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Blood, Vol. 112, Issue 4, 1205-1213, August 15, 2008
Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage Fc RI, FcRIII, and FcRIV
Blood Minard-Colin et al.
112: 1205
Supplemental materials for: Minard-Colin et al
Supplemental materials and methods Immunoglobulin gene rearrangements
Total RNA from B6 spleen cells and BL3750 cells (expanded 48 hours in vitro) was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). Random hexamer primers (Promega, Madison, WI) and Superscript II RNase H Reverse Transcriptase (Invitrogen) were used to generate cDNA as described.1 The following forward primers were used for kappa chains: VK1: 5′- ATG AAG TTG CCT GTT AGG CTG TTG GTG CTG - 3′, VK2: 5′- ATG GAG A/TCA GAC ACA CTC CTG C/TTA TGG GTG - 3′, VK3: 5′- ATG AGT GTG CTC ACT CAG GTC CTG GC/GG TTG - 3′, VK4: 5′- ATG AGG G/ACC CCT GCT CAG T/ATT T/CTT GGC/A T/ATC TTG - 3′,VK5: 5′– ATG ATG GAT TTT/A CAG GTG CAG ATT A/TTC AGC TTC - 3′, VK6: 5′- ATG AGG TG/TC C/TC/TT GT/CT C/GAG T/CTC/T CTG G/AGG - 3′, VK7: 5′- ATG GGC A/TTC AAG ATG GAG TCA CAG/T A/TC/TC/T CA/TG G -3′, VK8: 5′- ATG TGG GGA C/TCT T/GTT TT/CC C/AC/AT TTT TCA ATT G - 3′, VK9: 5′- ATG GTA/G TCC A/TCA C/GCT CAG TTC CTT G - 3′, VK10: 5′- ATG TAT ATA TGT TTG TTG TCT ATT TCT - 3′, VK11: 5′- ATG GAA GCC CCA GCT CAG CTT CTC TTC C - 3′, VK12: 5′- ATG AGT CCT GCC CAG TTC CTG TTT CTG- 3′. The following reverse primer was used for all kappa chains: Vc: 5′- ACT GGA TGG TGG GAA GAT GG - 3′. The following forward primers were used for heavy chains: MHV1: 5′- ATG AAA TGC AGC TGG GGC ATC/G TTC TTC – 3′, MHV2: 5′ – ATG GGA TGG AGC TG/AT ATC ATG/C T/CTC TT - 3′, MHV3: 5′- ATG AAG T/ATG TGG TTA AAC TGG GTT TTT -3′, MHV4: 5′- ATG A/GAC TTT GGG C/TTC AGC TTG A/GTT T - 3′, MHV5: 5′ – ATG GAC TCC AGG CTC AAT TTA GTT TTC CTT - 3′, MHV6: 5′ – ATG GCT GTC C/TTA/G GG/CG CTA/G CTC TTC TGC – 3′, MHV7: 5′- ATG GG/AA TTG AGC G/TGG A/GTC TTT C/ATC TT – 3′, MHV8: 5′- ATG AGA GTG CTG ATT CTT TTG TG - 3′, MHV9: 5′ – ATG GA/CT TGG GTG TGG AC/AC TTG CTA TTC CTG - 3′, MHV10: 5′- ATG GGC AGA CTT ACA TTC TCA TTC CTG - 3′, MHV11: 5′- ATG GAT TTT GGG CTG ATT TTT TTT ATTG - 3′, MHV12: 5′- ATG ATG GTG TTA AGT CTT CTG TAC CTG – 3′. The following reverse primer was used for all heavy chains: Cµ-in: 5′- GAG GGG GAA GAC ATT TGG GAA GGA CTG- 3′. PCR products were analyzed by gel electrophoresis. BL3750 PCR products were extracted from gels and purified using a QIAquick Gel Extraction Kit (Qiagen, Valencia, CA), and sequenced completely in both 5′ and 3′ directions using the Applied Biosystems Dye Terminator Cycle Sequencing System. REFERENCES 1. Engel P, Nojima Y, Rothstein D, et al. The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils and monocytes. J Immunol. 1993;150:4719-4732.
Files in this Data Supplement:
- Figure S1. BL3750 cells are monoclonal (JPG, 52.9 KB)
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(A) PCR amplification of heavy chain (VH) and light chain (VK) regions using spleen and BL3750 cells. (B) Predicted amino acid sequences for BL3750 cell heavy chain and light chain V regions. Regions representing primer sequences are underlined.
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