Large Ki-67⁺ tumor cells are present in lymph node sections and are found in close proximity to CD⁺ T cells. Confocal immunofluorescence microscopy of CLL lymph nodes. (A) View (original magnification ×200) of a CLL lymph node stained with Ki-67 and CD3. Ki-67⁺ cells were confirmed to be CLL cells using costaining with CD20 (data not shown). In this example, Ki-67⁺CD20⁺ cells had a patchy distribution. In areas of high Ki-67, staining with CD3 showed these areas also had a higher density of T cells. (Bi) View (original magnification ×630) of an area rich in proliferating CLL cells stained with CD20, Ki-67, and CD3. CD3⁺ cells are shown to be in intimate contact with Ki-67⁺ tumor cells. (Bii) View (original magnification ×400) of an area of lymph node stained with CD20 (green), CD3 (red), CD4 (green), and Ki-67 (blue). The CD3⁺ cells adjacent to CD20⁺Ki-67⁺ tumor cells show colocalization with CD4 (yellow). In all sections examined, more than 80% of these CD3⁺ cells colocalized with CD4. (C) Quantitative assessment of the spatial relationship between Ki-67⁺ tumor cells and CD3⁺ cells in serial views of 4 separate lymph node specimens confirms that proliferating tumor cells are significantly more probable to be touching an adjacent T cell compared with Ki-67⁻ cells in all cases (χ² test).