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For unknow reasons, the melanocyte stem cell (McSC) system fails earlier than other adult stem cell populations1, which leads to hair greying in most humans and mice2,3. Current dogma states that McSCs are reserved in an undifferentiated state in the hair follicle niche, physically segregated from differentiated progeny that migrate away following cues of regenerative stimuli4–8. Here we show that most McSCs toggle between transit-amplifying and stem cell states for both self-renewal and generation of mature progeny, a mechanism fundamentally distinct from those of other self-renewing systems. Live imaging and single-cell RNA sequencing revealed that McSCs are mobile, translocating between hair follicle stem cell and transit-amplifying compartments where they reversibly enter distinct differentiation states governed by local microenvironmental cues (for example, WNT). Long-term lineage tracing demonstrated that the McSC system is maintained by reverted McSCs rather than by reserved stem cells inherently exempt from reversible changes. During ageing, there is accumulation of stranded McSCs that do not contribute to the regeneration of melanocyte progeny. These results identify a new model whereby dedifferentiation is integral to homeostatic stem cell maintenance and suggest that modulating McSC mobility may represent a new approach for the prevention of hair greying.

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PDF file, 768K, IHC analysis on frozen BT sections was performed using anti-BDCA2 (brown) together with anti-CD3 (green, a) or anti- cytokeratine (green, b) antibodies (x10)

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3+ T cells are typical Treg based on their CD4+CD25highCD127lowFOXP3+ phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP+ dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3+, and CD8+ T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem. [Cancer Res 2009;69(5):2000–9]

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PDF file, 67K, Identification of pDC and mDC in blood and tissues.

PDF file, 64K, Breast TUMSN, but not BCCSN, inhibit IFN-α production by CpGA- or Flu-activated pDC in a dose-dependent manner.

Supplementary Data from Regulatory T Cells Recruited through CCL22/CCR4 Are Selectively Activated in Lymphoid Infiltrates Surrounding Primary Breast Tumors and Lead to an Adverse Clinical Outcome

PDF file, 46K, Purified healthy pDC were cultured with IL-3 either alone or in combination with 25% of TUMSN for 40 h and then incubated for 5 days with allogeneic naive CD4+ T cells.

PDF file, 114K, Overall survival and relapse-free survival of patients according to the presence of CD123+ pDC in primary breast carcinoma.

PDF file - 296K, Figure S1: ICOS is mainly expressed by TA-Treg. Figure S2: pDC expanded Treg sustain suppressive activity. Figure S3: ICOS-L is highly expressed on R848-activated HD-pDC. Figure S4: ICOS blockade reduces Treg but not Tconv proliferation induced by pDC without impacting mDC/T cell activation. Figure S5: Strong IL-10 secretion in culture of BT single cell suspension. Figure S6: Correlation between TA-pDC and TA-Treg percentages in fresh primary BT and in retrospective IHC cohort

PDF file - 103K, Table S1: Clinical characteristic of the cohort of BT patients enrolled for TMA analysis (n=120) Table S2: clones of mAbs used for the multi-parametric flow cytometry analyses

PDF file, 77K, Correlations between tumor characteristics and TA(p/m)DC.