Farage

Lack of Ig Polypeptide Chain Production in Farage Cells

To see whether Farage cells produce μ and Ig κ chains, the cells were metabolically labeled with ³⁵S-methionine and the cell lysates were immune precipitated using anti-μ and aIIti-κ sera. Daudi and DG-75 B-lymphomas known to express surface IgM (κ) served as positive controls. Fig. 1 shows that in contrast to Daudi, radioactively labeled μ and κ chains could not be detected in Farage cytoplasmic cell lysates even following a long exposure time. In Daudi cells (Fig. 1) and DG-75 (not shown), large amounts of labeled μ chains were detected by either anti-μ or anti-κ reagents, indicating that both cell types produced μ and κ polypeptide chains. On the other hand, labeled κ was not detected at all in DG-75, and was detected at a low quantity in Daudi cells, where it was present in immune precipitates formed with anti-κ and not when the anti-μ agent was used (Fig. 1). Our results show that the κ light chains in Daudi, (and even more so in DG75 cells), are poorly labeled probably due to lack of the amino acid methionine in their κ light chains.

Because of the κ-chain labeling problem, immunoblots were done on the cytoplasmic lysates of DG-75 and Farage cells using enhanced chemiluminescence. Fig. 1B shows that whereas DG-75 contained both μ and κ polypeptide chains, no Ig chains could be detected in Farage cells. A band of a somewhat higher molecular size than the μ chain appeared in Farage cell lysates. However, a similar band is also present in the K562 negative control. To find out whether Ig chains were abnormally directed to the perinuclear or nuclear compartments of Farage cells, the nuclear pellet formed following NP40 cell lysis was lysed by SDS and deoxycholate and then immunoprecipitated with various antisera. Whereas μ chains could be detected in the "nuclear lysates" of Daudi cells by either anti-μ or anti-κ sera, no labeled Ig chains were detected in similar lysates made from Farage cells (not shown). Examination of the culture medium of Daudi, DG75, and Farage cells showed that labeled IgM was secreted by DG-75 but not by Farage or Daudi cells (not shown). The latter finding fits the classification of DG-75 as a sporadic (American) type BL, representing a more advanced stage of the B-cell differentiation pathway, as compared to Daudi cells classified as an endemic (African) type BL.

Fig. 1 Farage cells do not synthesize Ig-polypeptide chains. (Baruch M, et al., 1996)

Generation of SLAMF1-Deficient Farage Cells

SLAMF1 was highly expressed in Farage cells, an EBV-positive B cell lymphoma cell line. To generate SLAMF1-deficient Farage cells, two sgRNAs targeting exon 3 of SLAMF1 were designed by using CRISPR/Cas9 technology (Fig. 2A). The two sgRNAs coupled with Cas9 expression vectors were transfected, and three cell lines named 3C, 8C, and 4E were obtained by serial dilution and mutational analysis (Fig. 2B). As shown in Fig. 2B, two bands were observed in 4E after PCR amplification, indicating a deletion of exon 3 in one allele (Fig. 2B). Sub-bands appeared after cleavage of heteroduplex in 3C, 8C and 4E, indicating the presence of at least one indel mutation (Fig. 2B). Whether the expression level of SLAMF1 was decreased at the cell surface was tested by using FACS analysis. SLAMF1 was almost negative in the three cell lines compared with SLAMF1 wild-type Farage cells (Fig. 2C). This result was further confirmed by western blot analysis with the anti-human SLAMF1 antibody, which was a different clone than that used for FACS analysis. SLAMF1 was not detected or remarkably decreased in mutant cell lines, whereas LMP1 had no discernable change between SLAMF1 wild-type and mutant cells (Fig. 2D).

Fig. 2 SLAMF1-deficient Farage cells were cloned by using CRISPR/Cas9. (Yoon H, et al., 2020)