DR3.DQ2 mice were orally dosed with 10 mg/kg of HB-298 (a fluorescent analog of an immunodominant 33 mer gluten peptide harboring multiple epitopes recognized by HLA-DQ2) either 2 or 24 hours prior to sacrifice. After euthanasia, mesenteric lymph nodes (MLNs) and Peyer’s patches (PP) were collected, snap-frozen, and stained with antibodies. (A and B) MLN and PP from DR3.DQ2 mice following HB-298 administration were costained with antibodies targeting CD103 (magenta) and HLA-DQ2 (SPV-L3, green). White arrows indicate HB-298 labeling of DQ2⁺CD103⁺ DCs. A focal image showed HB-298 colocalized with HLA-DQ2 (shown in yellow) (A). Quantification of labeled cells from MLN and PP confirmed that, whereas the prevalence of HB-298–labeled cells remain unchanged in the MLN at the 24-hour time point, analogously labeled cells in the PP had migrated out of this compartment by 24 hours. (C and D) CD11c⁺ and CD11c^– cells isolated from the MLNs of DR3.DQ2 and TG2-KO mice were incubated with 2 μM of HB-230 or HB-298 for 90 minutes. (C) Thereafter, MLN cells from DR3.DQ2 mice were stained with antibodies against HLA-DQ2 (SPV-L3, green), while cells from TG2-KO mice were stained with antibodies against mouse MHCII (I-A/I-E, green). Cell-surface gluten peptide presentation (indicated by a yellow cell boundary) was noted in HB-298–treated CD11c⁺ cells from DR3.DQ2 mice but not in HB-230–treated cells or in TG2-KO mice. (D) HB-298–treated MLN cells from DR3.DQ2 mice were also stained with anti-CD103 (green, upper panel) or anti-DQ2 (SPV-L3, green, lower panel) antibodies in CD11c⁺ cells from MLNs of DR3.DQ2 mice. Here, too, strong antigen presentation by CD103⁺ DCs was noted. Representative images were captured using a confocal microscope at 40× magnification. Data represent mean ± SEM.