Single-cell RNA sequencing for the effects of Commd3 deficiency and celastrol treatment on humoral immune responses

To profile the global effects of Commd3 deficiency on humoral immune responses, CD45+ cells from the draining lymph nodes (LNs) of CreERT2Commd3flox/+ and CreERT2Commd3flox/flox mice were subjected to single-cell RNA sequencing (scRNA-seq) at 7 days after immunization. We found that Commd3 deletion a week before immunization reduced only the proportion of germinal center B cells with minimal transcriptomic changes in B cell populations. Celastrol treatment in WT mice a week before immunization also selectively reduced the proportion of GC B cells with little impact on the transcriptomes, whereas none of the detectable immune cell populations were significantly affected by celastrol treatment in the immunized COMMD3C170A/C170A mice. Mice received subcutaneous injections of NP-CGG at both sides of the abdomen and tail base (100 μg NP-CGG in total), and the inguinal LNs were collected at 7 days after immunization. Live CD45+ cells sorted from the LNs were subjected to scRNA-seq. To test the effects of Commd3 deletion, CreERT2Commd3flox/+ and CreERT2Commd3flox/flox mice were treated with tamoxifen for 3 consecutive days from 9 days before immunization. To test the effects of celastrol treatment, WT or COMMD3C170A/C170A mice received intraperitoneal injections of vehicle [PBS containing 2% dimethyl sulfoxide] or celastrol (1 mg/kg, twice a week at 2- and 3-day intervals) from 7 days before immunization.