Volume 15, Issue 1, Pages 149-158 (July 2001) The Crohn's Disease-Associated Bacterial Protein I2 Is a Novel Enteric T Cell Superantigen  Harnisha Dalwadi, Bo Wei, Mitchell Kronenberg, Christopher L Sutton, Jonathan Braun  Immunity  Volume 15, Issue 1, Pages 149-158 (July 2001) DOI: 10.1016/S1074-7613(01)00164-9

Figure 1 I2 Sequence Is Present in Mouse Colon C57BL/6J genomic DNA was assayed by PCR for I2 (285 bp) or GAPDH (340 bp). The control I2 mimic product was 606 bp. (A) SPF mice. (B) Defined-flora mice Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 2 Recombinant I2 Stimulates Proliferation and IL-10 Secretion by CD4+ T Cells from SPF C57BL/6J Mice CD4+ T cells from SPF C57BL/6J mice were cultured with antigen-pulsed APCs (0, 2, 10, 15 μg/ml; GST, I2-GST, I1-GST). SEB (1 μg/ml) was used as the positive control (data not shown). (A) Proliferation (mean of triplicate samples). (B) Cytokine secretion (mean of duplicate samples). (C) Intracellular IL-10. Cultured cells were assayed for IL-10, CD4, and B220 by flow cytometry. Data for 2 μg/ml antigen are tabulated; similar results were observed with other antigen concentrations Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 3 I2 Response Was Dependent on MHC II-Mediated Recognition CD4+ T cells and antigen-pulsed APCs (I2-GST, 5 μg/ml) from SPF mice were cultured in the presence and absence of 10 μg/ml anti-I-Ab or anti-I-Ad. GST (5 μg/ml) and SEB (1 μg/ml) were negative and positive control antigens, respectively. Con A (1 μg/ml) was used as a model I-Ab-independent T cell mitogen. After 48 hr of incubation, proliferation was assayed by thymidine incorporation. (A) I2-GST, GST, SEB. (B) Con A Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 4 Response of Cells from Defined-Flora Mice Cell cultures from defined-flora mice were assayed for (A) proliferation of CD4+ T cells and (B) cytokine production. Data for 1 μg/ml antigen are tabulated; similar results were observed with other antigen concentrations Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 5 Activation of Naive and Memory CD4 T Cells upon I2 Stimulation Naive and memory CD4 T cells (see Experimental Procedures) from SPF mice were cocultured with antigen-pulsed APCs. (A) Proliferation, naive CD4+ T cells. (B) Proliferation, memory CD4+ T cells. (C) IL-10 production. Data for 5 μg/ml antigen are tabulated; similar results were observed with 1 μg/ml antigen Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 6 The I2 Response Does Not Require Antigen Processing (A and B) APCs, either normal or paraformaldehyde fixed, were antigen pulsed and cocultured with splenic CD4+ T cells. (C) A.E7 PCC-specific CD4 T cells were cocultured with antigen-pulsed APCs (normal or fixed). Proliferation was assayed for all groups by thymidine incorporation Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)

Figure 7 TCR-Vβ5.1 and 5.2 Expression Was Restricted in the Responding I2-GST-Specific T Cell Population CD4+ T cells from SPF and defined-flora C57BL/6J mice were cocultured with antigen-pulsed APCs (0.1 μg/ml; GST, I2-GST, SEB). After 48 hr, cultures were pulsed with 50 μg/ml BrdU and then assayed by flow cytometry. The data are represented as a percentage of BrdU+, CD4+, and Vβ+ cells. To do this, PERCP-negative cells and CD4+ cells were gated, and then the percentage of (BrdU+ and Vβ+ cells)/(the total number of Vβ+ cells) was calculated. The geometric mean of five experiments is shown here. (A) I2-GST, GST, no antigen (SPF mice). (B) SEB, no antigen, (SPF mice). (C) I2-GST, GST (defined-flora mice) Immunity 2001 15, 149-158DOI: (10.1016/S1074-7613(01)00164-9)