Th2 Differentiation of Naive CD4+ T Cells

Naive sorted ABM TCR-Tg CD4⁺CD25⁻ T cells (Th0) were isolated (CD4⁺CD25⁻ isolation kit, Milteny Biotec, Auburn, CA), and activated for 5 days with 1 μg/ml of plate-bound anti-CD3 and anti-CD28 antibodies (BD Biosciences) in presence of the same number of isolated (CD19⁺ isolation kit, Milteny Biotec) BM12 CD19⁺ B-cells (which are specifically recognized by the ABM TCR-Tg T-cells). Cultures were supplemented with 5 ng/ml mIL-4 (R&D System, Minneapolis, MN) and 1 μg/ml anti-IFN-γ-Ig (BD Bioscience) for Th2 differentiation. T-cells were the harvested and assessed by RT-PCR (40 (link)) for GATA3 expression (Th2 specific marker) to quantify level of differentiation (43 (link)).

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Vergani A., Gatti F., Lee K.M., D’Addio F., Tezza S., Chin M., Bassi R., Tian Z., Wu E., Maffi P., Nasr M.B., Kim J.I., Secchi A., Markmann J.F., Rothstein D.M., Turka L.A., Sayegh M.H, & Fiorina P. (2014). TIM4 regulates the anti-islet Th2 alloimmune response. Cell transplantation, 24(8), 1599-1614.

Publication 2014

CD19+ isolation kit mIL-4

Anti antibodies Anti cd3 B cells Cd25 Cd4 cells Gata3 Ifn γ Isolation Rt pcr T cells

Corresponding Organization : IRCCS Ospedale San Raffaele

Other organizations : University of Salento, Massachusetts General Hospital, Dana-Farber Cancer Institute, North Dakota State University, Vita-Salute San Raffaele University, University of Pittsburgh

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Variable analysis

independent variables

Plate-bound anti-CD3 and anti-CD28 antibodies
Isolated BM12 CD19+ B-cells
MIL-4
Anti-IFN-γ-Ig

dependent variables

GATA3 expression (Th2 specific marker)

control variables

CD4+CD25- Naive sorted ABM TCR-Tg T cells (Th0)

controls

Positive control: Th2 differentiation conditions (IL-4 and anti-IFN-γ-Ig)
Negative control: Not explicitly mentioned

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