Transforming Growth Factor-β1 Regulates the Expression of the High-Affinity Receptor for IgE on CD34+ Stem Cell-Derived CD1a+ Dendritic Cells In Vitro

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Transforming Growth Factor-β1 Regulates the Expression of the High-Affinity Receptor for IgE on CD34+ Stem Cell-Derived CD1a+ Dendritic Cells In Vitro Jean-Pierre Allam, Elisabeth Klein, Thomas Bieber, Natalija Novak Journal of Investigative Dermatology Volume 123, Issue 4, Pages 676-682 (October 2004) DOI: 10.1111/j.0022-202X.2004.23428.x Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 TGF-β1 decelerates cell proliferation and sustains cell immaturity. (A) The highest cell yield was detected in control culture (CON). TGF-β1low and TGF-β1high decreased total cell proliferation with the lowest cell yield under high TGF-β1high (n=26). (B) In contrast to control culture (CON), CD34pos stem cells were still detectable on day 9 in cultures with TGF-β1low and TGF-β1high (n=11). Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 FcεRI expression of CD34-derived CD1a+ DC during differentiation and on day 9. (A) FcεRI regularly appears on differentiating CD34-derived CD1a+ DC under all conditions peaking around day 5 and stabilizing under control condition (CON) and TGF-β1low (n=7). (B) Addition of IgE resulted in an increased FcεRI expression of CD34-derived CD1a+ DC under all culture conditions. On day 9, FcεRI could even be demonstrated on CD34-derived CD1a+ DC generated with TGF-β1high (n=5). Mean values±SEM of five experiments are shown in the upper right corner of the histogram including p values. Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Alteration of FcεRIα and FcεRIγ in response to different concentrations of TGF-β1. (A) Representative results of RT-PCR of highly enriched CD1a+ cells confirming the presence of FcεRIα and FcεRIγ (1=control condition, 2=TGF-β1low, 3=TGF-β1high, n=3) (B) Blocking endogenous TGF–β with an monoclonal anti-TGF-β antibody in control condition culture leads to a decreased surface FcεRI expression on CD34-derived CD1a+ DC on day 9 accompanied by a strong decrease of intracellular FcεRIα and FcεRIγ (n=7) Furthermore, control experiments revealed that supplementation of irrelevant IgG1 antibody did not alter surface expression of FcεRI (data not shown). (C) In contrast to CD34-derived CD1a+ DC generated under control culture (CON) and TGF-β1low, TGF-β1high downregulated FcεRI surface expression on CD34-derived CD1a+ DC by decreasing the intracellular pool of FcεRIγ. Intracellular FcεRIα was present under all conditions with the strongest detection in CD34-derived CD1a+ DC generated with TGF-β1low. Representative histograms of 12 experiments are shown. Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 High amounts of FcεRI+ LC were generated with TGF-β1low. The generation of CD34-derived CD1a+ DC and LC depended on exogenous TGF-β1 supplementation. The highest amounts of Langerin+ LC were generated under TGF-β1high followed by LC generated with TGF-β1low. Under control culture (CON) virtually no Langerin+ LC could be detected. A high percentage among the LC population expressed FcεRI especially in cultures with TGF-β1low (n=5). Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Phenotypical and morphological changes induced by TGF-β1. (A) Representative histograms of E-cadherin, Langerin, CD11b, CD14, CD206 and DC-SIGN on CD34-derived CD1a+ DC (n=5). (B) Representative phase contrast microscopic images of cells cultured with control condition, TGF-β1low and TGF-β1high and representative histograms co-stimulatory molecules and MHC class I and II complex, which were up-regulated through addition of exogenous TGF-β1 (TGF-β1low, TGF-β1high). Note the enhanced cluster formation in cultures supplemented with exogenous TGF-β. Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 TGF-β leads to enhanced allogeneic stimulatory capacity of CD34-derived CD1a+ DC. DC were co-cultured with autologous or allogeneic T cells for 5 d as described in Material and Methods. CD34-derived CD1a+ DC generated in the presence of exogenous TGF-β1 (TGF-β1low, TGF-β1high) lead to an increased stimulatory capacity towards allogeneic T cells (n=5). Journal of Investigative Dermatology 2004 123, 676-682DOI: (10.1111/j.0022-202X.2004.23428.x) Copyright © 2004 The Society for Investigative Dermatology, Inc Terms and Conditions