Dissecting CD8+ T cell pathology of severe SARS-CoV-2 infection by single-cell immunoprofiling.

Introduction: SARS-CoV-2 infection results in varying disease severity, ranging from asymptomatic infection to severe illness. A detailed understanding of the immune response to SARS-CoV-2 is critical to unravel the causative factors underlying differences in disease severity and to develop optimal vaccines against new SARS-CoV-2 variants. Methods: We combined single-cell RNA and T cell receptor sequencing with CITE-seq antibodies to characterize the CD8+ T cell response to SARS-CoV-2 infection at high resolution and compared responses between mild and severe COVID-19. Results: We observed increased CD8+ T cell exhaustion in severe SARS-CoV-2 infection and identified a population of NK-like, terminally differentiated CD8+ effector T cells characterized by expression of FCGR3A (encoding CD16). Further characterization of NK-like CD8+ T cells revealed heterogeneity among CD16+ NK-like CD8+ T cells and profound differences in cytotoxicity, exhaustion, and NK-like differentiation between mild and severe disease conditions. Discussion: We propose a model in which differences in the surrounding inflammatory milieu lead to crucial differences in NK-like differentiation of CD8+ effector T cells, ultimately resulting in the appearance of NK-like CD8+ T cell populations of different functionality and pathogenicity. Our in-depth characterization of the CD8+ T cell-mediated response to SARS-CoV-2 infection provides a basis for further investigation of the importance of NK-like CD8+ T cells in COVID-19 severity.

Distinctive differences in DNA double-strand break repair between normal urothelial and urothelial carcinoma cells.

Several lines of evidence suggest that defective repair of DNA double-strand breaks (DSB) contributes to genomic instability in human cancers, including urothelial carcinoma. In particular, extracts from urothelial cancers have been reported to repair DSBs preferentially by microhomology-mediated end-joining (MMEJ), considered as more error-prone than canonical non-homologous end-joining (NHEJ) predominating in normal urothelial cell extracts. However, it is not clear whether such differences are relevant to intact cells. We therefore transfected plasmids digested with different restriction enzymes to yield incompatible ends (blunt, 5'-protruding or 3'-protruding) into urothelial carcinoma cell lines or normal urothelial cells and characterized the recovered circular plasmids. All cells competently repaired DSBs in a standard cloning vector plasmid, processing 5'- as well as 3'-protruding ends. No significant differences in the extent of processing were detected and the junctions presented short microhomologies indicative of canonical NHEJ. However, dramatic and distinctive differences between normal and cancerous urothelial cells were seen in two different experiments. First, cancer cell lines processed a significantly higher fraction of plasmids cut with a single restriction enzyme that could have been repaired by direct ligation than normal cells. Secondly, for the repair of a large plasmid with incompatible ends containing a large fragment of human genomic DNA, normal cells used almost exclusively MMEJ exploiting a microhomology with the 3'-end of the break, whereas cancer cell lines often processed DNA despite suitable microhomologies. DNA repair of the small or large plasmid was almost abolished by siRNA knockdown of Ku70. These findings strongly suggest that urothelial carcinoma cells lack control mechanisms preventing overprocessing during NHEJ repair. This may account for previous findings that urothelial cancers contain unusually large chromosomal deletions. Moreover, in contrast to prevailing interpretations, our observations suggest that MMEJ, despite its error-proneness, in some instances may act as a failsafe mechanism against overprocessing during NHEJ.

Marked dependence on growth state of backup pathways of NHEJ.

PURPOSE: Backup pathways of nonhomologous end joining (B-NHEJ) enable cells to repair DNA double-strand breaks (DSBs) when DNA-PK-dependent NHEJ (D-NHEJ) is compromised. Recent evidence implicates growth signaling in the regulation of D-NHEJ. This study was intended to determine whether the ability to repair DSBs by B-NHEJ also depends on growth state. METHODS AND MATERIALS: LIG4(-/-) and wild type (WT) mouse embryo fibroblasts (MEFs) were used. Repair of DSBs was measured by pulsed-field agarose gel electrophoresis. G1 cells were selected by centrifugal elutriation. A plasmid assay was used to measure DNA end-joining activity in whole cell extracts. RESULTS: Wild-type MEFs efficiently repaired DSBs by D-NHEJ in either the exponential or plateau phase of growth. Because of their defect in ligase IV, which compromises D-NHEJ, LIG4(-/-) MEFs showed reduced repair capacity but were slowly able to rejoin a large proportion of DSBs via B-NHEJ. B-NHEJ was markedly reduced in the plateau phase of growth or at high radiation doses. Elutriated G1 cells from exponentially growing or plateau-phase LIG4(-/-) cultures showed a response similar to nonelutriated cells, ruling out that the effect simply reflects redistribution in the cell cycle. An in vitro assay, gauging the activity of B-NHEJ, showed a reduction in DNA end joining during the plateau phase that could be corrected by recombinant DNA ligase IIIalpha. CONCLUSIONS: Suppression of growth signaling markedly compromises DSB repair by B-NHEJ. This effect is associated with a reduction in DNA ligase III mediated DNA end joining.

