF and two non-CF) was compared with 35 controls (22 CF and 13 non-CF) distributed to three control groups, 17 CF subjects without NTM infection, nine subjects with NTM infection other than MABC, and nine subjects with tuberculosis. Cellular in vitro responses in the MABC group were stronger than in the control groups, especially toward the protein MAB_0405c (39 vs. 4 spots per 300,000 PBMC, p = 0.004; data represent mean values) in all patients and also in the subgroup of CF subjects (39 spots vs. 1 spot, p = 0.003). Receiver operating characteristic curve analysis indicated that spot numbers of at least 20 were highly predictive of MABC infection (all patients: area under curve 0.773, sensitivity 58%, and specificity 94%; CF patients: area under curve 0.818, sensitivity 60%, and specificity 100%). In conclusion, we identified MAB_0405c as a protein that may stimulate MABC-specific interferon-γ secretion and may add to the diagnosis of MABC infection in affected patients.

In kidney transplant (KTX) patients, immune responses after booster vaccination against SARS-CoV-2 are inadequately examined. We analyzed these patients a median of four months after a third/fourth vaccination and compared them to healthy controls. Cellular responses were analyzed by interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpot assays. Neutralizing antibody titers were assessed against SARS-CoV-2 D614G (wild type) and the variants alpha, delta, and omicron by a cell culture-based neutralization assay. Humoral immunity was also determined by a competitive fluorescence assay, using 11 different variants of SARS-CoV-2. Antibody ratios were measured by ELISA. KTX patients showed significantly lower SARS-CoV-2-specific IFN-γ responses after booster vaccination than healthy controls. However, SARS-CoV-2-specific IL-2 responses were comparable to the T cell responses of healthy controls. Cell culture-based neutralizing antibody titers were 1.3-fold higher in healthy controls for D614G, alpha, and delta, and 7.8-fold higher for omicron (p < 0.01). Healthy controls had approximately 2-fold higher concentrations of potential neutralizing antibodies against all 11 variants than KTX patients. However, more than 60% of the KTX patients displayed antibodies to variants of SARS-CoV-2. Thus, KTX patients should be partly protected, due to neutralizing antibodies to variants of SARS-CoV-2 or by cross-reactive T cells, especially those producing IL-2.

Protecting vulnerable groups from severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is mandatory. Immune responses after a third vaccination against SARS-CoV-2 are insufficiently studied in patients after hematopoietic stem-cell transplantation (HSCT). We analyzed immune responses before and after a third vaccination in HSCT patients and healthy controls. Cellular immunity was assessed using interferon-gamma (IFN-γ) and interleukin-2 (IL-2) ELISpots. Furthermore, this is the first report on neutralizing antibodies against 11 variants of SARS-CoV-2, analyzed by competitive fluorescence assay. Humoral immunity was also measured by neutralization tests assessing cytopathic effects and by ELISA. Neither HSCT patients nor healthy controls displayed significantly higher SARS-CoV-2-specific IFN-γ or IL-2 responses after the third vaccination. However, after the third vaccination, cellular responses were 2.6-fold higher for IFN-γ and 3.2-fold higher for IL-2 in healthy subjects compared with HSCT patients. After the third vaccination, neutralizing antibodies were significantly higher (p < 0.01) in healthy controls, but not in HSCT patients. Healthy controls vs. HSCT patients had 1.5-fold higher concentrations of neutralizing antibodies against variants and 1.2-fold higher antibody concentrations against wildtype. However, half of the HSCT patients exhibited neutralizing antibodies to variants of SARS-CoV-2, which increased only slightly after a third vaccination.

