Increased soluble HLA in COVID-19 present a disease-related, diverse immunopeptidome associated with T cell immunity.

HLA-presented antigenic peptides are central components of T cell-based immunity in infectious disease. Beside HLA molecules on cell surfaces, soluble HLA molecules (sHLA) are released in the blood suggested to impact cellular immune responses. We demonstrated that sHLA levels were significantly increased in COVID-19 patients and convalescent individuals compared to a control cohort and positively correlated with SARS-CoV-2-directed cellular immunity. Of note, patients with severe courses of COVID-19 showed reduced sHLA levels. Mass spectrometry-based characterization of sHLA-bound antigenic peptides, the so-called soluble immunopeptidome, revealed a COVID-19-associated increased diversity of HLA-presented peptides and identified a naturally presented SARS-CoV-2-derived peptide from the viral nucleoprotein in the plasma of COVID-19 patients. Of interest, sHLA serum levels directly correlated with the diversity of the soluble immunopeptidome. Together, these findings suggest an inflammation-driven release of sHLA in COVID-19, directly influencing the diversity of the soluble immunopeptidome with implications for SARS-CoV-2-directed T cell-based immunity and disease outcome.

Radiation-induced DNA damage and repair in peripheral blood mononuclear cells from Nijmegen breakage syndrome patients and carriers assessed by the Comet assay.

Nijmegen breakage syndrome (NBS) patients and carriers are predisposed to malignancy and are often treated with X-irradiation. In the present study, the single-cell gel electrophoresis (Comet) assay was used to examine radiation-induced DNA damage and repair in peripheral blood mononuclear cells from NBS patients (n=13) and carriers (n=36) of six unrelated families. Cells from apparently healthy donors (n=10) and from breast cancer patients with normal clinical radiosensitivity (n=10) served as controls. Cells were irradiated with 5 Gy of X-rays and assayed for initial DNA damage and for residual DNA damage after 40 min of repair; the kinetics of DNA repair also was estimated. In addition, the nuclear area of unirradiated cells was extracted from the Comet data. The initial radiation-induced DNA fragmentation indicated that cells from members of two out of six NBS families were significantly more sensitive to X-irradiation than cells from the controls. Cells from four NBS families had longer DNA repair half-time values, while cells from five NBS families had significantly increased residual DNA damage following repair. The mean nuclear area of unirradiated cells processed in the Comet assay was 1.3-fold higher in cells from all NBS families than in the controls (P<0.05). Notably, the Comet assay parameters (initial and residual DNA damage and the repair kinetics) of irradiated NBS cells predicted the carrier status of the majority (86%) of blindly tested individuals. The prediction of NBS status was higher if the nuclear area of unirradiated cells was used as the endpoint. The results of this study suggest that the impaired radiation response of NBS cells should be taken into account if radiotherapy of NBS patients and carriers is required.

Radiation induced DNA damage and damage repair in human tumor and fibroblast cell lines assessed by histone H2AX phosphorylation.

PURPOSE: To analyze the radiation-induced levels of gammaH2AX and its decay kinetics in 10 human cell lines covering a wide range of cellular radiosensitivity (SF2, 0.06-0.63). METHODS AND MATERIALS: Five tumor cell lines included Colo-800 melanoma, two glioblastoma (MO59J and MO59K), fibrosarcoma HT 1080, and breast carcinoma MCF7. Five primary skin fibroblasts lines included two normal strains, an ataxia telangiectasia strain, and two fibroblast strains from breast cancer patients with an adverse early skin reaction to radiotherapy. Cellular radiosensitivity was assessed by colony-forming test. Deoxyribonucleic acid damage and repair were analyzed according to nuclear gammaH2AX foci intensity, with digital image analysis. RESULTS: The cell lines tested showed a wide degree of variation in the background intensity of immunostained nuclear histone gammaH2AX, which was higher for the tumor cell lines compared with the fibroblast strains. It was not possible to predict clonogenic cell survival (SF2) for the 10 cell lines studied from the radiation-induced gammaH2AX intensity. In addition, the slopes of the dose-response (0-4 Gy) curves, the rates of gammaH2AX disappearance, and its residual expression (