Figure 1.

CD4^IL-10 Cells Kill Myeloid Cell Lines In Vitro

(A) Degranulation of CD4^IL-10 cells. CD4^IL-10 and CD4^ΔNGFR cells were co-cultured with CD3, CD14, U937, BV-173, Daudi, K562, and ALL-CM target cells at a 5:1 (E:T) ratio for 6 hr, and the frequency of CD107a⁺GzB⁺ cells was measured by FACS. One representative donor (left panels) and mean ± SEM of n = 20 for CD4^IL-10 and CD4^ΔNGFR cells cultured alone or with ALL-CM or U937 cell lines, n = 4 for CD4^IL-10 cells and n = 2 for CD4^ΔNGFR cells co-cultured with CD3⁺ cells, n = 8 for CD4^IL-10 and CD4^ΔNGFR cells co-cultured with CD14⁺ cells, n = 5 for CD4^IL-10 and CD4^ΔNGFR cells co-cultured with the BV-173 cell line, n = 12 for CD4^IL-10 and CD4^ΔNGFR cells co-cultured with the Daudi cell line, and n = 17 for CD4^IL-10 cells and CD4^ΔNGFR cells co-cultured with the K562 cell line (right panels) are reported. p, Mann-Whitney test. (B) Cytotoxicity of CD4^IL-10 cells. Cytotoxicity against U937, BV-173, Daudi, K562, and ALL-CM cell lines was determined by ⁵¹Cr-release assay. Mean ± SEM of cytotoxicity performed in duplicated is reported; n = 4 independent donors for CD4^IL-10 and CD4^ΔNGFR cells against ALL-CM and U937 cell lines, tested in two independent experiments, and n = 2 for CD4^IL-10 and CD4^ΔNGFR cells against BV-173, Daudi, and K562 cell lines tested in one experiment. p, two-sided Mann-Whitney test. (C) CD4^IL-10 cells killed myeloid cell lines. CD4^IL-10 and CD4^ΔNGFR cells were co-cultured with CD14, U937, BV-173, K562, THP-1, and ALL-CM cells at a 1:1 ratio for 3 days. Residual leukemic cell lines (CD45^lowCD3⁻) were counted by FACS, and cytolysis mediated by CD4^IL-10 cells was measured as elimination index (see Materials and Methods) for each target cells. Analysis was performed in five independent experiments. Dots represent the elimination index of CD4^IL-10 cells generated from different healthy donors, and lines represent mean values of the elimination index. Gray area indicates the threshold of cytotoxicity.