Fig. 9
From: Increased yields and biological potency of knob-into-hole-based soluble MHC class II molecules
The KIH Fc affords increased biological potency and stabilizes “empty” MHCII. a Representative pMHCII tetramer/eGFP (TCR) FACS dot plots for Jurkat cells expressing hCD4 and an IGRP13–25/DR3-specific TCR or mCD4 and a BDC2.5mi/IAg7-specific TCR (negative control). b Representative pMHCII tetramer/eGFP (TCR) dot plots for the Jurkat cells in A, but stained with KIH-based tetramers lacking (left) or carrying a CT (right). c Introduction of a CT into KIH-based human pMHCII does not alter their reactivity with a MHCII conformational epitope-specific mAb, as measured by ELISA. Data correspond to mean ± SEM of triplicates. d Luciferase activity induced by NPs coated with c-jun/c-fos-based (‘conv’), CT IGRP13–25/DR3 pMHCs vs. NPs coated with non-CT, KIH-based IGRP13–25/DR3 coated at three different valencies on Jurkat cells co-expressing hCD4, an IGRP13–25/DR3-specific TCR and NFAT-luciferase. Data correspond to mean ± SEM of triplicates. e Luciferase activity induced by NPs coated with c-jun/c-fos-based/CT (‘conv’) or KIH-based/CT IGRP13–25/DR3 monomers vs. their non-CT counterparts on Jurkat cells co-expressing hCD4, an IGRP13–25/DR3-specific TCR and NFAT-luciferase. Data correspond to mean ± SEM of triplicates. f SDS-PAGE of CT leucine-zippered (1) or KIH-based (2) Gliadin62–72/DQB1*0201/DQA1*0501 monomers. βME, beta-mercaptoethanol. g Representative pMHCII tetramer/CD4 dot plots for Jurkat cells expressing hCD4 and an IGRP13–25/DR3-specific (top) or mCD4 and a BDC2.5mi/IAg7-specific TCR (bottom) stained with c-jun/c-fos-based (‘conv’) IGRP13–25/DR3 tetramer (peptide-linked; left) or tetramers generated using peptide-loaded empty DR3-KIH monomers (right). h Representative pMHCII tetramer/eGFP (TCR) plots for Jurkat cells expressing hCD4 and PDC-E2122–135/DRB4-specific TCR (top left), a PDC-E2249–262/DRB4-specific TCR (top right) or an IGRP13–25/DR3-specific TCR (bottom; negative control). i Signal amplification of KIH-based tetramer binding using anti-hFc. Human PBMCs (106) were spiked with cells from a hIGRP13–25/DR3-specific T-cell clone (top) or an irrelevant (PPI(76–90(88S)/DR4-specific) clone (104) (bottom). Cells were treated with Dasatinib (right) or left untreated (left) and then stained with PE-labeled tetramers and PE-labeled anti-IgG. Values on the plots correspond to the geometric mean fluorescence intensity for pMHC tetramer staining. The staining patterns shown in (a), (b), (g), and (h) are representative of at least two independent experiments. Source data for panels (c–e) are provided as a Source Data File
