Program: Oral and Poster Abstracts
Session: 203. Lymphocytes, Lymphocyte Activation and Immunodeficiency, including HIV and Other Infections: Poster III
Methods:CD19-null (ΔCD19) derivatives of Nalm6 and 697 B-ALL cell lines were generated using the CRISPR/Cas9 approach. We then reconstituted them with full-length and Δex2 CD19 isoforms, expressed either on their own or as CD19-GFP fusions. Additionally, CD19 N-terminus mutations (ΔSP [no signal peptide], N86A, C97A and N86A/C97A) were introduced in full-length CD19. Transduced cells were analyzed by flow cytometry, confocal microscopy, and western blotting. Glycosylation of the mutants was verified using treatment with swainsonine (Golgi glycosylation inhibitor) and an in vitro de-glycosylation assay.
Results:As expected, deletion of the N-terminal signal peptide responsible for the endoplasmic reticulum translocation led to impaired surface expression. Surprisingly, deletion of exon 2 sequences had a similar effect: although up to 10% of Δex2 CD19 was found on the plasma membrane (where it enhanced pre-B-cell receptor signaling), the remainder was largely cytosolic. Furthermore, while the N86A substitution in full-length CD19 did not significantly affect its surface localization, substituting Cys-97 with Ala fully recapitulated the Δex2 cytosolic phenotype. In fact, the C97A mutant appeared to be stuck in the ER and never reach Golgi, since its electrophoretic mobility was not affected by swainsonine. In contrast, its glycosylation in ER was unperturbed, as evidenced by in vitro de-glycosylation assays.
Conclusion: The immunoglobulin-like loop connecting Cys-38 and Cys-97 of CD19 is required for its plasma membrane localization. One possible mechanism is the N-termini-mediated interaction with CD81, which is known to be needed to transport CD19 to the cell surface (Matsumoto et al 1993).
Maude SL, Frey, N, Shaw, PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, and Grupp SA. Chimeric antigen receptor T cells for sustained remissions in leukemia. N Engl J Med2014 371:1507-1517.
Sotillo E, Barrett D, Bagashev A, Black K, Lanauze C, Oldridge D, Sussman R, Harrington C, Chung EY, Hofmann TJ, Maude SL, Martinez NM, Raman P, Ruella M, Allman D, Jacoby E, Fry T, Barash Y, Lynch KW, Mackall C, Maris J, Grupp SA, and Thomas-Tikhonenko A. Alternative splicing of CD19 mRNA in leukemias escaping CART-19 immunotherapy eliminates the cognate epitope andcontributes to treatment failure. 2015 AACR Annual Meeting, Philadelphia.
Zhou LJ, Ord DC, Hughes AL and Tedder TF, Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain. J Immunol. 1991 147(4):1424-32
Matsumoto AK, Martin DR, Carter RH, Klickstein LB, Ahearn JM, and Fearon DT Functional dissection of the CD21/CD19/TAPA-1/Leu-13 complex of B lymphocytes. J Exp Med. 1993 178(4):1407-17.
Disclosures: No relevant conflicts of interest to declare.
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