Severe-AIM patients have the greatest expansion of IAV/EBV cross-reactive CD8 T cells in short-term in vitro cultures. Examination of functional cross-reactivity in the same samples as in Fig. 5 by gating on the cognate tetramer + cells in each culture and showing an overlay of IFN-γ or MIP-1β histogram values for each peptide pulse also demonstrates that the severe-AIM patient (i) had the greatest number of functional cross-reactive responses to IAV-M1 and EBV-BM or EBV-BR responses but not other peptides compared to those of a mild-AIM patient (ii) and an HD-SP (iii). A cognate peptide pulse can result in such strong ligation of the TCR that it downregulates the TCR and thus tetramer binding is hampered. IAV-M1-, EBV-BM-, and EBV-BR-specific short-term in vitro cultures generated from sorted CD8 T cells of representative severe-AIM (E-1302) (i) and mild-AIM (E-1392) (ii) patients and HD-SP (D002) (iii) were costained with cognate (same as the culture-stimulating peptide) tetramer and pulsed with cognate, cross-reactive, and control peptides IFN-γ and MIP-1β production was determined. Download FIG S2b, TIF file, 44.7 MB.Ī severe-AIM patient had enhanced functionally cross-reactive CD8 T-cell responses in a short-term in vitro culture compared to those of a mild-AIM patient or HD-SP, as shown in histograms. Red indicates blocked tetramers, and blue indicates blocking tetramers. These data indicate that BR tetramer blocked cross-reactive M1 tetramer binding. However, in the presence of BR costaining, cross-reactive M1 tetramer + cells declined to 14.3% with a 16.5-fold decline in MFI compared to a frequency of 24% with single M1 tetramer or costaining with a tyrosinase-specific tetramer. (iii) In the BR-stimulated culture, there was an outgrowth of cross-reactive M1 cells with double M1 + BR + tetramer + cells at 2.3%. Costaining with M1 and BM tetramers did result in 0.16% double tetramer + cells, and BM tetramer + cells declined to a total of 0.66% compared to 1% with single BM tetramer or costaining with a tyrosinase-specific tetramer. (ii) Culturing of CD8 T cells with M1 peptide promoted the growth of a smaller population of cross-reactive BM-specific cells. This indicates that the M1 tetramer was blocking BM tetramer binding on the cross-reactive cells. There was no blocking of the cross-reactive M1 tetramer binding by BM tetramer. However, upon costaining with M1- and BM-specific tetramers, the total BM tetramer + cell percentage declined to 54% and the MFI dropped 11-fold compared to 60% with single BM tetramer or in the presence of tyrosinase-specific tetramer. (i) Culturing of CD8 T cells with BM peptide resulted in the proliferation of cross-reactive IAV-M1-specific cells (14%) in a severe-AIM patient (E-1325) at visit 8. Representative examples of costaining with two tetramers simultaneously showing blocking of tetramer binding when CD8 T-cell cross-reactivity was present in short-term-cultured cells. (iii) In a severe-AIM patient (E-1382) later during the acute phase of infection (visit 5), we observed different blocking patterns upon costaining with two tetramers compared to visit 2 staining, suggesting that the cross-reactive TcR repertoires were evolving over time. There was no blockade between EBV-lytic epitope-specific tetramers. Also, in the presence of BR tetramer, the total M1 tetramer + cell level declined to 0.13% compared to 0.24% in the presence of a tyrosinase-specific tetramer. BM tetramer + cells declined to 2.59% in the presence of M1 tetramer from 3.66% when BM tetramer was used alone. M1 tetramer + cells declined to 0.08% compared to 0.25% in the presence of M1 tetramer alone or a tyrosinase-specific tetramer. (ii) In a severe-AIM patient (E-1382), costaining of CD8 T cells directly ex vivo with M1 and BM tetramers showed a mutual blockade. There was also a blockade of the two EBV-specific tetramers, BM and BR. A similar blockade of M1 tetramer was observed in the presence of BR tetramer costaining. However, in the presence of BM tetramer, M1 tetramer + cells declined to 0.06% compared to 0.25% in the presence of single M1 tetramer or the non-cross-reactive control tyrosinase-specific tetramer. (i) Costaining of freshly isolated CD8 T cells derived from a mild-AIM patient (E-1303) with M1 and BM tetramers showed 0.02% double tetramer + cells. Representative examples of costaining with two tetramers simultaneously, resulting in blocking of tetramer binding when CD8 T-cell cross-reactivity is present directly ex vivo.
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