Low levels of DNA ligases III and IV sufficient for effective NHEJ.

Cells of higher eukaryotes rejoin double strand breaks (DSBs) in their DNA predominantly by a non-homologous DNA end joining (NHEJ) pathway that utilizes the products of DNA-PKcs, Ku, LIG4, XRCC4, XLF/Cernunnos, Artemis as well as DNA polymerase lambda (termed D-NHEJ). Mutants with defects in these proteins remove a large proportion of DSBs from their genome utilizing an alternative pathway of NHEJ that operates as a backup (B-NHEJ). While D-NHEJ relies exclusively on DNA ligase IV, recent work points to DNA ligase III as a component of B-NHEJ. Here, we use RNA interference (RNAi) to further investigate the activity requirements for DNA ligase III and IV in the pathways of NHEJ. We report that 70-80% knock down of LIG3 expression has no detectable effect on DSB rejoining, either in D-NHEJ proficient cells, or in cells where D-NHEJ has been chemically or genetically compromised. Surprisingly, also LIG4 knock down has no effect on repair proficient cells, but inhibits DSB rejoining in a radiosensitive cell line with a hypomorphic LIG4 mutation that severely compromises its activity. The results suggest that complete coverage for D-NHEJ or B-NHEJ is afforded by very low ligase levels and demonstrate residual end joining by DNA ligase IV in cells of patients with mutations in LIG4.

DNA ligase III as a candidate component of backup pathways of nonhomologous end joining.

Biochemical and genetic studies support the view that the majority of DNA double-strand breaks induced in the genome of higher eukaryotes by ionizing radiation are removed by two pathways of nonhomologous end joining (NHEJ) termed D-NHEJ and B-NHEJ. Whereas D-NHEJ depends on the activities of the DNA-dependent protein kinase and DNA ligase IV/XRCC4, components of B-NHEJ have not been identified. Using extract fractionation, we show that the majority of DNA end joining activity in extracts of HeLa cells derives from DNA ligase III. DNA ligase III fractionates through two columns with the maximum in DNA end joining activity and its depletion from the extract causes loss of activity that can be recovered by the addition of purified enzyme. The same fractionation protocols provide evidence for an additional factor strongly enhancing DNA end joining and shifting the product spectrum from circles to multimers. An in vivo plasmid assay shows that DNA ligase IV-deficient mouse embryo fibroblasts retain significant DNA end joining activity that can be reduced by up to 80% by knocking down DNA ligase III using RNA interference. These in vivo and in vitro observations identify DNA ligase III as a candidate component for B-NHEJ and point to additional factors contributing to NHEJ efficiency.

Alternative splicing of transcripts of the transposon Restless is maintained in the foreign host Neurospora crassa and can be modified by introducing mutations at the 5' and 3' splice sites.

The primary transcript of the transposon Restless from Tolypocladium inflatum undergoes an unusual mechanism of alternative splicing by employing either of two 3' "CAG" splice sites. These are separated by only four nucleotides, thus generating two different splice products, which differ in their coding capacity. To analyse whether this alternative splicing occurs in its natural host exclusively, we introduced the transposon into the heterologous host Neurospora crassa. In addition to the wild -type transposon sequence, transposon sequences mutagenised in vitro with modified 5' and 3' intron splice sites were generated. RNA was isolated from transformants and RT-PCR was performed with specific oligonucleotides flanking the intron sequence. Alternative splicing was analysed, employing a simple test procedure based on the convenient presence of a BamHI restriction site between both splice sites. The ratio of alternative splicing seems to be influenced by both the 5' and the 3' splice site, as mutations at either position influence the ratio of alternative splice products. At the 5' splice site, mutating the first "C" has a strong effect on the ratio of alternative splicing, while mutating the second "C" has little or no effect. Similarly, at the 3' splice site, only mutations at the first 3' "CAG" change the ratio of alternative splicing. It appears that alternative splicing of the Restless intron is not host-specific, but is influenced by the intron splice site sequences themselves.

Ds-like restless deletion derivatives occur in Tolypocladium inflatum and two foreign hosts, Neurospora crassa and Penicillium chrysogenum.

Single copies of the transposon Restless from Tolypocladium inflatum were introduced into Neurospora crassa and Penicillium chrysogenum. Excision of Restless from its donor site was investigated in N. crassa and in P. chrysogenum using direct selective conditions. In N. crassa, forward selection was also analyzed. Deleted Restless elements were frequently obtained in addition to the expected complete removal of Restless from its donor site. Similar deleted elements were also identified in T. inflatum employing a PCR amplification strategy. These deleted Restless copies strongly resemble maize Ds elements of various types, and direct repeated sequences of 3 to 16 bp were found to flank the truncated regions. In addition Ds1-like Restless elements were identified that carried foreign sequences between the inverted repeats. We discuss how Ds-like Restless elements might be generated by inaccurate excision from an active transposon copy.