MR1Ts recognized by the hpMR1+EC tetramer are more likely to be TRAV1-2−. MR1Ts recognized by the hpMR1+EC tetramer are more likely to be TRAV1-2−. PBMCs.

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MR1Ts recognized by the hpMR1+EC tetramer are more likely to be TRAV1-2−. MR1Ts recognized by the hpMR1+EC tetramer are more likely to be TRAV1-2−. PBMCs from 15 donors were stained as described in Fig. 1, and the population of tetramer+ cells was analyzed for phenotypic MAIT cell markers. (A) TRAV1-2 staining of tetramer+ cells for three representative donors. Black indicates TRAV1-2+ cells, and red indicates TRAV1-2− cells, with the gate defined by the population of CD3+CD4−TRAV1-2+CD161+ cells, as shown in fig. S2B. (B) The graphs depict the frequency of CD3+CD4− tetramer+TRAV1-2− cells for all 15 PBMC donors. Right: Frequencies of TRAV1-2− tetramer+ cells in left graphs have been paired for each donor. (C) Frequency of CD26+CD161+ cells among tetramer+ cells between the tetramers. (D and E) PBMCs from each donor were simultaneously stained in panels with or without the tetramer to analyze tetramer inhibition of TRAV1-2 staining. (D) Frequency of CD26+CD161+TRAV1-2+ cells for two representative donors. (E) Left: Tetramer inhibition of TRAV1-2 staining is calculated for all donors using the method in (D). Right: For each donor, the inhibition of TRAV1-2 staining by tetramer was used to calculate what proportion of the TRAV1-2− events depicted in (B) are not explained by tetramer inhibition of TRAV1-2 staining. (F) TRAV1-2− MR1T clones from D520 (E10, F1, F7, and G3) and a TRAV1-2+ MR1T clone (D481C7) were analyzed by flow cytometry for tetramer and TRAV1-2 staining. (G) MR1-dependent activation of TRAV1-2− MR1T clones by human DCs infected with M. smegmatis (Msm) was measured by IFN-γ ELISPOT assay using the anti-MR1 blocking antibody (aMR1 ab). (F and G) Results are representative of three independent experiments. Error bars represent means and SD from technical replicates. Melanie J. Harriff et al. Sci. Immunol. 2018;3:eaao2556 Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).