After one hour at 4C, cells were washed twice with FACS buffer and then fixed with 1% PFA before analysis on a BD LSRFortessa flow cytometer. severe COVID-19, thereby identifying a potential target for novel therapeutic interventions. relationship between auto-reactive IgM and COVID-19 pathophysiology, we first examined post-mortem pulmonary tissue to determine IgM distribution and presence. Immunohistochemical staining of parafin-embedded lung tissue revealed vastly greater IgM binding to alveolar septa and luminal surfaces of three COVID-19 non-survivors, compared to three COVID-19 unfavorable control patients for whom lung tissue was available from cancer-related resection (Physique 3a). It should be noted that some modest IgM deposition in the COVID-19 unfavorable patient controls was expected, as auto-reactive IgMs can develop during lung malignancy progression17 and/or following radiation therapy18. While we cannot formally rule out that this IgM detected in COVID-19+ lung tissue are reactive against SARS-CoV-2 surface antigens, the observed staining patterns are not consistent with the distribution patterns observed for SARS-CoV-2 antigens such as the Spike protein19,20. Importantly, the considerable IgM staining patterns are at levels at least three times higher than COVID-19 unfavorable controls (Physique 3b), and are not described for other causes of acute respiratory distress21. Further histological analysis revealed, in the lung of severe COVID-19 patients, significant alveolar damage and patchy hemorrhage, alongside considerable inflammatory infiltrate breaching the alveolar lumen. Previous studies have linked alveolar damage to dysregulated cytokine release and neutrophil extracellular traps seeded by resident macrophages22C25. Yet, these observations could also be linked to auto-reactive IgM, through the capacity of these immunoglobulins to fix match and induce cytotoxicity. Indeed, staining for match component 4 (C4d), a marker of match activation, showed a two-fold increase in COVID-19 patients compared to unfavorable controls (Physique 3c), indicating frequent match fixation. Complement-dependent cytotoxicity (CDC) and match deregulation have been proposed to play a functions in the pathogenesis of ARDS26. Additionally, as there is considerable pulmonary microangiopathy observed in severe COVID-19 patients27,28, it is conceivable that CDC can precede or even cause the damage to the pulmonary endothelium. Given the observed IgM and C4d binding to pulmonary targets and to confirm that the auto-reactive IgM can mediate CDC, we next tested plasma samples from severe COVID-19 patients for their capability of fixing match and inducing Tolfenamic acid cytotoxicity em in vitro /em . To this end, we investigated individual plasma samples that showed greater than 10% binding to the respective cell type in the screening assay. Interestingly, we consistently observed higher rates of CDC in cells of pulmonary origin (Physique 3d-h). In addition, while non-COVID-19 ICU patient plasma samples induced limited or no cell death, most COVID-19 ICU patients plasma samples induced cell death at frequencies proportional to their measured level of cell binding (Physique 3i). Collectively, these data indicate that auto-reactive IgM present in plasma from severe COVID-19 patients can fix match and induce cytotoxicity. The identification of auto-reactive IgM as a potential contributing factor to the pathogenesis of severe COVID-19 has two immediate implications. First, this observation may explain how COVID-19 is usually disproportionately more serious in the elderly29, who typically manifest higher plasma levels of circulating auto-reactive antibodies30. This phenomenon would be Rabbit polyclonal to PDCL2 exacerbated by decreases in functional Tolfenamic acid T follicular helper cells that promote antibody class switching31, a process associated with better disease outcomes32. Given that IgM levels peak within a week of the clinical onset of COVID-19 and persist at comparable levels for weeks thereafter34, the elderly face a protracted period where there is usually steadfast secretion of auto-reactive IgM that maintain relatively low Tolfenamic acid affinity for the same epitope without either switching to alternate antibody class types or undergoing somatic hypermutation and affinity maturation. In this perspective, Tolfenamic acid the elderly may be more prone to severe COVID-19 due to a more protracted exposure to the cytopathic effects of auto-reactive IgM. Second of all, it is conceivable that this type of immunopathology can be limited by therapeutic interventions that inhibit the IgM-complement axis. In the immediate.