Detection of T cells whose TCR is being engaged by MHCp in vivo. (A) Increased APA1/1 reactivity on encountering the antigen in vivo. Draining lymph nodes from OT-I mice injected subcutaneously with 400 μg OVA or vehicle alone (PBS) were collected 6 hours after injection, fixed, and immunostained with both the anti-CD3 antibody 145-2C11 and APA1/1. Low-power magnification (5 ×) of the whole lymph node is shown. Total MFIs for green and red channels in 3 experiments were 67 318.5 ± 6824 (CD3, PBS), 74 371 ± 1839 (CD3, OVA), 25 011 ± 6237 (APA1/1, PBS), and 74 223 + 9658 (APA1/1, OVA). (B) APA1/1 staining is concentrated at the IS. High-power magnification (100 ×) of the lymph node from OVA-injected mice shown in panel A, illustrating that APA1/1 reactivity concentrates at the T-cell/APC contact sites (arrows). T cells are labeled with anti-CD3ζ in blue, APA1/1 in red, and APCs bearing the CD11c marker (DCs) in green. Higher magnifications of areas rich in APA1/1-positive synapses (white square insets) are shown to the right. The white circle inset denotes a T-cell-rich area (stained with anti-CD3ζ) in which there are no APA1/1-positive synapses. In 81% of the APA1/1⁺ cells, the APA1/1 epitope was exposed at a T-cell/APC contact site where the APC bears the H-2K^b/OVA complex (n = 199). Although 36% of the lymph node T cells were estimated to express the OT-I TCR by flow cytometry, 21% of the OVA-exposed lymph node T cells were APA1/1 positive (n = 369). Images were acquired with a Zeiss Radiance 2000 microscope using a 63×/1.4 oil Plan-Apochromatic lens.