Bam32: a novel mediator of Erk activation in T cells

Abstract

Bam32 (B lymphocyte adapter molecule of 32 kDa) is an adapter protein expressed in some hematopoietic cells including B and T lymphocytes. It was previously shown that Bam32-deficient mice have defects in various aspects of B cell activation including B cell receptor (BCR)-induced Erk activation, BCR-induced proliferation and T-independent antibody responses. In this study, we have examined the role of Bam32 in T cell activation using Bam32-deficient mice. By comparing CD4(+) T cells from lymph nodes of wild-type and Bam32-deficient mice, we found that Bam32 was required for optimal TCR-induced Erk activation, cytokine production, proliferation and actin-mediated spreading of CD4(+) T cells. These results indicate a novel pathway to Erk activation in T cells involving the adapter protein Bam32.

Fig. 1

Bam32 expression in human cell lines and human peripheral T cells. RNA was extracted from the following cell lines: Jurkat (origin—T cell leukemia), Daudi (origin—Burkitt’s lymphoma) and HEK293 (origin—kidney epithelium) or from sorted human peripheral blood (PB) T cells. After RT, cDNA was analyzed by PCR for expression of Bam32 and GAPDH.

Fig. 2

Bam32 contribution to proliferation and cytokine production in CD4⁺ T cells. CD4⁺ T cells were purified from lymph node cells from 8– to 11-week-old C57BL/6 (B6) and Bam32^−/− mice (>90% purity). Cells were then plated in wells pre-coated with the following concentrations of anti-CD3 and anti-CD28: (1) PBS control, (2) 1 µg ml⁻¹ anti-CD3, (3) 2.5 µg ml⁻¹ anti-CD3, (4) 5 µg ml⁻¹ anti-CD3, (5) 10 µg ml⁻¹ anti-CD3, (6) 5 µg ml⁻¹ anti-CD28, (7) 1 µgml⁻¹ anti-CD3 + 5 µgml⁻¹ anti-CD28, (8) 2.5 µgml⁻¹ anti-CD3 + 5 µgml⁻¹ anti-CD28, (9) 5 µgml⁻¹ anti-CD3 + 5 µgml⁻¹ anti-CD28 and (10) 10 µg ml⁻¹ anti-CD3 + 5 µg ml⁻¹ anti-CD28. After 48 h incubation, proliferation was measured by ³H-thymidine incorporation. Cytokine production was quantified by SearchLight protein array analysis of culture supernatants. Triplicate wells were analyzed individually for proliferation and culture supernatants were pooled for cytokine analysis. Results are representative of four experiments.

Fig. 3

Signal transduction in Bam32^−/− CD4⁺ T cells. (A) TCR-induced calcium flux in Bam32^−/− CD4⁺ T cells. Lymph node cells from 11-week-old Bam32^+/+ and Bam32^−/− littermates were loaded with indo-1 and surface stained for CD4 and CD8. Biotinylated anti-CD3 (0.5 µg ml⁻¹) and anti-CD4 (10 µg ml⁻¹) were added at 30 s and streptavidin (80 µg ml⁻¹) at 60 s (represented by AB and SA arrowheads). CD4 CD8 dot plots are shown and calcium kinetic data are shown for gated CD4⁺ cells over 6 min. (B) TCR-induced Erk, Jnk and p38 MAPK activation in Bam32^−/− CD4⁺ T cells. CD4⁺ T cells were purified from lymph nodes of C57BL/6 (B6) and Bam32^−/− mice (8 weeks) by negative magnetic bead separation. For Erk and Jnk analyses, CD4⁺ T cells were then stimulated using 2.5 µg ml⁻¹ plate-bound anti-CD3 for the indicated periods of time (minutes). For p38 analysis, rested cells were stimulated with 5 µg ml⁻¹ plate-bound anti-CD3 + 5 µg ml⁻¹ anti-CD28. Total cell lysates were then analyzed by western blotting for the indicated proteins or phospho-proteins. Graphs show the ratio of the levels of phospho-protein to total protein as determined by densitometry. Data are representative of at least three experiments. (C) Lymph node cells from 7– to 8-week-old Bam32^+/+ and Bam32^−/− mice were stimulated with anti-CD3 + anti-CD4 in solution, fixed, permeabilized, stained and analyzed by flow cytometry. Histograms are shown for gated CD4⁺ lymph node cells. Stimulation conditions were as follows: red line, no stimulation; dark blue line, 1 min stimulation; black line, 2 min stimulation; green line, 5 min stimulation; light blue line, 15 min stimulation and magenta line, phorbol myristate acetate stimulation. Orange line indicates staining with a negative control antibody. On the right, mean fluorescence intensity (MFI) of antibody-stimulated versus unstimulated cells is graphed over time.

Fig. 4

TCR-mediated spreading ofBam32^−/− CD4⁺ Tcells. CD4⁺ Tcells were purified from lymph nodes of 8– to 11-week-old Bam32^−/− (A) and C57BL/6 (B) mice by negative magnetic bead selection (>95% purity). Tcells were dropped onto chamber surfaces pre-coated with anti-CD3 (10 µgml⁻¹) and anti-CD28 (10 µg ml⁻¹). After 15 min, cells were fixed and stained with phalloidin and anti-phospho-ZAP-70. Interference reflection microscopic (IRM) and confocal fluorescent images were acquired using a 63× objective with or without optical zoom (upper and lower images, respectively) and the area of spread cells was quantitated. (C) The mean and standard error of the mean of the areas are presented for C57BL/6 (n = 95 cells) and Bam32^−/− (n = 71 cells). Using a Student’s t-test for unpaired data with unequal variance, the two means differ at a probability value of <0.0001.

Fig. 5

TCR down-modulation in Bam32^−/− CD4⁺ T cells. Lymph node T cells from C57BL/6 (B6) and Bam32^−/− mice were stimulated with plate-bound anti-CD3 for the number of hours indicated. Surface TCRβ was quantitated by flow cytometry and % down-modulation calculated as described in Methods