SECTM1A costimulates whereas SECTM1B inhibits T cell activation.
A. 48+ T cells cultured on 2C11 coated plates in the presence of solid-phase mCD7-Fc or mSECTM1B-Fc fusion protein. Campath-1H antibody was used to equilibrate each well to an equal protein concentration. Results representative of five separate experiments. * = p<0.05, ** = p<0.005 by Student's t test. B. ELISA for IL-2 in supernatants of CBA/Ca CD4+ T cells stimulated for 48 hours in the presence of plate-bound 2C11 and solid phase SECTM1B-Fc, CD7-Fc or Campath-1H. Results are representative of three experiments. SECTM1B inhibition significant at 20,50 and 100 ug plated fusion protein assessed by Student's t test. C. Proliferation assay with CD4+ T cells cultured on 2C11 coated plates in the presence of solid-phase mSECTM1B-Fc fusion protein or Campath-1H as control. Anti-mCD28 or neutralising anti-TGFβ (1D11) was added to some wells at 1 ug/ml. Where indicated, IL-2 was added at 10 U/mL. * = p<0.05, ** = p<0.005 by Student's t test. Results are representative of two experiments. D. Proliferation assay with CD4+ T cells. The supernatants from the cell cultures described in ‘Figure C’ were added to fresh cultures of CBA.CA CD4+ T cells cultured with plate-bound 2C11. Proliferation was assessed at 48 hours. Results representative of two separate experiments. Differences not significant by Student's t test. E. Left panel; Cell surface expression of CD69 on CD4+ T cells (approximately 85% pure) cultured for 48 hours on 2C11(1 ug/ml) coated 96 well plates in the presence of plate-bound CP1H, SECTM1B-Fc or SECTM1A-Fc. All fusion proteins coated at 10 ug/ml. Results representative of three separate experiments. Right Panel; Pooled data from three experiments showing percentage change in CD69 on activated CD4+ T cells. Cells were cultured as above in the presence of 10 ug/ml plate bound fusion proteins and the percentage change in mean fluorescence intensity for CD69 above or below the CP1H control is shown. Statistical significance was measured using Student's T test. F. Proliferation assay with CD4+ T cells cultured on 2C11 coated plates in the presence of solid-phase mSECTM1A-Fc fusion protein or SECTM1A-ExIV-Fc. Proliferation was measured at 48 hours. Results representative of five separate experiments. * = p<0.05, ** = p<0.005 by Student's t test.