Effect of BALB/c cells, cytokine blockade, and cognate antigen on the deletion and anergy of transferred SEB-activated KJ1-26.1⁺ T cells.

(A) Deletion: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Experimental mice were then observed (control, n = 12), or injected with SEB alone (n = 8) or in combination with rhuTNFR:Fc (anti-TNF, n = 5), anti–IFN-γ (anti-IFN, n = 3), CFA (CFA, n = 3), or CFA + ovalbumin (CFA/OVA, n = 3) (see below). SCID mice reconstituted with OVA-SCID PLNCs and about 1 × 10⁶ SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 3) were injected with SEB in parallel. Ten days after adoptive transfer, the percentage and absolute numbers of KJ1-26.1⁺ and CD4⁺Vβ8⁺KJ1-26.1⁻ cells in the spleens and lymph nodes (the latter defined as the difference between CD4⁺Vβ8⁺ cells and KJ1-26.1⁺cells) were determined. Circulating TNF-α and IFN-γ were blocked by intraperitoneal injections of rhuTNFR:Fc31 (200 μg) or anti–IFN-γ ascites28 61 (250 μL) every 2 days from day −1. The effect of simultaneous stimulation with cognate antigen was determined in SCID mice immunized 24 hours earlier with CFA with or without chicken ovalbumin. Deletion of KJ1-26.1⁺ cells in the presence of BALB/c cells was prevented by decreasing the posttransfer SEB responsiveness of cotransferred BALB/c cells by treating donors with SEB prior to cell harvest, by decreasing overall activation levels with anticytokine antibodies, or by increasing the relative strength of activation of KJ1-26.1⁺ T cells by simultaneous stimulation with SEB and cognate antigen. The means ± SE of n animals are shown. The initial ratio of KJ1-26.1⁺/CD4⁺Vβ8⁺KJ1-26.1⁻cells at time of transfer was about 1:1, with about 1 × 10⁶ of each of KJ1-26.1⁺ and CD4⁺Vβ8⁺KJ1-26.1⁻ cells injected. Without SEB stimulation, T-cell numbers were independent of the use of anticytokine reagents or of CFA (not shown). (B) Anergy: Mixtures of OVA-SCID PLNCs and naive BALB/c spleen cells were transferred into SCID mice. Ten days later, reconstituted control mice (control, n = 7) and animals injected with SEB alone (n = 7) or in combination with rhuTNFR:Fc (anti-TNF, n = 7), or anti–IFN-γ (anti-IFN, n = 3), were injected intraperitoneally with 3 mg chicken ovalbumin. SCID mice reconstituted with OVA-SCID PLNCs and approximately 1 × 10⁶ SEB-tolerant BALB/c spleen cells (tolerant Vβ8, n = 4) were injected in parallel. Three days later, total numbers of spleen and lymph node KJ1-26.1⁺ cells were determined and used to calculate the proliferation index (defined as the ratio of the cell numbers before and 48 hours after injection of chicken ovalbumin). OVA-dependent anergy of KJ1-26.1⁺ cells transferred together with BALB/c cells and stimulated with SEB was decreased in the presence of SEB-tolerant BALB/c cells and by TNF-α and IFN-γ blockade. The means ± SE of n animals are shown. (C) KJ1-26.1⁺ T cells are rescued from deletion by stimulation with cognate antigen. BALB/c and KJ1-26.1⁺ cells were transferred into SCID mice, some of which had been injected subcutaneously 24 hours earlier with chicken ovalbumin in CFA (lower panels). Mice were observed (control, upper panels) or injected with SEB (SEB and SEB OVA, middle and lower panels, respectively) and secondary lymphoid organs were analyzed 10 days later. The percentages of CD4⁺KJ1-26.1⁺ T cells (left panels) and CD4⁺Vβ8⁺KJ1-26.1⁻ T cells (right panels) are shown. The reduction in transferred CD4⁺KJ1-26.1⁺ T cells that is observed following SEB stimulation (SEB, 0.7%), is prevented by stimulation with the cognate antigen ovalbumin (OVA SEB, 13.2%). One of 6 representative experiments is shown.