Fig. 2.

Macrophages can stimulate T cells for at least 7 days in vitro. Timeline key: bold line, hours of co-culture of pOVA-loaded APC with naive CD4⁺ T cells; dashed line, T cell culture in the absence of pOVA-loaded macrophages; arrow, hour of reagent addition to culture. The total hours of T cell culture before harvest are indicated at the right end of the timeline. (A) Naive DO11.10 CD4⁺ T cells were co-cultured with pOVA (50 μg/ml)-loaded macrophages for 144 h (6 days). Then new CFSE-labeled DO11.10 T cells were added to the ongoing co-culture (bold histogram). In parallel, some of the new T cells were co-cultured with macrophages that had not been loaded with pOVA, nor previously cultured with T cells (thin histogram). After 24 h, CD69 expression on CFSE-labeled T cells was measured by FCM. (B) Unstimulated naive DO11.10/RAG2^−/− CD4⁺ T cells (thin histogram) were either co-cultured with pOVA (50 μg/ml)-loaded APC for 96 h (dotted histograms), or co-cultured with pOVA (50 μg/ml)-loaded APC for 18 h, followed by continued culture in the presence of anti-I-A^d until 96 h (bold histograms). Surface clonotype TCR (KJ126) expression was measured by FCM.