Tumor-infiltrating immune cells in pA20-36– or pCNT-treated A20 B lymphoma–engrafted mice. (A) The pA20-36 treatment affects the number of tumor-infiltrating CD4⁺, CD8⁺ T, and Treg cells. Cell suspensions were prepared from tumor tissues at day 14 after subcutaneously tumor injection and analyzed by flow cytometry on staining with specific antibodies. Values are the mean ± SD (n = 4/group); statistical analysis was performed by Student t test. (B) Tumor B220⁺ cells show an increased expression of the activation markers on pA20-36 treatment. Expression of activation markers in CD11c⁺ (top), CD11b⁺ (middle), and B220⁺ (bottom) antigen-presenting cells derived from tumor masses of pA20-36– or pCNT-treated A20 B lymphoma–engrafted mice as assessed by flow cytometry. Histograms are representative of sample from 5 mice with similar staining profiles. (C) Tumor-infiltrating CD8⁺ T cells from pA20-36–treated mice show an activated phenotype. Expression of activation markers in CD4⁺ (top) and CD8⁺ (bottom) T cells from tumor masses of pA20-36– or pCNT-treated A20 B lymphoma–engrafted mice as assessed by flow cytometry. Histograms are representative of sample from 5 mice with similar staining profiles. (D) Granzyme-positive CD8⁺ T cells infiltrating tumor masses of pA20-36– or pCNT-treated A20 B lymphoma–engrafted mice. Sections of fresh tumor tissues were incubated with antibody to CD8, anti–granzyme B, and with TOPO-3. Pictures were captured with a Leica TCS SP2 confocal microscope with a HCX PL APO 63.0×/1.40 oil UV objective (NA 1.40) in glycerol and acquired with Leica Confocal Software Version 2.61. Image manipulation was performed with Adobe Photoshop CS. Scale bar = 14.2 μm. CTLA4 indicates cytotoxic T-lymphocyte–associated antigen 4.