Journal: Clinical and Experimental Immunology

Article Title: Monocytes show immunoregulatory capacity on CD4 + T cells in a human in‐vitro model of extracorporeal photopheresis

doi: 10.1111/cei.13232

Figure Lengend Snippet: Percentages of regulatory T cells (Tregs), pro‐ and anti‐inflammatory T cell subsets after co‐culture with ECP‐treated monocytes of healthy donors. (a) In‐vitro ECP‐treated or untreated monocytes of healthy donors were co‐cultured with autologous CD4+ T cells with anti‐CD3 antibody (100 ng/ml) at a monocyte : T cell ratio of 1 : 4 for 5 days. Induction of CD3+CD4+CD25+forkhead box protein 3 (FoxP3)+ Tregs (n = 8), proinflammatory CD3+CD4+interferon (IFN)‐γ+ T helper type 1 (Th1) cells (n = 27), CD3+CD4+interleukin (IL)‐17A+ Th17 cells (n = 27), CD3+CD4+IL‐17A+IFN‐γ+ Th17/Th1 cells (n = 27), CD3+CD4+IL‐2+ T cells (n = 19) and anti‐inflammatory CD3+CD4+IL4+ Th2 cells (n = 18) was assessed by intracellular flow cytometry analysis. (b–f) Necessity of cell‐contact or cytokine environment for changes in T cell subsets was analysed by incubation of ECP untreated or treated monocytes for 24 h and subsequent addition of either monocytes and 100 ng/ml anti‐CD3 antibody (cell‐contact) or supernatant of monocytes, 100 ng/ml anti‐CD3 antibody and 1 μg/ml anti‐CD28 antibody (supernatant) to CD4+ T cells (n = 7 or n = 5). Co‐cultures were investigated after 5 days. ECP untreated or treated monocytes directly co‐cultured with anti‐CD3 antibody and CD4+ T cells were used as reference (reference). ****P < 0·0001, **P < 0·01, *P < 0·05.

Article Snippet: CD4 + T cells and CD14 + CD16 + monocytes were isolated by magnetic cell isolation (MACS) using CD4 microbeads and the Pan Monocyte Isolation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Co-Culture Assay, In Vitro, Cell Culture, Flow Cytometry, Incubation

Journal: Clinical and Experimental Immunology

Article Title: Monocytes show immunoregulatory capacity on CD4 + T cells in a human in‐vitro model of extracorporeal photopheresis

doi: 10.1111/cei.13232

Figure Lengend Snippet: Influence of in‐vitro ECP‐treated monocytes on autologous T cell proliferation. ECP untreated or treated monocytes were co‐cultured with CD4+ T cells from the same donor with anti‐CD3 antibody (100 ng/ml) for 5 days after in‐vitro ECP. (a,b) Proliferation rates of CFSE‐labelled CD3+CD4+ T cells were assessed via flow cytometry (histogram shows one donor of 31, n = 31). (c) Necessity of cell‐contact or cytokine environment for changes in proliferation was analysed (n = 4). Therefore, ECP untreated or treated monocytes were cultured for 24 h exclusively. In cell‐contact‐dependent approaches freshly isolated CD4+ T cells were cultured with precultured monocytes at a monocyte : T cell ratio of 1 : 4 in the presence of 100 ng/ml anti‐CD3 antibody for 5 days (cell‐contact). For cytokine‐mediated approaches supernatant of precultured monocytes were added to freshly isolated CD4+ T cells with 100 ng/ml anti‐CD3 antibody and 1 μg/ml anti‐CD28 antibody (supernatant). Approaches where ECP untreated or treated monocytes were directly co‐cultured with 100 ng/ml soluble anti‐CD3 antibody and CD4+ T cells were used as reference (reference). Co‐cultures were investigated after 5 days. ****P < 0·0001, **P < 0·01.

Article Snippet: CD4 + T cells and CD14 + CD16 + monocytes were isolated by magnetic cell isolation (MACS) using CD4 microbeads and the Pan Monocyte Isolation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: In Vitro, Cell Culture, Flow Cytometry, Isolation

Journal: Clinical and Experimental Immunology

Article Title: Monocytes show immunoregulatory capacity on CD4 + T cells in a human in‐vitro model of extracorporeal photopheresis

