Plasmablasts in COVID-19: Dual Role as Immune Effectors or Amplifiers of Inflammation
Plasmablasts in COVID-19: Dual Role as Immune Effectors or Amplifiers of Inflammation
All patients with a laboratory-confirmed diagnosis of COVID-19 who were hospitalized at the CHU Lille and had available lymphocyte immunophenotyping performed between April 8th and May 1st, 2020, were retrospectively enrolled. Patients had to have symptomatic SARS-Cov2 infection confirmed by positive nasopharyngeal swab polymerase chain reaction. Samples used for lymphocyte immunophenotyping were stored for B-cell isolation and DNA extraction. Data on demographics, clinical manifestations, routine laboratory features, and therapeutic management, and outcomes were collected. The alpha (B.1.1.7) SARS-CoV-2 variant was dominant in France during this period. Among the patients in cohort 2, we retrospectively constituted a cohort named 2b, by collecting 41 samples from 23 patients with blood samples available according to maximum severity and time since onset of disease to perform RNA sequencing on PB cells and other B cells.  Total RNAs were extracted using the NucleoSpin® RNA kit (MACHEREY-NAGEL) as per the manufacturer's instructions. The samples exhibited heterogeneity in total RNA quantity. To address this, we concentrated the samples with the lowest RNA concentrations using a SpeedVac and limited the input to a maximum of 50 ng for the most concentrated ones (n=2) to avoid an excessive concentration range. After concentration, the estimated total RNA amounts ranged from 0.1 ng to 50 ng in a volume of 4 µL. From 4 µL of total RNA, we added 1 µL of ERCC spike-in control. Library preparation was then initiated using oligo(dT) priming, following the QuantSeq FFPE 3' mRNA FWD protocol with UDI (Unique Dual Indexes) from Lexogen, incorporating the low-input specific workflow. Unique Molecular Identifiers (UMIs) were introduced to facilitate the removal of PCR duplicates during analysis. After generating the double-stranded cDNA library, we purified it using magnetic beads and amplified it. Library amplification was performed with 22 cycles. Libraries with the lowest yields (less than 600 pM in the 200 bp–1000 bp range) were re-amplified using the reamplification add-on kit for Illumina (LEXOGEN) with six to eight additional cycles. Final library concentrations ranged from 222 pM to 11,187 pM (200 bp–1000 bp interval). After equimolar pooling of the libraries, the pool was repurified to eliminate primer dimers. 