The regulation of NK cell detachment from target cells as a key factor for serial killing and effector function

Natural Killer cells efficiently recognize and kill infected or malignant cells and can eliminate several of these cells one after the other in a process called serial killing. The activation of NK cells and how they form conjugates with target cells is well studied, but for the efficient killing of multiple cells, NK cells also have to detach from the previous target cell. The process of detachment is still poorly understood; therefore analyzing the regulation of this process in more detail is important for the investigation of NK cell biology. I used flow cytometry based assays to analyze the adhesion and the detachment of NK cells and target cells. The data show, that the detachment was different between fresh, IL-2 activated and cultured NK cells while the formation of conjugates was almost unchanged. Interestingly, blocking NK cell cytotoxicity inhibited detachment. Reducing target cell death by increasing their resistance against NK cells also reduced the detachment of NK cells and increased the production of the cytokines IFN-γ and TNF-α. Induction of NK cell independent target cell death had the opposite effect. The loss of ligands for activating NK cell receptors and the majority of adhesion molecules on the target cell membrane during cell death could be responsible for the NK cell detachment. Additionally, NK cells seem to sense soluble factors released by dying cells, like apoptotic bodies, which accelerated the detachment. This demonstrates, that the detachment of NK cells is a highly regulated process, which depends on the activation status of the NK cell, ongoing receptor-ligand interactions and changes in the protein profile of the cell membrane after death of the target cell. Regulation of detachment is not only necessary for the efficiency of serial killing but also influences other NK cell effector functions like cytokine production

Detection of pre-existing SARS-CoV-2-reactive T cells in unexposed renal transplant patients

Background: Recent data demonstrate potentially protective pre-existing T cells reactive against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in samples of healthy blood donors, collected before the SARS-CoV-2 pandemic. Whether pre-existing immunity is also detectable in immunosuppressed patients is currently not known. Methods: Fifty-seven patients were included in this case-control study. We compared the frequency of SARS-CoV-2-reactive T cells in the samples of 20 renal transplant (RTx) patients to 20 age/gender matched non-immunosuppressed/immune competent healthy individuals collected before the onset of the SARS-CoV-2 pandemic. Seventeen coronavirus disease 2019 (COVID-19) patients were used as positive controls. T cell reactivity against Spike-, Nucleocapsid-, and Membrane-SARS-CoV-2 proteins were analyzed by multi-parameter flow cytometry. Antibodies were analyzed by neutralization assay. Results: Pre-existing SARS-CoV-2-reactive T cells were detected in the majority of unexposed patients and healthy individuals. In RTx patients, 13/20 showed CD4(+) T cells reactive against at least one SARS-CoV-2 protein. CD8(+) T cells reactive against at least one SARS-CoV-2 protein were demonstrated in 12/20 of RTx patients. The frequency and Th1 cytokine expression pattern of pre-formed SARS-CoV-2 reactive T cells did not differ between RTx and non-immunosuppressed healthy individuals. Conclusions: This study shows that the magnitude and functionality of pre-existing SARS-CoV-2 reactive T cell in transplant patients is non-inferior compared to the immune competent cohort. Although several pro-inflammatory cytokines were produced by the detected T cells, further studies are required to prove their antiviral protection

Case Report: Co-occurrence of tubulitis and SARS-CoV-2 specific T-cells in a kidney transplant recipient

BackgroundKidney transplantation is associated with an increased risk of severe COVID-19 disease. Additionally, cells of the kidney express ACE-2 making them a potential target of the SARS-CoV-2 virus. Both uncontrolled viral replication and T-cell receptor (TCR) mediated cellular immunity towards the infected cells could lead to tissue destruction in the kidney. In cases where pathological findings are not always capable of providing definitive diagnosis, insights into the TCR repertoire could offer valuable information. Here we present a case of potentially infection driven tubulitis in a kidney transplant patient.MethodsThe source of kidney infiltrating T-cells was assessed through next generation TCR sequencing. Using cells from the living donor and overlapping peptide pool of SARS-CoV-2 S-, N-, and M-protein (Wuhan variant), antigen specific T-cells were isolated from peripheral blood by overnight stimulation and subsequent isolation using antibodies and magnetic beads against CD154 and CD137. The clonotypes of these two samples were compared to the clonotypes in a single kidney biopsy cylinder.ResultsWe found that 11.1% of the repertoire of the kidney infiltrating T cells were identical to SARS-CoV-2 specific T-cells in the periphery, and only 3.1% of the repertoire was identical to allo-specific TCRs. We also observed substantial overlap between the TCR repertoires of virus-specific and donor-specific T cells, with high similarity and even identical TCR sequences present in both populations. The TCRs with dual specificity constituted a larger proportion of the allo-specific than the virus specific population. These results indicate that SARS-CoV-2 specific T-cells may directly spill into an allo-specific T cell response and that either class of T-cells may cause the observed tubulitis.ConclusionTCR-seq of whole biopsies is a method to evaluate the ingragraft TCR repertoire can complement routine pathology and provide further insights into the mechanisms underlying a diagnosis

