The Roles of Costimulation and Fas in T Cell Apoptosis and Peripheral Tolerance  Luk Van Parijs, Alexander Ibraghimov, Abul K. Abbas  Immunity  Volume 4, Issue 3, Pages 321-328 (March 1996) DOI: 10.1016/S1074-7613(00)80440-9

Figure 1 In Vitro Activation of Wild-Type and Fas-Deficient T Cells in the Presence of Competent APCs Can Be Blocked with mCTLA-4Ig Naive CD4+ T cells (a) or Th1 lines (b) derived from wild-type and lpr/lpr 2B4 mice were cultured in triplicate with PCC (81–104) peptide in the presence of mitomycin C-treated H-2k spleen cells, alone, or with 2 μg/ml mCTLA-4Ig or control immunoglobulin. Proliferation was assessed by [3H]thymidine incorporation at 72 hr (a) or 36 hr (b). The error bars represent standard errors. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 2 In Vitro Activation of Wild-Type and Fas-Deficient T Cells in the Presence of PAF APCs Requires a CD28-Mediated Signal Naive CD4+ T cells (a) or Th1 lines (b) derived from wild-type and lpr/lpr 2B4 mice were cultured in triplicate with PCC (81–104) peptide in the presence of PAF H-2k spleen cells, alone, or with anti-CD28 or control antibody (1:1000 dilution of hybridoma ascites). Proliferation was assessed by [3H]thymidine incorporation at 72 hr (a) or 48 hr (b). The error bars represent standard errors. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 3 PCD in Naive T Cells Is Regulated by Costimulation and Not by Fas Naive CD4+ T cells derived from wild-type and lpr/lpr 2B4 mice were cultured in duplicate (a) with or without PCC (81–104) peptide in the presence of mitomycin C-treated H-2k spleen cells, alone, or with 2 μg/ml mCTLA-4Ig or control immunoglobulin, or (b) with increasing concentrations of soluble anti-CD3, together with anti-CD28 or control antibody (1:1000 dilution of hybridoma ascites). After 48 hr, cell death was assessed by double staining for the transgenic TCR (recognized by the A2B4 monoclonal antibody) and propidium iodide incorporation. The error bars represent standard errors. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 4 AICD in Activated T Cells Is Regulated by Fas and Not by Costimulation Wild-type and lpr/lpr 2B4 Th1 lines were generated as described and cultured in duplicate in medium containing 20 U/ml IL-2 (a) with or without immobilized anti-CD3 (1 μg/ml) and anti-CD28 (1:1000 dilution of hybridoma ascites), 2 μg/ml mCTLA-4Ig, or control immunoglobulin, or (b) with increasing concentrations of soluble anti-CD3, together with anti-CD28 or control antibody. After 24 hr, cell death was assessed by double staining for the transgenic TCR (recognized by the A2B4 monoclonal antibody) and propidium iodide incorporation. The error bars represent standard errors. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 5 bcl-xL and FasL mRNA Induction Differs in CD28 Dependence and in Its Correlation with Different Forms of Cell Death The expression of bcl-xL and FasL mRNA in naive or activated Th1 2B4/+ T cells was assayed by RT–PCR following a 24 hr culture with or without soluble anti-CD3 (1 μg/ml) and anti-CD28 (1:1000 dilution of hybridoma ascites). The percentage of apoptotic 2B4/+ T cells in parallel cultures was assessed either at 24 hr (2B4 Th1 line) or 48 hr (naive 2B4 T cells) and gave the following results: naive 2B4 T cells: media, 38%; anti-CD3, 32%; anti-CD3/anti-CD28, 7%; 2B4 Th1 line: media, 27%; anti-CD3, 45%; anti-CD3/anti-CD28, 52%. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 6 Superantigen-Mediated Deletion Is Defective in the Periphery, but Not the Thymus, of Fas-Deficient Mice Wild-type and lpr mice were treated intraperitoneally with 2 μg SEB on days 1, 3, and 5. On day 8, the percentage of Vβ8+ and Vβ6+ CD4+ T cells was assessed in the spleen (a) and the thymus (b) by two- or three-color flow cytometry. Data from two mice was pooled for each treatment, and the error bars represent the standard error. Results are from one representative experiment out of four. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)

Figure 7 Peripheral Unresponsiveness Can Be Induced in Fas-Deficient Mice MRL +/+ (a) and lpr/lpr (b) mice were treated intraperitoneally with 1 mg OVA, emulsified in IFA. The mice were challenged 10 days later by injecting 200 μg OVA, emulsified in CFA, into the footpad. On day 20, the inguinal and popliteal lymph nodes were harvested, and the lymph node cells restimulated in vitro with OVA, as shown. Proliferation was assessed at 72 hr. Cytokine production was assessed at 48 hr for IL-2 or 72 hr for IL-4 and IFNγ. Data were pooled from 2–4 mice per group, and the error bars represent the standard error. Results are from one representative experiment out of four. Immunity 1996 4, 321-328DOI: (10.1016/S1074-7613(00)80440-9)