Hematopoietic stem cell transplant (HSCT) recipients have a high risk of developing primary varicella-zoster virus (VZV) infection and reactivation. VZV vaccination may prevent infection and reactivation. In the current study, recipients of allogeneic HSCT (34 females, 45 males) were vaccinated with adjuvanted, recombinant zoster vaccine Shingrix™, which contains the VZV glycoprotein E. Cellular immunity against various VZV antigens was analyzed by interferon-gamma ELISpot. Peripheral blood mononuclear cells (PBMC) of recipients with versus without prior shingles (n = 36 and n = 43, respectively) showed approximately twofold higher VZV-specific responses prior to and post vaccination. After the first and second vaccination, ELISpot responses towards the glycoprotein E were significantly higher in males versus females (median of spots increment 18 versus 1 and 17 versus 4, respectively, p ≤ 0.02 each). Multivariate analysis showed that shingles and sex both impacts significantly on VZV immunity. Whereas vaccination-induced changes could hardly be detected after stimulation with a whole VZV antigen, there was a significant increase in responses towards glycoprotein E after vaccination (p < 0.005). These data indicate that vaccination with Shingrix™ augmented cellular, VZV-specific immunity in HSCT recipients. Shingles and male sex could both be identified as factors leading to increased immunity.

Solid organ transplant recipients have an up to ninefold higher risk of varicella–zoster virus (VZV) reactivation than the general population. Due to lifelong immunosuppressive therapy, vaccination against VZV may be less effective in kidney transplant (KTX) recipients. In the current study, twelve female and 17 male KTX recipients were vaccinated twice with the adjuvanted, recombinant zoster vaccine Shingrix™, which contains the VZV glycoprotein E (gE). Cellular immunity against various VZV antigens was analyzed with interferon-gamma ELISpot. We observed the strongest vaccination-induced changes after stimulation with a gE peptide pool. One month after the second vaccination, median responses were 8.0-fold higher than the responses prior to vaccination (p = 0.0006) and 4.8-fold higher than responses after the first vaccination (p = 0.0007). After the second vaccination, we observed an at least twofold increase in ELISpot responses towards gE peptides in 22 out of 29 patients (76%). Male sex, good kidney function, early time point after transplantation, and treatment with tacrolimus or mycophenolate were correlated significantly with higher VZV-specific cellular immunity, whereas diabetes mellitus was correlated with impaired responses. Thus, our data indicate that vaccination with Shingrix™ significantly augmented cellular, VZV gE-specific immunity in KTX recipients, which was dependent on several covariates.

Several polymorphisms within Fc receptors (FCR) have been described, some of which correlate with allograft function. In the current study, we determined three Fcγ receptor and five Fcα receptor dimorphisms in 47 kidney transplant recipients who had been vaccinated against Streptococcus pneumoniae. We analyzed if FCR genotypes correlated with pneumococcal antibodies and their serotype-specific opsonophagocytic function, tested prior to and at months 1 and 12 post-vaccination. In parallel, we assessed antibodies against HLA and MICA and determined kidney function. We observed that IgG2 antibodies against pneumococci at months 1 and 12 after vaccination and IgA antibodies at month 1 differed significantly between the carriers of the three genotypes of FCGR3A rs396991 (V158F, p = 0.02; 0.04 and 0.009, respectively). Moreover, the genotype of FCGR3A correlated with serotype-specific opsonophagocytic function, reaching statistical significance (p < 0.05) at month 1 for 9/13 serotypes and at month 12 for 6/13 serotypes. Heterozygotes for FCGR3A had the lowest antibody response after pneumococcal vaccination. On the contrary, heterozygotes tended to have more antibodies against HLA class I and impaired kidney function. Taken together, our current data indicate that heterozygosity for FCGR3A may be unfavorable in kidney transplant recipients.

