FIG 1 .

Evidence of increased IAV/EBV CD8 T-cell cross-reactivity in PBMCs of severe-AIM patients by tetramer costaining directly ex vivo. (a) One way to determine the frequency of cross-reactive CD8 T cells in peripheral blood sorted CD8 T cells is costaining of cells with different tetramer pairs as shown in this representative example, which shows that during the acute phase of AIM at visit 1 (v1), a severe-AIM donor (E-1382) has more IAV-M1+EBV-BM tetramer⁺ CD8 T cells than a mild-AIM donor (E-1392) and HD-SP (D-002). (b) This same severe-AIM patient at v2 showed a competitive reaction and mutual tetramer blocking of IAV-M1 and EBV-BM tetramer binding upon costaining with these two tetramers. Costaining with IAV-M1 and EBV-BR tetramers did not result in double-tetramer-positive cells, but the level of IAV-M1 tetramer⁺ cells declined compared to that of IAV-M1 single-tetramer-staining cells. There was no blockade upon costaining with EBV-BM and EBV-BR or IAV-M1 and control tyrosinase_369–377 tetramers. In these studies, the exact same tetramers were used for the single-color control and double-tetramer staining. The values in parentheses are the MFIs of the populations indicated. (c) Significant IAV-M1-specific tetramer blockade by EBV-BM-specific tetramer and EBV-BR-specific tetramer was detected ex vivo in severe-AIM patients (n = 11) compared to mild-AIM patients (n = 7). On the x axis, the red text indicates the tetramer-specific response that was being blocked by the other tetramer. Below the line is the double-tetramer combination used for costaining. The estimated blocking level for each tetramer was calculated by the formula [(costained tetramer A MFI/alone tetramer A MFI) × (costained tetramer A%/alone tetramer A%)] × 100. The gating strategies used are shown in Fig. S1.