doi: 10.1111/cei.13232

Figure Lengend Snippet: Comparison of T cell subsets and proliferation rates after ex‐vivo and in‐vitro ECP of patient samples. The samples were collected from the apheresis bag during ECP procedure before addition of UVADEX® (ECP–, ex vivo) and after ultraviolet A (UVA) irradiation (ECP+, ex vivo). Monocytes from patients were isolated, co‐cultured with freshly isolated CD4+ T cells from healthy donors at a monocyte : T cell ratio of 1 : 4 with 100 ng/ml anti‐CD3 antibody and analysed 5 days after ECP. Furthermore, monocytes from patient samples before addition of UVADEX® were isolated and treated with the ECP in‐vitro model (ECP– and ECP+, in vitro). Afterwards, monocytes were co‐cultured with CD4+ T cells from healthy donors and anti‐CD3 antibody (100 ng/ml) and investigated after 5 days. Induction of CD3+CD4+CD25+forkhead box protein 3 (FoxP3)+ regulatory T cells (Tregs) (n = 4*), proinflammatory CD3+CD4+interferon (IFN)‐γ+ T helper type 1 (Th1) cells (n = 6*), CD3+CD4+interleukin (IL)‐17A+ Th17 cells (n = 6*), CD3+CD4+IL‐17A+IFN‐γ+ Th17/Th1 cells (n = 6*), CD3+CD4+IL‐2+ T cells (n = 5*) and anti‐inflammatory CD3+CD4+IL4+ Th2 cells (n = 5*) in approaches with (a) ex‐vivo‐ and (b) in‐vitro‐treated monocytes was assessed by intracellular flow cytometry analysis. (c) Determination of T cell proliferation rates after co‐culture with ex‐vivo‐ and in‐vitro‐treated monocytes via CFSE labelling and flow cytometry analysis (n = 3). *One of 4 or 6 data points after ex‐vivo ECP treatment is missing due to coagulation of sample material. **P < 0·01, *P < 0·05.

Article Snippet: CD4 + T cells and CD14 + CD16 + monocytes were isolated by magnetic cell isolation (MACS) using CD4 microbeads and the Pan Monocyte Isolation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Comparison, Ex Vivo, In Vitro, Irradiation, Isolation, Cell Culture, Flow Cytometry, Co-Culture Assay, Coagulation

Journal: Clinical and Experimental Immunology

Article Title: Monocytes show immunoregulatory capacity on CD4 + T cells in a human in‐vitro model of extracorporeal photopheresis

doi: 10.1111/cei.13232

Figure Lengend Snippet: Characterization of in‐vitro ECP‐treated monocytes. ECP untreated or treated monocytes were cultured after in‐vitro ECP for 2 days and (a) absolute cell counts referred to originally seeded cell numbers directly after ECP treatment (day 0) were determined (n = 9). (b) Determination of proportions of living, early apoptotic, late apoptotic and dead cells was performed by calcium‐dependent staining of ECP untreated or treated monocytes with annexin V after 2 days and successive flow cytometry analysis (n = 6). (c) Moreover, ECP untreated or treated monocytes were co‐cultured with CFSE‐labelled CD4+ T cells from the same donor in different monocyte : T cell ratios with anti‐CD3 antibody (100 ng/ml) for 5 days after in‐vitro ECP and proliferation rates of CD3+CD4+ T cells were assessed via flow cytometry (n = 3–5). Induced percentages of (d) CD14+CD209‐human leucocyte antigen D‐related (HLA‐DR)+CD86+ monocytes and (e) CD14+CD209+HLA‐DR+CD86+ macrophages (n = 15) as well as (f) HLA‐DR‐CD33+CD14+monocytic myeloid derived suppressor cells (M‐MDSCs) (n = 16) were assessed via flow cytometry analysis 2 days after in‐vitro ECP. ****P < 0·0001, **P < 0·01, *P < 0·05.

Article Snippet: CD4 + T cells and CD14 + CD16 + monocytes were isolated by magnetic cell isolation (MACS) using CD4 microbeads and the Pan Monocyte Isolation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: In Vitro, Cell Culture, Staining, Flow Cytometry, Derivative Assay

Journal: Clinical and Experimental Immunology

Article Title: Monocytes show immunoregulatory capacity on CD4 + T cells in a human in‐vitro model of extracorporeal photopheresis

doi: 10.1111/cei.13232

Figure Lengend Snippet: Investigation of the connection between programmed cell death ligand 1 (PD‐L1) and PD‐L2 and T cell proliferation. ECP untreated or treated monocytes were cultured for 2 days and proportions of living (a) CD14+PD‐L1+ and (b) CD14+PD‐L2+ monocytes were determined via flow cytometry. Furthermore, ECP untreated or treated monocytes were co‐cultured with CD4+ T cells from the same donor with anti‐CD3 antibody (100 ng/ml) for 5 days after in‐vitro ECP and proportions of living (c) CD3+CD4+PD‐L1+ and (d) CD3+CD4+PD‐2+ monocytes were determined via flow cytometry. (e) To block PD‐L1‐PD‐1 and PD‐L2‐PD‐1 interactions 25 µg/ml of pembrolizumab, anti‐PD‐L1 or anti‐PD‐L2 antibody was added to co‐cultures of either untreated or ECP‐treated monocytes with CFSE‐labelled CD4+ T cells from the same donor with anti‐CD3 antibody (100 ng/ml) stimulation for 5 days after in‐vitro ECP. Proliferation rates of CD3+CD4+ T cells were assessed via flow cytometry. **P < 0·01, *P < 0·05.

Article Snippet: CD4 + T cells and CD14 + CD16 + monocytes were isolated by magnetic cell isolation (MACS) using CD4 microbeads and the Pan Monocyte Isolation Kit, according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Cell Culture, Flow Cytometry, In Vitro, Blocking Assay