Cellular immunotherapy using chimeric antigen receptors (CARs) so far has almost exclusively used autologous peripheral blood-derived T cells as immune effector cells. However, harvesting sufficient numbers of T cells is often challenging in heavily pre-treated patients with malignancies and perturbed hematopoiesis and perturbed hematopoiesis. Also, such a CAR product will always be specific for the individual patient. In contrast, NK cell infusions can be performed in non-HLA-matched settings due to the absence of alloreactivity of these innate immune cells. Still, the infused NK cells are subject to recognition and rejection by the patient’s immune system, thereby limiting their life-span in vivo and undermining the possibility for multiple infusions. Here, we designed genome editing and advanced lentiviral transduction protocols to render primary human NK cells unsusceptible/resistant to an allogeneic response by the recipient’s CD8+ T cells. After knocking-out surface expression of HLA class I molecules by targeting the B2M gene via CRISPR/Cas9, we also co-expressed a single-chain HLA-E molecule, thereby preventing NK cell fratricide of B2M-knockout (KO) cells via “missing self”-induced lysis. Importantly, these genetically engineered NK cells were functionally indistinguishable from their unmodified counterparts with regard to their phenotype and their natural cytotoxicity towards different AML cell lines. In co-culture assays, B2M-KO NK cells neither induced immune responses of allogeneic T cells nor re-activated allogeneic T cells which had been expanded/primed using irradiated PBMNCs of the respective NK cell donor. Our study demonstrates the feasibility of genome editing in primary allogeneic NK cells to diminish their recognition and killing by mismatched T cells and is an important prerequisite for using non-HLA-matched primary human NK cells as readily available, “off-the-shelf” immune effectors for a variety of immunotherapy indications in human cancer.

Tumor immune escape is associated with both, the expression of immune checkpoint molecules on peripheral immune cells and soluble forms of the human leukocyte antigen-G (HLA-G) in the blood, which are consequently discussed as clinical biomarker for disease status and outcome of cancer patients. HLA-G preferentially interacts with the inhibitory receptor immunoglobulin-like transcript (ILT) receptor-2 in the blood and can be secreted as free soluble molecules (sHLA-G) or via extracellular vesicles (EV). To investigate the contribution of these two forms to the expression of checkpoint molecules in peripheral blood, we primed peripheral blood mononuclear cells with purified soluble sHLA-G1 protein, or EV preparations derived from SUM149 cells transfected with membrane-bound HLA-G1 or control vector prior to anti-CD3/CD28 T cell activation. Our study demonstrated that priming of PBMC with sHLA-G1 protein prior to 48 h activation resulted in enhanced frequencies of ILT-2 expressing CD8+ T cells, and in an upregulation of immune checkpoint molecules CTLA-4, PD-1, TIM-3, and CD95 exclusively on ILT-2 positive CD8+ T cells. In contrast, when PBMC were primed with EV (containing HLA-G1 or not) upregulation of CTLA-4, PD-1, TIM-3, and CD95 occurred exclusively on ILT-2 negative CD8+ T cells. Taken together, our data suggest that priming with sHLA-G forms induces a pronounced immunosuppressive/exhausted phenotype and that priming with sHLA-G1 protein or EV derived from HLA-G1 positive or negative SUM149 cells affects CD8+ T cells complementary by targeting either the ILT-2 positive or negative subpopulation, respectively, after T cell activation.

HLA-E is a member of the non-classical HLA molecules and by interaction with activating or inhibitory receptors of NK and T cells, HLA-E can lead to immune activation or suppression context-dependently. Recently, the non-classical HLA molecules gain more attention in the setting of allogeneic hematopoietic stem cell transplantation (HSCT). Most studies so far have focused on the two most frequent genotypes (HLA-E*01:01 and HLA-E*01:03) and investigated their potential association with clinical endpoints of HSCT, like graft-versus-host disease (GvHD), relapse, and overall survival (OS). However, these studies have produced inconsistent results regarding the role of HLA-E and the clinical endpoints after HSCT. We therefore here investigate the amount of soluble HLA-E (sHLA-E) in patients following HSCT and relate this to the clinical endpoints after HSCT. In univariate analysis, we observe a significant association of reduced levels of sHLA-E with severe acute GvHD, extended chronic GvHD and with inferior OS. Using receiver operating characteristic analyses specific thresholds obtained 1, 2, or 3 month(s) after HSCT were identified being indicative for severe acute GvHD, extended chronic GvHD, or inferior OS. In sub-group analyses, this effect can be confirmed in patients not treated with ATG, but is derogated in ATG-treated patients. Notably, we could not detect any association of the course of sHLA-E levels post-HSCT with the three most frequent HLA-E genotypes (HLA-E*01:03/*01:03, HLA-E*01:01/*01:01, HLA-E*01:01/*01:03). However, with regard to 5-year-OS there was an association of HLA-E*01:03 homozygosity with inferior OS. Taking ATG-treatment, recipient and donor HLA-E genotypes into consideration among other well-known risk factors, the sHLA-E status was found as an independent predictor for the development of extended cGvHD and inferior OS following HSCT irrespective of the sHLA-E thresholds. These findings shed some light on the possible impact of reduced sHLA-E levels after HSCT on GvHD and OS. Thus, sHLA-E appears to be a novel promising candidate for the prediction of clinical HSCT outcome with regards to extended cGvHD and OS.