Immune Response in Moderate to Critical Breakthrough COVID-19 Infection After mRNA Vaccination

SARS-CoV-2 variants of concern (VOCs) can trigger severe endemic waves and vaccine breakthrough infections (VBI). We analyzed the cellular and humoral immune response in 8 patients infected with the alpha variant, resulting in moderate to fatal COVID-19 disease manifestation, after double mRNA-based anti-SARS-CoV-2 vaccination. In contrast to the uninfected vaccinated control cohort, the diseased individuals had no detectable high-avidity spike (S)-reactive CD4+ and CD8+ T cells against the alpha variant and wild type (WT) at disease onset, whereas a robust CD4+ T-cell response against the N- and M-proteins was generated. Furthermore, a delayed alpha S-reactive high-avidity CD4+ T-cell response was mounted during disease progression. Compared to the vaccinated control donors, these patients also had lower neutralizing antibody titers against the alpha variant at disease onset. The delayed development of alpha S-specific cellular and humoral immunity upon VBI indicates reduced immunogenicity against the S-protein of the alpha VOC, while there was a higher and earlier N- and M-reactive T-cell response. Our findings do not undermine the current vaccination strategies but underline a potential need for the inclusion of VBI patients in alternative vaccination strategies and additional antigenic targets in next-generation SARS-CoV-2 vaccines

Low avidity circulating SARS-CoV-2 reactive CD8+ T cells with proinflammatory TEMRA phenotype are associated with post-acute sequelae of COVID-19

The role of adaptive SARS-CoV-2 specific immunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although a growing population of convalescent COVID-19 patients with manifestation of PASC is observed. We analyzed the SARS-CoV-2-specific immune response, via pseudovirus neutralizing assay and multiparametric flow cytometry in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. Although frequencies of SARS-CoV-2-reactive CD4+ T cells were similar between the studied cohorts, a stronger SARS-CoV-2 reactive CD8+ T cell response, characterized by IFNγ production and predominant TEMRA phenotype but low functional TCR avidity was detected in PASC patients compared to controls. Of interest, high avidity SARS-CoV-2-reactive CD4+ and CD8+ T cells were comparable between the groups demonstrating sufficient cellular antiviral response in PASC. In line with the cellular immunity, neutralizing capacity in PASC patients was not inferior compared to controls. In conclusion, our data suggest that PASC may be driven by an inflammatory response triggered by an expanded population of low avidity SARS-CoV-2 reactive pro-inflammatory CD8+ T cells. These pro-inflammatory T cells with TEMRA phenotype are known to be activated by a low or even without TCR stimulation and lead to a tissue damage. Further studies including animal models are required for a better understanding of underlying immunopathogensis. Summary: A CD8+ driven persistent inflammatory response triggered by SARS-CoV-2 may be responsible for the observed sequelae in PASC patients

High incidence and viral load of HHV-6A in a multi-centre kidney transplant cohort

