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Effector Memory CD4 T cells engage the innate immune system through CD40 and TNFR to trigger cytokine storm and autoimmune pathology II

ABSTRACT: Introduction: DCs sense and respond to a variety of pathogen-dependent and independent features. We found that DCs can directly sense the presence of immunological memory in the form of effector memory T cells via TNFRSF signaling. Expression of CD40L and TNF by effector memory T cells specifically led to a robust inflammatory signature in interacting DCs. Method: Bone marrow derived DCs (BMDCs) were differentiated in the presence of GMCSF for 7 days. WT naïve T cells were polarized to Th0 (platebound anti-CD3 and anti-CD28 and soluble IL-2) for 5 days, followed by 2 days of rest in low IL-2 containing media to mimic generation of effector memory T cells. CD11c+ BMDCs were sorted via AutoMacs and activated with effector memory T cells (ratio of 1:4) in the presence or absence of soluble anti-CD3 (200ng/mL) for 3 hours. Cells were pretreated with blocking and neutralizing antibodies for CD40L and TNF (20ug/mL for 30 minutes) when indicated. Cells were blocked with FC block and stained with flourophores to distinguish DCs from T cells. DCs were FACS sorted and lysed in Trizol. Conclusion: Effector memory T cells activate interacting BMDCs via CD40 and TNF signaling. When these pathways are blocked, BMDCs fail to upregulate the transcriptional program associated with effector memory T cell induced activation.

ORGANISM(S): Mus musculus

PROVIDER: GSE184608 | GEO | 2021/11/30

REPOSITORIES: GEO

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Project description:Dendritic cells (DCs) are a special class of leukocytes able to activate both innate and adaptive immune responses. They interact with microbes through germline-encoded pattern-recognition receptors (PRRs), which recognize molecular patterns expressed by various microorganisms. Upon antigen binding, PRRs instruct DCs for the appropriate priming of natural killer cells, followed by specific T-cell responses. Once completed the effector phase, DCs reach the terminal differentiation stage and eventually die by apoptosis. We have observed that following lipopolysaccharide (LPS)-stimulation the initiation of the apoptotic pathway in DCs is due the activation of NFAT proteins. Indeed, LPS induces Src-family kinase and phospholipase C (PLC)Î³2 activation, influx of extracellular Ca2+ and calcineurin-dependent nuclear NFAT translocation. The initiation of this pathway is independent of TLR4 engagement, and dependent exclusively on CD14. According with this observation CD14-deficient DCs do not die following LPS stimulation. Nevertheless, CD14-deficient DC death following LPS activation can be restored by co-stimulating DCs with LPS and thapsigargin. Thapsigargin empties the intracellular calcium stores by blocking calcium pumping into the sarcoplasmic and endoplasmic reticulum and thereby activates plasma membrane calcium channels. This, in turn, allows an influx of calcium into the cytosol and NFAT activation. To identify the NFAT controlled apoptosis genes in LPS activated DCs we have performed a kinetic microarray analysis (0, 48 and 60 h) in conditions allowing or inhibiting NFAT activation. Four genes have been selected: Nur77, Gadd45g, Ddit3/Gadd153/Chop-10 and Tia1. To identify apoptosis genes selectively modulated by NFAT, a comparative kinetic (time points 0, 48 and 60 h) microarray analysis was performed in the following conditions: 1) wild type bone marrow derived DCs (wtBMDCs) stimulated with LPS; 2) CD14-deficient BMDCs stimulated with LPS; 3) wtBMDCs stimulated with LPS in presence of thapsigargin; 4) CD14-deficient BMDCs stimulated with LPS in presence of thapsigargin.

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Effector Memory CD4 T cells engage the innate immune system through CD40 and TNFR to trigger cytokine storm and autoimmune pathology II

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