Extracellular vesicles (EVs) mediate targeted cellular interactions in normal and pathophysiological conditions and are increasingly recognised as potential biomarkers, therapeutic agents and drug delivery vehicles. Based on their size and biogenesis, EVs are classified as exosomes, microvesicles and apoptotic bodies. Due to overlapping size ranges and the lack of specific markers, these classes cannot yet be distinguished experimentally. Currently, it is a major challenge in the field to define robust and sensitive technological platforms being suitable to resolve EV heterogeneity, especially for small EVs (sEVs) with diameters below 200 nm, i.e. smaller microvesicles and exosomes. Most conventional flow cytometers are not suitable for the detection of particles being smaller than 300 nm, and the poor availability of defined reference materials hampers the validation of sEV analysis protocols. Following initial reports that imaging flow cytometry (IFCM) can be used for the characterisation of larger EVs, we aimed to investigate its usability for the characterisation of sEVs. This study set out to identify optimal sample preparation and instrument settings that would demonstrate the utility of this technology for the detection of single sEVs. By using CD63eGFP-labelled sEVs as a biological reference material, we were able to define and optimise IFCM acquisition and analysis parameters on an Amnis ImageStreamX MkII instrument for the detection of single sEVs. In addition, using antibody-labelling approaches, we show that IFCM facilitates robust detection of different EV and sEV subpopulations in isolated EVs, as well as unprocessed EV-containing samples. Our results indicate that fluorescently labelled sEVs as biological reference material are highly useful for the optimisation of fluorescence-based methods for sEV analysis. Finally, we propose that IFCM will help to significantly increase our ability to assess EV heterogeneity in a rigorous and reproducible manner, and facilitate the identification of specific subsets of sEVs as useful biomarkers in various diseases.

Introduction: Response to platinum-based therapy is a major prognostic factor in epithelial ovarian cancer (EOC) and reliable prognostic biomarkers are urgently needed to identify patients at high risk. Since ligands of the Programmed Death Receptor-1 (PD-L1 and PD-L2) play a crucial role within the tumor microenvironment for tumorigenesis, we investigated levels of sPD-L1 and sPD-L2 in liquid biopsies of serum samples, and correlated the results with the clinical status, presence of circulating tumor cells (CTCs) and disease outcome in primary EOC patients.Methods: sPD-L1 and sPD-L2 were determined by ELISA in patients (N = 83) and healthy females (N = 29). Gene expression analysis of EpCAM, MUC-1, CA-125, and ERCC1 was performed by RT-PCR after CTCs enrichment.Results: sPD-L1 was significantly (p = 0.0001) increased and sPD-L2 decreased (p = 0.003) in EOC patients compared to controls. While enhanced sPD-L1 was associated with residual tumor burden (p = 0.022), reduced sPD-L2 levels were related to platinum-resistance (p < 0.01) and the presence of ERCC1+ CTCs (p < 0.0001). High sPD-L1 levels were associated with a reduced 5 year overall survival (OS, p = 0.003) and progression-free survival (PFS, p = 0.019). Strikingly, sPD-L1 levels >6.4 pg/ml were indicative of a reduced OS (p = 0.035) and PFS (p = 0.083) in platinum-sensitive patients, while OS and PFS in platinum-resistant patients did not differ when patients were stratified to this cut-off.Conclusions: Our study highlights sPD-L1 and sPD-L2 as complementary biomarkers reflecting clinical status, treatment response and disease outcome of EOC patients. Especially, sPD-L1 may facilitate the identification of high-risk patients with unfavorable disease outcomes despite platinum-sensitivity arguing for additional therapeutic approaches. As sPD-L1 and sPD-L2 are easily accessible via liquid biopsy, the inclusion of sPD-L1 and sPD-L2 in addition to CTC investigation as markers for risk assessment during patient therapy planning and follow-up appears to be a valuable approach.