Human herpesvirus 6 (HHV-6) is a common opportunistic pathogen in kidney transplant recipients. Two distinct species of HHV-6, HHV-6A and HHV-6B, have been identified, of which the latter seems to be dominant. However, it is unclear whether they increase the likelihood of other viral reactivations. We characterized a multi-centre cohort of 93 patients along nine study visits for viral load. We tested for the following viruses: HHV-6A and HHV-6B, the herpesviruses cytomegalovirus (CMV) and Epstein-Barr virus (EBV) and the polyomavirus BK (BKV). We detected HHV-6A viral load in 48 (51.6%) patients, while the incidence of HHV-6B was much lower, being detected in 6 (6.5%) patients. The incidence of HHV-6A was higher than of BKV, CMV and EBV. HHV-6A also demonstrated higher viral loads than the rest of viruses. There was a non-significant trend of association between HHV-6A and HHV-6B as co-infection, whereas no increased incidence of other viruses among patients with HHV-6A reactivation was observed. There was no negative effect of high HHV-6A (>10,000 copies/ml) load on markers of renal graft and hepatic function or blood count twelve months post-transplant. In contrast to previously published data, our results show a clear dominance of HHV-6A in peripheral blood when compared to HHV-6B, with higher incidence and viral load levels. Despite the high HHV-6A loads observed, we did not identify any negative effects on posttransplant outcome

In-depth analysis of T cell immunity and antibody responses in heterologous prime-boost-boost vaccine regimens against SARS-CoV-2 and Omicron variant

With the emergence of novel Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs), vaccination studies that elucidate the efficiency and effectiveness of a vaccination campaign are critical to assess the durability and the protective immunity provided by vaccines. SARS-CoV-2 vaccines have been found to induce robust humoral and cell-mediated immunity in individuals vaccinated with homologous vaccination regimens. Recent studies also suggest improved immune response against SARS-CoV-2 when heterologous vaccination strategies are employed. Yet, few data exist on the extent to which heterologous prime-boost-boost vaccinations with two different vaccine platforms have an impact on the T cell-mediated immune responses with a special emphasis on the currently dominantly circulating Omicron strain. In this study, we collected serum and peripheral blood mononuclear cells (PBMCs) from 57 study participants of median 35-year old’s working in the health care field, who have received different vaccination regimens. Neutralization assays revealed robust but decreased neutralization of Omicron VOC, including BA.1 and BA.4/5, compared to WT SARS-CoV-2 in all vaccine groups and increased WT SARS-CoV-2 binding and neutralizing antibodies titers in homologous mRNA prime-boost-boost study participants. By investigating cytokine production, we found that homologous and heterologous prime-boost-boost-vaccination induces a robust cytokine response of CD4+ and CD8+ T cells. Collectively, our results indicate robust humoral and T cell mediated immunity against Omicron in homologous and heterologous prime-boost-boost vaccinated study participants, which might serve as a guide for policy decisions

The protein interaction of mitochondrial transcription factors A and B2 is associated with 30-day survival in critical COVID-19

IntroductionRepair of mitochondrial damage seems pivotal for clinical recovery and determining outcome in patients with critical COVID-19. However, reliable biomarkers for non-invasively assessing mitochondrial repair in peripheral blood of critically ill COVID-19 patients are currently lacking. Accordingly, we sought to assess different surrogates of mitochondrial repair in peripheral blood and correlate these measurements with clinical outcome in patients with critical COVID-19.MethodsIn this prospective multicentric cohort study, 88 critically ill COVID-19 patients were enrolled across three German intensive care units. Gene products of mitochondrial quality control (MFN2, PINK, TFAM, TFB2M) and the mtDNA copy number were measured in peripheral blood mononuclear cells. Furthermore, the protein interactions between TFAM and TFB2M were quantified. Patients were stratified regarding 30-day mortality. ResultsTranscript levels of the assessed mRNA markers of mitochondrial quality control were not associated with clinical outcome. In contrast, more than 10.7 protein interactions per cell were associated with a 74% 30-day survival (37 out of 50), while 10.7 or fewer protein interactions per cell were associated with a 32% 30-day survival (12 out of 38; p < 0.001). Furthermore, multivariable Cox regression analysis revealed TFAM-TFB2M protein interaction as an independent predictor for 30-day survival (HR: 3.2; 95% CI: 1.6 to 6.5; p < 0.001). DiscussionOur findings indicate that TFAM-TFB2M protein interactions, identified as a novel biomarker, are strongly and independently associated with 30-day survival in critical COVID-19. Therefore, our data suggest a significant impact of mitochondrial repair and quality control on clinical outcome in critical COVID-19