IntroductionA single center open label phase II randomised control trial was done to assess the pathogen and host-intrinsic factors influencing clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT), in addition to standard of care (SOC) therapy in severe COVID-19 patients, as compared to patients only on SOC therapy.MethodsConvalescent plasma was collected from patients recovered from COVID-19 following a screening protocol which also included measuring plasma anti SARS-CoV2 spike IgG content. Retrospectively, neutralizing antibody content was measured and proteome was characterized by LC-MS/MS for all convalescent plasma units that were transfused to patients. Severe COVID-19 patients with evidence for acute respiratory distress syndrome (ARDS) with PaO2/FiO2 ratio 100-300 (moderate ARDS) were recruited and randomised into two parallel arms of SOC and CPT, N=40 in each arm. Peripheral blood samples were collected on the day of enrolment (T1) followed by day3/4 (T2) and day 7 (T3). RT-PCR and sequencing was done for SARS-CoV2 RNA isolated from nasopharyngeal swabs collected at T1. A panel of cytokines and neutralizing antibody content were measured in plasma at all three timepoints. Patients were followed up for 30 days post-admission to assess the primary outcomes of all cause mortality and immunological correlates for clinical benefits.ResultsWhile across all age-groups no statistically significant clinical benefit was registered for patients in the CPT arm, significant immediate mitigation of hypoxia, reduction in hospital stay as well as survival benefit was recorded in severe COVID-19 patients with ARDS aged less than 67 years receiving convalescent plasma therapy. In addition to its neutralizing antibody content a prominent effect of convalescent plasma on attenuation of systemic cytokine levels possibly contributed to its benefits.ConclusionPrecise targeting of severe COVID-19 patients is necessary for reaping the clinical benefits of convalescent plasma therapy.Clinical trial registrationClinical Trial Registry of India No. CTRI/2020/05/025209

Transition from resting to cell cycle in response to antigenic stimulation is an essential step for naïve CD8+ T cells to differentiate to effector and memory cells. Leaving the resting state requires dramatic changes of chromatin status in the key cell cycle inhibitors but the details of these concerted events are not fully elucidated. Here, we showed that Ezh2, an enzymatic component of polycomb repressive complex 2 (PRC2) catalyzing the trimethylation of lysine 27 on histone 3 (H3K27me3), regulates activation induced naïve CD8+ T cells proliferation and apoptosis. Upon deletion of Ezh2 during thymocyte development (Ezh2fl/flCd4Cre+ mice), naive CD8+ T cells displayed impaired proliferation and increased apoptosis in response to antigen stimulation. However, naive CD8+ T cells only had impaired proliferation but no increase in apoptosis when Ezh2 was deleted after activation (Ezh2fl/flGzmBCre+ mice), suggesting cell cycle and apoptosis are temporally separable events controlled by Ezh2. We then showed that deletion of Ezh2 resulted in the increase in expression of cyclin-dependent kinase inhibitors Cdkn2a (p16 and Arf) and Cdkn1c (p57) in activated naïve CD8+ T cells as the consequence of reduced levels of H3K27me3 at these two gene loci. Finally, with real time imaging, we observed prolonged cell division times of naïve CD8+ T cells in the absence of Ezh2 post in vitro stimulation. Together, these findings reveal that repression of Cdkn1c and Cdkn2a by Ezh2 plays a critical role in execution of activation-induced CD8+ T cell proliferation.

Components of the conserved engulfment pathways promote programmed cell death in Caenorhabditis elegans (C. elegans) through an unknown mechanism. Here we report that the phagocytic receptor CED-1 mEGF10 is required for the formation of a dorsal–ventral gradient of CED-3 caspase activity within the mother of a cell programmed to die and an increase in the level of CED-3 protein within its dying daughter. Furthermore, CED-1 becomes enriched on plasma membrane regions of neighbouring cells that appose the dorsal side of the mother, which later forms the dying daughter. Therefore, we propose that components of the engulfment pathways promote programmed cell death by enhancing the polar localization of apoptotic factors in mothers of cells programmed to die and the unequal segregation of apoptotic potential into dying and surviving daughters. Our findings reveal a novel function of the engulfment pathways and provide a better understanding of how apoptosis is initiated during C. elegans development.

A single center open label phase 2 randomised control trial (Clinical Trial Registry of India No. CTRI/2020/05/025209) was done to assess clinical and immunological benefits of passive immunization using convalescent plasma therapy. At the Infectious Diseases and Beleghata General Hospital in Kolkata, India, 80 patients hospitalized with severe COVID-19 disease and fulfilling the inclusion criteria (aged more than 18 years, with either mild ARDS having PaO2/FiO2 200–300 or moderate ARDS having PaO2/FiO2 100–200, not on mechanical ventilation) were recruited and randomized into either standard of care (SOC) arm (N = 40) or the convalescent plasma therapy (CPT) arm (N = 40). Primary outcomes were all-cause mortality by day 30 of enrolment and immunological correlates of response to therapy if any, for which plasma abundance of a large panel of cytokines was quantitated before and after intervention to assess the effect of CPT on the systemic hyper-inflammation encountered in these patients. The secondary outcomes were recovery from ARDS and time taken to negative viral RNA PCR as well as to report any adverse reaction to plasma therapy. Transfused convalescent plasma was characterized in terms of its neutralizing antibody content as well as proteome. The trial was completed and it was found that primary outcome of all-cause mortality was not significantly different among severe COVID-19 patients with ARDS randomized to two treatment arms (Mantel-Haenszel Hazard Ratio 0.6731, 95% confidence interval 0.3010-1.505, with a P value of 0.3424 on Mantel-Cox Log-rank test). No adverse effect was reported with CPT. In severe COVID-19 patients with mild or moderate ARDS no significant clinical benefit was registered in this clinical trial with convalescent plasma therapy in terms of prespecified outcomes.