Summary: Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1(−/−) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression. : In this article, Klump and colleagues demonstrate that the human homeotic selector protein HOXB4 promotes ESC-derived hematopoiesis by inducing hemogenic endothelium formation, in vitro. It propels hematopoietic specification by upregulating the transcription of genes essential for hematopoietic development, such as those encoding members of the so-called heptad transcription factors. Keywords: HOXB4, hematopoietic stem cells, hemangioblast, hemogenic endothelium, hematopoietic specification, EHT, RUNX1, pluripotent stem cells

In organ transplant recipients, the rate of invasive pneumococcal diseases is 25 times greater than in the general population. Vaccination against S. pneumoniae is recommended in this cohort because it reduces the incidence of this severe form of pneumococcal infection. Previous studies indicate that transplant recipients can produce specific antibodies after pneumococcal vaccination. However, it remains unclear if vaccination also induces specific cellular immunity. In the current study on 38 kidney transplant recipients, we established an interferon-γ ELISpot assay that can detect serotype-specific cellular responses against S. pneumoniae. The results indicate that sequential vaccination with the conjugated vaccine Prevenar 13 and the polysaccharide vaccine Pneumovax 23 led to an increase of serotype-specific cellular immunity. We observed the strongest responses against the serotypes 9N and 14, which are both components of Pneumovax 23. Cellular responses against S. pneumoniae correlated positively with specific IgG antibodies (r = 0.32, p = 0.12). In conclusion, this is the first report indicating that kidney transplant recipients can mount specific cellular responses after pneumococcal vaccination. The ELISpot we established will allow for further investigations. These could help to define, for example, factors influencing specific cellular immunity in immunocompromised cohorts or the duration of cellular immunity after vaccination.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the greatest medical challenge. Although crucial to the future management of the pandemic, the factors affecting the persistence of long-term SARS-CoV-2 immunity are not well understood. Therefore, we determined the extent of important correlates of SARS-CoV-2 specific protection in 200 unvaccinated convalescents after COVID-19. To investigate the effective memory response against the virus, SARS-CoV-2 specific T cell and humoral immunity (including virus-neutralizing antibodies) was determined over a period of one to eleven months. SARS-CoV-2 specific immune responses were present in 90% of individual patients. Notably, immunosuppressed patients did not have long-term SARS-CoV-2 specific T cell immunity. In our cohort, the severity of the initial illness influenced SARS-CoV-2 specific T cell immune responses and patients’ humoral immune responses to Spike (S) protein over the long-term, whereas the patients’ age influenced Membrane (M) protein-specific T cell responses. Thus, our study not only demonstrated the long-term persistence of SARS-CoV-2 specific immunity, it also determined COVID-19 severity and patient age as significant factors affecting long-term immunity.

Chronic kidney disease may alter antiviral T cell immunity. In the current study, we assessed in 63 patients prior to kidney transplantation how humoral and cellular immunity against cytomegalovirus (CMV) correlated using an interferon (IFN)-γ ELISpot (T-Track® CMV, Mikrogen, Neuried, Germany). The cohort comprised 24 patients with negative and 39 with positive CMV IgG. Whereas none of the patients with negative CMV IgG showed detectable responses to the T-Track® CMV, 26 out of 39 patients with positive CMV IgG had positive ELISpot responses. The median response to CMV pp65 in the CMV seronegative group was 0 spot forming units (SFU) per 200,000 PBMC (range 0–1) and in the seropositive group 43 SFU (range 0–750). Thus, 13 out of 39 patients with positive CMV serostatus (33%) had undetectable T cell immunity and may be at an increased risk of CMV reactivation. CMV pp65-specific ELISpot responses were 29.3-fold higher in seropositive patients with vs. without dialysis and 5.6-fold higher in patients with vs. without immunosuppressive therapy, but patients with dialysis and immunosuppressive therapy showed, as expected, lower responses to phytohemagglutinin, the positive control. This finding may be caused by (subclinical) CMV-DNAemia and a “booster” of CMV-specific T cells.

