NK cells dramatically increase inside Ag104 L^d LIGHT tumor. (A) Intratumor NK cells dramatically increased at an early stage of tumor growth, CD8⁺ T cells increased at a late stage. Tumor tissues from Ag104 L^d- or Ag104L^d LIGHT-bearing mice were collected at days 10, 14, 18, and 22. Single cells were obtained by collagenase D digestion and stained with anti–NK1.1-conjugated PE and anti–CD8-conjugated APC. The lymphocyte area was gated for analysis. Data are representative of at least 3 independent experiments. (B) Dynamic change of increased infiltration of NK and CD8⁺ cells inside the tumor by immunohistochemical staining. Frozen sections of the indicated tumor tissues were stained with NK-specific receptor marker anti-Ly49G2, anti-CD8, or together. Results shown represent 1 of 3 independent experiments. (C) NK cells distribute closely to CD8⁺ T cells inside Ag104L^d LIGHT tumor tissues. NK and CD8⁺ T cells separate at different zones in spleens of Ag104L^d LIGHT tumor. Data shown represent 1 of 3 independent experiments. Images were acquired through an Olympus BX41 microscope equipped with a 20 ×/0.50 objective lens and a 10 ×/0.22 eyepiece (Olympus America, Melville, NY), for a total original magnification of × 200. An Axiocam camera and Axiovision 3.0 software (Carl Zeiss Microimaging, Thornwood, NY) were used to capture and process images.