CCR6⁺CD25⁺ T cells are generated in vivo after activation of CCR6^-CD4⁺ T cells. FACS analysis is shown for CFDA-labeled CD4⁺ T cells. CD4⁺ T cells were isolated from TCR transgenic mice and depleted of CCR6⁺ cells by MACS. The CCR6^-CD4⁺ cells were then labeled with CFDA and adoptively transferred into syngenic hosts. Two different model systems were used, based either on the recognition of MBP Ac1-11 (donor, TG4; recipient, B10.PL; A-C) or of OVA323-339 (donor, DO11.10; recipient, BALB/c; D-E). T cells were activated by priming with the respective peptide antigen and lymph node cells were analyzed 9 days later by FACS. (A) Antigen-specific induction of proliferation by TG4 T cells. Dot plots of the CFDA staining versus side scatter (SSC) are shown for lymph node cells of B10.PL mice that received CFDA-labeled CCR6^-CD4⁺ T cells from TG4 mice. The cells were analyzed 9 days after transfer and derived either from primed mice (right panel) or from unprimed control mice (left panel). (B) Induction of CCR6 expression. Dot plots are gated on CD4⁺ cells and exclude all CFDA^- cells so that they represent only transferred TG4 T cells. Staining of CD4 versus CCR6 is shown for cells isolated on day 9 from primed mice (right panel) or from nonprimed control mice (left panel). Numbers represent the percentages of CCR6⁺ and CCR6^- cells of the respective populations. (C) Delayed expression of CCR6. Lymph node cells isolated on day 9 from peptide primed mice were stained with αCD4, αCD25, and αCCR6. The dot plots show CFDA⁺ CD4⁺ cells after staining with an isotype control or antibodies specific for CCR6, CD5, or CD54. Staining is shown for the population gated on CD4⁺CD25^- (left panels) and CD4⁺CD25⁺ (right panels). Vertical lines separate daughter generations of dividing cells. One of 4 independent experiments is shown. (D) Induction of CCR6 expression in adoptively transferred DO11.10 T cells. Cells were isolated 9 days after the priming of BALB/c mice that previously received CFDA-labeled CCR6^-CD4⁺ T cells from DO11.10. Staining is shown for CFDA versus isotype control (upper panels) or CCR6 (lower panels). Dot plots represent the CFDA⁺ populations gated on CD25^-CD4⁺ cells (left panels) and on CD25⁺CD4⁺ cells (right panels). (E) Fraction of CCR6⁺ cells in dividing T cells after in vivo priming. The plot represents the relative fraction of CCR6⁺ T cells in the daughter generations of dividing CD25^-CD4⁺ T cells (○) and CD25⁺CD4⁺ T cells (•). The plot is generated from the data shown in panel D; dashed lines represent trend lines. One of 2 independent experiments is shown. More than 10⁶ CD4⁺ T cells were analyzed in each experiment.