The cellular response to SARS-CoV-2 vaccination and infection in allogeneic hematopoietic stem cell transplant (HSCT) recipients is not yet clear. In the current study, HSCT recipients prior to and post vaccination were tested for SARS-CoV-2-specific humoral and cellular immunity. Antibodies against spike (S) 1 were assessed by Anti-SARS-CoV-2 IgG ELISA (Euroimmun). Cellular immunity was analyzed by an in house interferon-gamma ELISpot and T-SPOT.COVID (Oxford Immunotec), using altogether seven SARS-CoV-2-specific antigens. In 117 HSCT patients vaccinated twice, SARS-CoV-2 IgG antibodies were significantly higher than in HSCT controls pre vaccination (p < 0.0001). After the second vaccination, we observed a median antibody ratio of 4.7 and 68% positive results, whereas 35 healthy controls reached a median ratio of 9.0 and 100% positivity. ELISpot responses in patients were significantly (p < 0.001) reduced to ≤33% of the controls. After the second vaccination, female HSCT patients and female healthy controls showed significantly higher antibody responses than males (6.0 vs. 2.1 and 9.2 vs. 8.2, respectively; p < 0.05). Cellular immunity was diminished in patients irrespective of sex. In conclusion, especially male HSCT recipients showed impaired antibody responses after SARS-CoV-2 vaccination. Changing the vaccine schedule or composition could help increase vaccine responses.

Herd immunity is essential to control severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), especially in immunocompromised patients. Convalescent individuals should be vaccinated later due to vaccine shortage, as studies show that neutralizing antibodies generated during infection are stable for at least 6 months. Cellular immunity is also detectable for months. However, there is evidence of cross-reactivity of T cells with human endemic coronaviruses (HCoVs). Here, we show that cross-reactivity—which may prevent the specific detection of SARS-CoV-2-specific T cell responses—can be avoided if cells are stimulated with the N-terminus of the spike protein in IFN-γ ELISpot. In contrast to previous studies, we examined T-cell responses against all four known HCoVs using IFN-γ ELISpot in 19 convalescent volunteers and 10 fully vaccinated volunteers. In addition, we performed Spearman analyses to detect cross-reactivity of T cells. We observed no correlation between T-cell responses against SARS-CoV-2 and human endemic coronaviruses, either in the whole cohort or in the individual groups. The use of the respective stimuli could lead to a more accurate assessment of cellular immunity in recovered individuals. This testing procedure could help to define the best time point at which convalescents should receive SARS-CoV-2 vaccination.

Kidney transplantation is the therapy of choice for patients with end stage renal disease. Due to immunosuppressive treatment, patients are at risk for opportunistic infections. Cytomegalovirus (CMV) reactivation is highly relevant in kidney transplant recipients because it occurs—depending on the serological constellation of the donor and recipient—in more than half of the patients and influences patient outcome. Patients with CMV reactivation show decreased allograft and overall survival. Previous studies could demonstrate that transplant patients often show weak CMV-specific immunity. Besides immunosuppressive treatment, additional mechanisms may reduce CMV-specific immunocompetence such as enhanced negative costimulation. Hence, the aim of this study was to investigate if the function of CMV-specific cells of kidney transplant recipients could be restored by a modulation of costimulatory molecules. To address this question, lymphocytes of kidney transplant patients were stimulated with CMV-specific antigens and incubated with programmed death-ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), or B- and T-lymphocyte attenuator (BTLA) antibodies. Afterwards, the IFN-γ, IL-21, and IL-17A production was measured by the ELISpot assay. It could be shown that a blockade of the ligand PD-L1 resulted in an increased CMV-specific IFN-γ, IL-21, and IL-17A secretion. The blockade of the receptor PD-1 distinctly enhanced the production of IL-21. BTLA antibodies, however, led only to a marginal increase of CMV-specific IFN-γ and of IL-21 production. Experiments in healthy controls could confirm the results of the kidney transplant recipients. Furthermore, they could demonstrate that treatment with the immunosuppressive drug tacrolimus resulted in decreased CMV-specific IFN-γ and of IL-21 production. Thus, our study could show for the first time that the blockade of the PD-L1/PD-1 pathway also modulates CMV-specific Th21 and Th17 cell function in kidney transplant recipients. Further studies are mandatory to clarify the role of Th21 and Th17 cells in CMV control of these patients.

Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs.

HLA expression levels have been suggested to be genetically controlled by single nucleotide polymorphisms (SNP) in the untranslated regions (UTR), and expression variants have been associated with the outcome of chronic viral infection and hematopoietic stem cell transplantation (HSCT). In particular, the 3′UTR rs9277534-G/A SNP in HLA-DPB1 has been associated with graft-versus-host-disease after HSCT (Expression model); however its relevance in different immune cells and its mode of action have not been systematically addressed. In addition, there is a strong though not complete overlap between the rs9277534-G/A SNP and structural HLA-DPB1 T cell epitope (TCE) groups which have also been associated with HSCT outcome (TCE Structural model). Here we confirm and extend previous findings of significantly higher HLA-DPB1 expression in B cell lines, unstimulated primary B cells, and monocytes homozygous for rs9277534-G compared to those homozygous for rs9277534-A. However, these differences were abrogated by interferon-γ stimulation or differentiation into dendritic cells. We identify at least seven 3′UTR rs9277534-G/A haplotypes differing by a total of 37 SNP, also characterized by linkage to length variants of a short tandem repeat (STR) in intron 2 and TCE group assignment. 3′UTR mapping did not show any significant differences in post-transcriptional regulation assessed by luciferase assays between two representative rs9277534-G/A haplotypes for any of eight overlapping fragments. Moreover, no evidence for alternative splicing associated with the intron 2 STR was obtained by RT-PCR. In an exemplary cohort of 379 HLA-DPB1 mismatched donor-recipient pairs, risk prediction by the Expression model and the Structural TCE model was 36.7% concordant, with the majority of discordances due to non-applicability of the Expression model. HLA-DPB1 from different TCE groups expressed in the absence of the 3′UTR at similar levels by transfected HeLa cells elicited significantly different mean alloreactive CD4+ T-cell responses, as assessed by CD137 upregulation assays in 178 independent cultures. Taken together, our data provide new insights into the cell type-specific and mechanistic basis of the association between the rs9277534-G/A SNP and HLA-DPB1 expression, and show that, despite partial overlap between both models in HSCT risk-prediction, differential alloreactivity determined by the TCE structural model occurs independently from HLA-DPB1 differential expression.

To estimate protection from cytomegalovirus (CMV) replication after solid organ transplantation, CMV serology has been considered insufficient and thus CMV immunity is increasingly assessed by cellular in vitro methods. We compared two commercially available IFN-γ ELISpot assays (T-Track CMV and T-SPOT.CMV) and an IFN-γ ELISA (QuantiFERON-CMV). Currently, there is no study comparing these three assays. The assays were performed in 56 liver transplant recipients at the end of antiviral prophylaxis and one month thereafter. In CMV high- or intermediate-risk patients the two ELISpot assays showed significant correlation (p < 0.0001, r > 0.6) but the correlation of the ELISpot assays with QuantiFERON-CMV was weaker. Results of both ELISpot assays were similarly predictive of protection from CMV-DNAemia ≥500 copies/mL [CMV pp65 T-SPOT.CMV at the end of prophylaxis: area under curve (AUC) = 0.744, cut-off 142 spot forming units (SFU), sensitivity set to 100%, specificity 46%; CMV IE-1 T-Track CMV at month 1: AUC = 0.762, cut-off 3.5 SFU, sensitivity set to 100%, specificity 59%]. The QuantiFERON-CMV assay was inferior, reaching a specificity of 23% when setting the sensitivity to 100%. In conclusion, both CMV-specific ELISpot assays appear suitable to assess protection from CMV infection/reactivation in liver transplant recipients.

