Monocytes, macrophages and dendritic cells (DCs) are developmentally related regulators of the immune system that share the monocyte-macrophage DC progenitor (MDP) as a common precursor. Unlike differentiation into DCs, the distal pathways for differentiation into monocytes and monocyte-derived macrophages are not fully elucidated. We have now demonstrated the existence of a clonogenic, monocyte- and macrophage-restricted progenitor cell derived from the MDP. This progenitor was a Ly6C(+) proliferating cell present in the bone marrow and spleen that generated the major monocyte subsets and macrophages, but not DCs or neutrophils. By in-depth quantitative proteomics, we characterized changes in the proteome during monocyte differentiation, which provided insight into the molecular principles of developing monocytes, such as their functional maturation. Thus, we found that monocytes and macrophages were renewed independently of DCs from a committed progenitor.

Regulatory T cells (Tregs) differentiate in the thymus, but the mechanisms that control this process are not fully understood. We generated a comprehensive quantitative and differential proteome of murine Tregs and conventional T cells. We identified 5225 proteins, 164 of which were differentially expressed in Tregs. Together with the comparative analysis of proteome and gene expression data, we identified TCF7 as a promising candidate. Genetic elimination of transcription factor 7 (TCF7) led to increased fractions of Tregs in the thymus. Reduced levels of TCF7, found in the heterozygote, resulted in a greater potential for Treg precursors to differentiate into the Treg lineage. In contrast, activation of TCF7 through β-catenin had the opposite effect. TCF7 levels influenced the required TCR signaling strength of Treg precursors, and TCF7 deficiency broadened the repertoire and allowed lower TCR affinities to be recruited into the Treg lineage. FOXP3 was able to repress TCF7 protein expression. In summary, we propose a regulatory role for TCF7 in limiting access to the Treg lineage.

Genetically encoded sensors based on intramolecular FRET between CFP and YFP are used extensively in cell biology research. Flow cytometry has been shown to offer a means to measure CFP-YFP FRET; we suspected it would provide a unique way to conduct multiplexed measurements from cells expressing different FRET sensors, which is difficult to do with microscopy, and that this could be used for screening. We confirmed that flow cytometry accurately measures FRET signals using cells transiently transfected with an ERK activity reporter, comparing responses measured with imaging and cytometry. We created polyclonal long-term transfectant lines, each expressing a different intramolecular FRET sensor, and devised a way to bar-code four distinct populations of cells. We demonstrated the feasibility of multiplexed measurements and determined that robust multiplexed measurements can be conducted in plate format. To validate the suitability of the method for screening, we measured responses from a plate of bacterial extracts that in unrelated experiments we had determined contained the protein kinase C (PKC)-activating compound teleocidin A-1. The multiplexed assay correctly identifying the teleocidin A-1-containing well. We propose that multiplexed cytometric FRET measurements will be useful for analyzing cellular function and for screening compound collections.

Quantitative proteomics identifies proteins bound to the Foxp3 gene promoter Promoter-binding proteins are suppressing Foxp3 expression TCF1-deficient animals have more Foxp3expressing CTLA4 À CD25 À CD4 + T cells TCF1 suppresses Foxp3 expression in activated non-T reg cells

Peripheral immune regulation depends on the generation of thymic-derived regulatory T (tTreg) cells to maintain self-tolerance and to counterbalance overshooting immune responses. The expression of the Treg lineage defining transcription factor Foxp3 in developing tTreg cells depends on TCR signaling during the thymic selection process of these T cells. In this study, we surprisingly identify Foxp3+ immature thymocytes at the double-negative (DN) stage in transcription factor 7 (Tcf7)-deficient mice. These Foxp3+ cells did not express a TCR (β or γδ chains), CD3 or CD5 and therefore these cells were true DN cells. Further investigation of this phenomenon in a transgenic TCR model showed that Foxp3-expressing DN cells could not respond to TCR stimulation in vivo. These data suggest that Foxp3 expression in these DN cells occurred independently of TCR signaling. Interestingly, these Foxp3+ DN cells were located in a transition state between DN1 and DN2 (CD4-CD8-CD3-TCR-CD44highCD25low). Our results indicate that Tcf7 is involved in preventing the premature expression of Foxp3 in DN thymocytes.

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