Immunohistochemical analysis and EM of tumors. Serial sections of representative tumors (n = 8) of Th1 cell–treated and control mice from day 18. (A) Tumors were stained with monoclonal anti-CD4 antibody (i,ii) showing a strong increase in CD4⁺ cells in tumors of Th1 cell–treated mice. indicates CD4⁺ cells. Staining with H&E (iii,iv) showed no major differences in the morphology of the tumors. Necrosis (N) was detected in tumors of Th1 cell–treated and PBS-treated mice. Ki-67 staining (v,vi) showed no obvious differences in the proliferation rate of the tumors. indicates Ki-67–positive cells. Pictures, ×100 magnification. Inlays, ×200 original magnification. (B) Quantitative analysis of (Av,vi). Ki-67⁺ cells of 500 total cells in tumor sections (n = 60) of Th1 cell–treated and PBS-treated mice were counted. Error bars denote SEM. (C) Tumors were stained with monoclonal anti–PECAM-1 antibody (i-iv) showing homogenous vascularization in both groups. However, minor changes in vessel morphology (white arrows) and a minor decrease of tumor vessel density were detectable in tumors of Th1 cell–treated mice (ii,iv) compared with control mice (i,iii). Staining of tumors with monoclonal anti–VEGFR-2 antibody (v,vi) revealed no obvious change in the receptor expression pattern between both groups. Pictures, ×200 original magnification. (D) EM of blood vessels. (i) Blood vessels with a bulged EC body (E), tight junctions (), and the basal lamina (labeled with *). (ii) Blood lacuna without limiting ECs, but with extracellular matrix–like collagen fibrils (*) that are located between tumor cells and blood cells. (iii) Blood vessel with leukocyte transendothelial migration. (iv) Higher magnification of panel iii, showing a transcellularly transmigrated leukocyte (L) still covered by a basal lamina (). (v) Obliterated vessel. The left arrow labels a tight junction; the right arrow, the rest of the lumen. (vi) Vessels in adjacent muscle of Th1 cell–treated tumors appear normal. In summary, blood vessels of PBS-treated (i,ii) tumors were seen as normal tumor vessels. In contrast, degenerated ECs were observed more frequently in tumors of Th1 cell–treated mice (iii-v). In addition, strong inflammatory processes (iii,iv) and more obliterated vessels (v) were seen in tumors of Th1 cell–treated mice. Experiments were performed as described in “Methods.”