Journal: ImmunoHorizons

Article Title: Antigen-driven Convergent Evolution of Polysaccharide-specific “DH-less” B Cells in Glycoconjugate Immunized Mice

doi: 10.4049/immunohorizons.2400055

Figure Lengend Snippet: Clonal expansion of PCP14-specific B cells in mice immunized with PCP14-CRM197. Sequence alignment of the Ig H and L chain genes expressed in hybridomas derived from clonally related B cells with their germline counterpart are shown. Clone 1 comprising B07B02, F03A11, and F04F09 and clone 2 with members D03E02 and B12G10 are presented in ( A ) and ( B ), respectively. The nucleotide sequence of germline VH, DH, JH, VL, and JL gene segments used are shown on top. The suffixes “-H” and “-L” denote H and L chain genes expressed in the anti-PCP14 hybridomas, respectively. The FR, CDR, and codon positions are according to the IMGT numbering system. The amino acid translation (in single-letter code; in italics) is given below the codons. Nucleotide identity with the corresponding nucleotide in the germline gene is indicated by a dash (-). The figure shows only those codon positions where mutation(s) have occurred. Replacement and silent mutations are shown in upper and lowercase, respectively. CDR3 sequence is, however, presented in full. P- and N-nucleotide addition(s) are indicated, and the nucleotides lost due to exonuclease activity in the CDR3s during the V(D)J recombination are shown as missing. Deduced genealogical trees for clones 1 ( C ) and 2 ( D ) are shown as schematic diagrams. The number of nucleotide changes observed in the expressed H chain gene is written first, followed by the number of changes in the expressed L chain gene (italicized). HP denotes the hypothetical intermediate precursor. V(D)J rearrangements used in the hypothetical germline precursor and the isotypes of the expressed H and L chains are also indicated.

Article Snippet: The Ig nucleotide sequences were analyzed using MacVector (with Assembler) software (version 17.0.5; MacVector Inc., USA).

Techniques: Sequencing, Derivative Assay, Mutagenesis, Activity Assay, Clone Assay

Journal: ImmunoHorizons

Article Title: Antigen-driven Convergent Evolution of Polysaccharide-specific “DH-less” B Cells in Glycoconjugate Immunized Mice

doi: 10.4049/immunohorizons.2400055

Figure Lengend Snippet: Convergent evolution observed in DH-less PCP14 reactive B cells. ( A ) The HCDR3 sequence ARWDY was identical in a subset of PCP14-specific hybridomas despite (i) using different VH, DH, and JH gene segments, (ii) difference in the N- and P-nucleotides, (iii) extent of exonuclease chewing at the V–D and V–J junctions in PCP14-specific hybridomas, and (iv) the fact they were generated from different mice. *The VH gene segment used could be either VH1-4 or VH1-7. # The VH gene segment used could be either VH1-4, VH1-7, or VH1-26. P- and N-nucleotides are shown in red and green, respectively. Additional DH-less anti-PCP14 mAbs are shown in ( B ). Two anti-CRM197 mAbs that lacked DH derived amino acids are presented in ( C ). The codons between the conserved cysteine (codon position 104 in VH) and conserved tryptophan (codon position 118 in JH) are shown. The nucleotide sequence of the HCDR3 portion of the IgH transcript expressed in the hybridoma and the corresponding germline VH, DH, and JH gene segments is shown in bold type. P- and N-nucleotide addition(s) are indicated on top of the alignment. Identity with the corresponding nucleotide in the germline gene segment is indicated with a dash (-). Silent mutations are shown in lowercase letters. The amino acid translation is shown in single-letter code below the codons (in italics). It may be noted that some nucleotides are missing at the V–D and D–J junctions due to exonuclease activity. The frequency (Fq.) of occurrence, dose regimen (Rg.), and the mouse ID (Mo. ID.) from which the hybridoma was derived are shown on the right.

Article Snippet: The Ig nucleotide sequences were analyzed using MacVector (with Assembler) software (version 17.0.5; MacVector Inc., USA).

Techniques: Sequencing, Generated, Derivative Assay, Activity Assay