28 To analyze the dynamics of T cell recruitment and expansion at the single cell level during an in vivo response, we used CFSE, a nontoxic fluorochrome that is retained in cell membranes. After division, daughter cells fluoresce with only 1/2 the intensity of the parent cell. This permits discrimination of dividing and non-dividing cells, and quantification of the number of mitoses each cell has undergone (JCI, 100:3173). Ovalbumin TCR transgenic cells were labeled with CFSE, and 5×106 cells were adoptively transferred to congenic mice. Mice were immunized with OVA in CFA, treated with 1 dose of control Ig or CTLA4Ig, and sacrificed on days 1.5, 3 or 5. Cells were stained for OVA-TCR, CD4, and TOPRO-3 (a vital dye), and analyzed by FACS. T cell division was seen within 36 hours of immunization. Dividing cells were found in both regional and distant nodes. In regional nodes, we observed a 16-fold increase in transgenic cells. Analysis of CFSE levels showed that 60% of the recruited cells underwent cell division, indicating that a significant number of antigen specific cells were recruited, yet did not divide. Those which did proliferate, generated a mean of 21 daughter cells per dividing precursor. In distant nodes, a similar fraction (60%) divided, although the "strength" of the response was weaker, with only 4-fold overall expansion and 5 daughter cells per dividing precursor. Treatment with CTLA4Ig reduced the precursor frequency of proliferating cells from 60% to 25%, and reduced the mean number of daughter cells generated per precursor in regional and distant lymph nodes to 5.5 and 1.9 respectively.FigureThese data describe for the first time the quantitative proliferative characteristics of T cells during an in vivo immune response. Blockade of costimulation limits both the frequency of dividing cells and their proliferative potential. However, even under optimal conditions up to 40% of recruited antigen-reactive T cells fail to divide, indicating additional checkpoints upon T cell responses.