The activation, by antigen, of naïve TCR transgenic CD4 T cells cultured at physiological, rather than artificially high, frequencies more accurately reflects the in vivo activation of normal numbers of naïve CD4+ T cells

The majority of in vitro studies investigating the activation of naïve TCR transgenic T cells routinely employ an artificially high frequency of such cells. To assess whether employing high frequencies of TCR transgenic cells in vitro accurately reflects the in vivo activation of a normal number of T cells, we cultured between 300 and 3 × 106 Rag2−/− DO11.10 T cells per well under otherwise identical conditions. We find that those T cells cultured at low frequencies proliferate more and are more potently activated, as assessed by the expression of CD44 and CD62L, each giving rise to a much larger number of cytokine producing cells, comparable to the number generated in vivo when a normal number of CD4+ T cells are activated. The effect of T cell frequency on the level of their activation was not due to differences in MHCII or CD80/86 expression by B cells, the major APC population present, nor to increased death of B cells in high frequency cultures. Taken together, our observations illustrate the necessity of culturing naïve TCR transgenic CD4+ T cells at a physiological frequency if one is to more accurately recapitulate the in vivo activation of naïve CD4+ T cells.

► We culture 300–3 × 106 Rag2−/− DO11.10 CD4+ T cells with APC and peptide antigen. ► The overall level of T cell activation decreases with increasing T cell frequency. ► This decrease is not due to any observed differences at the APC level. ► This decrease is likely due to increased intraclonal competition.