Figure 2

Polyreactive antibodies bind primarily to late apoptotic cells and to multiple sites on and within apoptotic T cells.

(a) Human T cells exposed to UV light were sorted into live (Annexin V⁻/PI⁻), early (Annexin V⁺/PI⁻) and late (Annexin V⁺/PI⁺) apoptotic populations. The binding profile of monoreactive 8512 and polyreactive 2E4, ZH-6 and ZH-20 antibodies showed that the polyreactive antibodies bound primarily to late apoptotic cells. (b) UV-induced late apoptotic human T cells were incubated with polyreactive 2E4 and then stained with FITC-labeled antibody to CD3 (green) to identify the plasma membrane and Draq 5 (blue) to identify the nucleus. PE-labeled anti-immunoglobulin (red) was used to co-localize polyreactive 2E4 with these structures based upon bright field similarity analysis by ImageStream. 2E4/Draq 5, 2E4/CD3 and the 2E4/Draq 5/CD3 co-localization was quantified based on the fraction of each population present in a total of 10,000 cell images obtained from each of the samples collected. Representative images show co-localization (merge) of 2E4 with nucleus (Draq 5: row 1); 2E4 with plasma membrane (CD3: row 2); and 2E4 with cytoplasm, nucleus and plasma membrane (Draq 5 and CD3: row 3). Controls show staining in the absence of 2E4 (row 4). Experiments (panels a and b) were repeated at least three times and each time with T cells from different donors.