Expression of ultralong complementarity determining region 3 and development of IgM and IgG B cell receptor repertoires in Holstein heifer calves

Abstract There are currently knowledge gaps regarding the development of the bovine B cell repertoire especially for cells expressing B cell receptors with heavy chain ultralong complementarity determining region 3 (CDR3). The objective of this study was to assess percentages of B cells with ultralong CDR3s, and immunoglobulin heavy chain gene usage in Holstein calves from birth to 10 mo of age. Blood was collected from 7 heifer calves on the following days of life: 0 (prior to ingestion of colostrum), 2 (after colostrum ingestion), 42, 56, 90, and 285. IgM+ and IgG+ B cells were collected using magnetic-bead separation. RNA was extracted, cDNA was produced, and IgM and IgG sequences were amplified using polymerase chain reactions. Amplicons were sequenced using long-read Oxford Nanopore sequencing on R10.4 flow cells. Differences in the percentages of B cells with ultralong CDR3s were assessed using non-parametric Wilcoxon signed-rank tests. The mean percentage of productive IgM ultralong CDR3 sequences at birth was 3.27%, which decreased by day 42 and remained low until there was a significant increase between day 90 and ∼day 285 (5.68% ± 1.51 standard error of the mean, P = 0.03). The mean percentage of productive IgG ultralong CDR3 sequences was low at birth (0.89%) and remained low until there was a significant increase between day 90 and ∼day 285 (6.86% ± 0.99, P = 0.03). These increases in the percentages of sequences with ultralong CDR3 may be indicative of increased antigenic exposure and concurrent maturation of immune functionality.