Fig. 5. VLRB N8 recognizes a tyrosine sulfation–dependent antigen on HLA-I.

(A) KMS-11 cells were cultured in the presence of the indicated concentrations of NaClO₃ for 48 hours followed by flow cytometric assessment of VLRB N8 and HLA-I reactivity. A representative experiment is depicted in the top panel, and VLRB N8/HLA-I ratios from five independent experiments are shown in the bottom bar diagram, depicted as means ± SD. Statistical significance was determined using one-way ANOVA with Dunnett’s post test (n = 5). (B) Inhibition of VLRB N8 recognition of HLA-I on BJAB cells following PMA and ionomycin stimulation. Cells were stimulated for 1 hour with PMA and ionomycin, and VLRB N8 and HLA-I binding were assessed following a 36-hour culture with the indicated concentrations of NaClO₃. Means ± SD of VLRB N8 signals normalized to HLA-I are shown. Statistical significance was determined using two-way ANOVA test with Dunnett’s post test (n = 4). (C) shRNA-mediated down-regulation of transduced BJAB cells was verified by qRT-PCR. Transcript levels of TPST1 (left) and TPST2 (right) of the indicated cell populations are depicted as means ± SD (n = 3). Statistical significance was determined using a Student’s t test. (D) shRNA-transduced BJAB cells were stimulated with anti-Ig (20 μg/ml), followed by the assessment of VLRB N8 and anti–HLA-I recognition. Numbers indicate the mean fold induction of HLA-I normalized VLRB N8. Statistical significance for induced VLRB N8 binding was determined using a one-way ANOVA test with Tukey’s post test (n = 9). (E) Tyrosine sulfation of HLA-I following antigen receptor engagement. A representative autoradiogram (left) of anti–HLA-I immunoprecipitates of unstimulated and stimulated BJAB cells and the quantitation (right) of six independent experiments are shown. ³⁵SO₄ incorporation is shown with arbitrary units (AU). Statistical significance was determined using paired Student’s t test (n = 6). (F) TPST1 and TPST2 transcript analysis of tonsillar B cell populations. Means ± SD of qRT-PCR of TPST1 or TPST2 normalized to HPRT from five independent tonsil specimens are shown. Statistically significant differences of P < 0.05 are indicated by *P < 0.05, **P < 0.01, ***P < 0.001; n.s., P > 0.05.