Aggressive B-cell lymphomas account for the majority of non-Hodgkin lymphomas (B-NHL). NK cells govern the responses to anti-CD20 monoclonal antibodies and have emerged as attractive targets for immunotherapy in subtypes of B-NHL. NKG2C and its cognate ligand HLA-E represent key molecules for fine-tuning of NK cell-mediated immune responses. Here, we investigated the impact of genetic variants of NKG2C and HLA-E on clinical outcomes of 441 B-NHL patients. Homozygous deletion of NKG2C (NKG2C−/−) was three-fold increased in patients compared to 192 healthy controls. Among studied patients, NKG2C−/− was more abundant in International Prognostic Index (IPI) high-risk patients compared to patients with a lower IPI (p = 0.013). Strikingly, NKG2C−/− was associated with a significantly reduced 2-year PFS (progression-free survival) (p = 0.0062) and represented an independent risk factor for 2-year PFS in multivariate analysis (p = 0.005). For HLA-E, the cognate ligand of NKG2C, the HLA-E*01:01 allele frequency was increased in B-NHL patients compared to controls (p = 0.033) and was associated with complete remission in univariate (p = 0.034) and multivariate (p = 0.018) analysis. Our data suggest that NKG2C and HLA-E genotyping is a promising tool for both defining risk groups of aggressive B-NHL and predicting response to immune therapeutic approaches.

Extracellular vesicles (EVs), such as exosomes and microvesicles, are released by different cell types and participate in physiological and pathophysiological processes. EVs mediate intercellular communication as cell-derived extracellular signalling organelles that transmit specific information from their cell of origin to their target cells. As a result of these properties, EVs of defined cell types may serve as novel tools for various therapeutic approaches, including (a) anti-tumour therapy, (b) pathogen vaccination, (c) immune-modulatory and regenerative therapies and (d) drug delivery. The translation of EVs into clinical therapies requires the categorization of EV-based therapeutics in compliance with existing regulatory frameworks. As the classification defines subsequent requirements for manufacturing, quality control and clinical investigation, it is of major importance to define whether EVs are considered the active drug components or primarily serve as drug delivery vehicles. For an effective and particularly safe translation of EV-based therapies into clinical practice, a high level of cooperation between researchers, clinicians and competent authorities is essential. In this position statement, basic and clinical scientists, as members of the International Society for Extracellular Vesicles (ISEV) and of the European Cooperation in Science and Technology (COST) program of the European Union, namely European Network on Microvesicles and Exosomes in Health and Disease (ME-HaD), summarize recent developments and the current knowledge of EV-based therapies. Aspects of safety and regulatory requirements that must be considered for pharmaceutical manufacturing and clinical application are highlighted. Production and quality control processes are discussed. Strategies to promote the therapeutic application of EVs in future clinical studies are addressed.

Early detection of cancer holds high promise for reducing cancer-related mortality. Detection of circulating tumor-specific nucleic acids holds promise, but sensitivity and specificity issues remain with current technology. We studied cell-free RNA (cfRNA) in patients with non-small cell lung cancer (NSCLC; n = 56 stage IV, n = 39 stages I-III), pancreatic cancer (PDAC, n = 20 stage III), malignant melanoma (MM, n = 12 stage III-IV), urothelial bladder cancer (UBC, n = 22 stage II and IV), and 65 healthy controls by means of next generation sequencing (NGS) and real-time droplet digital PCR (RT-ddPCR). We identified 192 overlapping upregulated transcripts in NSCLC and PDAC by NGS, more than 90% of which were noncoding. Previously reported transcripts (e.g., HOTAIRM1) were identified. Plasma cfRNA transcript levels of POU6F2-AS2 discriminated NSCLC from healthy donors (AUC = 0.82 and 0.76 for stages IV and I−III, respectively) and significantly associated (p = 0.017) with the established tumor marker Cyfra 21-1. cfRNA yield and POU6F2-AS transcript abundance discriminated PDAC patients from healthy donors (AUC = 1.0). POU6F2-AS2 transcript was significantly higher in MM (p = 0.044). In summary, our findings support further validation of cfRNA detection by RT-ddPCR as a biomarker for early detection of solid cancers.