Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19

To understand the fine differential elements that can lead to or prevent acute res-piratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investi-gate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes asso-ciated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expan-sion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

Automated summary

To investigate the immune response architecture in COVID-19 patients and understand the factors that can cause or prevent ARDS, we analyzed the B cell responses using flow cytometry and Ig repertoire analysis. Flow cytometry with FlowSOM analysis revealed changes associated with COVID-19 inflammation, including an increase in double-negative B cells and ongoing plasma cell differentiation. We also found the expansion of two disconnected B cell repertoires. Analysis of Ig repertoire patterns showed an early expansion of IgG1 clonotypes with atypical CDR3 characteristics, which correlated with ARDS severity. Additionally, we observed a convergent response with progressively increasing somatic hypermutation and normal or short CDR3 in anti-SARS-CoV-2 clonotypes, which persisted until the memory B cell stage after recovery.