1. Arp2/3-mediated formation of nuclear actin networks is essential for CD4+ T cell effector functions.
Arp2/3-mediated formation of nuclear actin networks is essential for CD4+ T cell effector functions. (A and B) Presence of the Arp2/3 inhibitor CK-869 during 10 min of PMA/Iono stimulation inhibits nuclear actin network formation in primary human CD4+ T cells. (A) Representative images of primary human CD4+ T cells expressing nuc.lifeact.GFP. (B) Occurrence of nuclear F-actin relative to the PMA/Iono control (mean ± SD of cells from four different donors with 30 cells evaluated each per condition). (C) Primary human CD4+ T cells isolated from three healthy donors as depicted in fig. S7A were either left unstimulated or stimulated by PMA/Iono in the presence or absence of CK-869 for 24 hours. Shown are cytokine concentrations detected in the supernatants of the respective treatments that were potently blocked by the presence of CK-869 during T cell stimulation. n.d., not detected. (D) Schematic overview and experimental workflow of the in vivo analysis of T cell help. Transferred cells expressed transgenic BCR and TCR recognizing HEL and OVA antigens, respectively. (E) Cytokine production of murine CD4+ T cells expressing nuc.dnArp2.mCherry relative to mCherry control shown as log2 fold changes for cells from three animals. (F and G) Expression of nuc.dnArp2.mCherry in CD4+ T lymphocytes impairs production of antigen-specific antibodies. CD4+ T cells were transduced to express mCherry or nuc.dnArp2.mCherry, and mCherry-positive cells were adoptively transferred together with B cells into recipient mice, which were later immunized with HEL-OVA antigen (see legend to fig. S10 for details). At the indicated time points after HEL-OVA immunization, IgG and IgM antibody production was quantified (F and G). At day 12 or 14 after immunization, cells were isolated from the lymph node of recipient animals to determine the frequency of mCherry-positive cells among the lymphocyte population (H). Ex vivo proliferation assay of murine OT-II cells expressing mCherry or nuc.dnArp2.mCherry activated by coculture with SW-HEL B cells with different concentrations of HEL-OVA (I). Statistical significance relative to DMSO-treated (B) or mCherry (F) controls was assessed by one-sample t test (B) or Mann-Whitney test (F; mean ± SD from six mice per group and two independent experiments). Scale bar, 5 μm. Arrowheads indicate examples of nuclear F-actin filaments.
N. Tsopoulidis et al. Sci. Immunol. 2019;4:eaav1987
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