Computational protocol: Accumulation of Self Reactive Naïve and Memory B Cell Reveals Sequential Defects in B Cell Tolerance Checkpoints in Sjögren’s Syndrome

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Protocol publication

[…] Aliquots of VH, Vκ and Vλ chain second PCR products were sequenced with the respective reverse primer (Beckman Coulter Genomics) and the sequences were analyzed by IgBlast (http://www.ncbi.nlm.nih.gov/igblast/) to identify germline V(D)J gene segments with highest homology. IgH complementary determining region (CDR)3 length and the number of positively (Histidine(H), Arginine (R), Lysine (K)) and negatively charged (Aspartate (D), Glutamate (E)) amino acids were determined as previously described , . Out of 96 single naïve B cell sorted from each of the 4 SS patients, we obtained successful sequences from a total of 119 different VH and JH regions (SS3 = 19, SS5 = 53, SS12 = 17, SS13 = 30), 73 Vκ and Jκ regions (SS3 = 17, SS5 = 31, SS12 = 2, SS13 = 23), and 36 Vλ and Jλ regions (SS3 = 13, SS5 = 10, SS12 = 10, SS13 = 3) (). For the memory unswitched B cells, we obtained successful sequences from a total of 131 different VH and JH regions (SS3 = 33, SS4 = 18, SS5 = 11, SS6 = 18, SS12 = 26, SS13 = 25), 74 Vκ and Jκ regions (SS3 = 23, SS4 = 8, SS5 = 8, SS6 = 11, SS12 = 10, SS13 = 14), and 37 Vλ and Jλ regions (SS3 = 8, SS4 = 6, SS5 = 2, SS6 = 5, SS12 = 5, SS13 = 11) (). Finally, for the memory switched B cells, we obtained successful sequences from a total of 103 VH and JH regions (SS3 = 24, SS4 = 21, SS5 = 8, SS6 = 6, SS9 = 20, SS12 = 19, SS13 = 5), 41 Vκ and Jκ regions (SS3 = 14, SS4 = 3, SS5 = 7, SS6 = 4, SS9 = 3, SS12 = 9, SS13 = 1), and 32 Vλ and Jλ regions (SS3 = 10, SS4 = 7, SS5 = 0, SS6 = 2, SS9 = 4, SS12 = 4, SS13 = 5) (). VH, Vκ and Vλ sequences from SS patients were compared with VH, Vκ and Vλ sequences from either freshly obtained IgD+ naïve B cells () or from a re-analysis of sequences from memory unswitched and memory switched B cells from HD previously published , and kindly provided by Prof Hedda Wardemann at Max Planck Institute for Infection Biology (Berlin, Germany).Clones with matching productive VH and VL products were used for downstream cloning and recombinant antibody expression. All productive VH and VL products from naïve B cells displayed unmutated germ line sequences (data not shown).Immunoglobulin Analysis Tool (IgAT) software was used to calculate the probability of antigen-driven selection within the Ig repertoire of memory unswitched and switched B cells . IgAT uses the algorithms generated by Chang and Casali and by Lossos et al. in order to identify sequences that are indicative of an antigen-driven selection. […]

Pipeline specifications

Software tools IgBLAST, IgAT
Application Rep-seq analysis
Organisms Homo sapiens
Diseases Wernicke Encephalopathy