Transforming growth factor β (TGFβ) is an important differentiation factor for cytotoxic T lymphocytes (CTLs) and alters the expression levels of several of homing-receptors during infection. SMAD4 is part of the canonical signaling network used by members of the transforming growth factor family. For this study, genetically-modified mice were used to determine how SMAD4 and TGFβ receptor II (TGFβRII) participate in transcriptional-programing of pathogen-specific CTLs. We show that these molecules are essential components of opposing signaling mechanisms, and cooperatively regulate a collection of genes that determine whether specialized populations of pathogen-specific CTLs circulate around the body, or settle in peripheral tissues. TGFb uses a canonical SMAD-dependent signaling pathway to down-regulate Eomesodermin (EOMES), KLRG1 and CD62L, while CD103 is induced. Conversely, in vivo and in vitro data show that EOMES, KLRG1, CX3CR1 and CD62L are positively-regulated via SMAD4, while CD103 and Hobit are downregulated. Intravascular staining shows that signaling via SMAD4 promotes formation of terminally-differentiated CTLs that localize in the vasculature. Our data shows that inflammatory molecules play a key role in lineage-determination of pathogen-specific CTLs, and use SMAD-dependent signaling to alter the expression levels of multiple homing-receptors and transcription factors with known functions during memory formation.
Sequencing data have been deposited in GEO under accession codes GSE151637Figure 3-source data 1 contain the numerical data used to generate the figures
Transcriptional profile of SMAD4-deficient cells is similar to TRMNCBI Gene Expression Omnibus, GSE151637.
Molecular signature of brain resident memory CD8+ T cellsNCBI Gene Expression Omnibus, GSE39152.
RNA-Seq of CD8+ T cell subsets during LCMV infectionNCBI Gene Expression Omnibus, GSE107281.
Hobit and Blimp1 instruct a universal transcriptional program of tissue-residency in lymphocytesNCBI Gene Expression Omnibus, GSE70813.
Comparative analysis reveals a role for TGF-β in shaping the residency-related transcriptional signature in tissue-resident memory CD8+ T cellsNCBI Gene Expression Omnibus, GSE125471.
SMAD4 CHip-seqNCBI Gene Expression Omnibus, GSE135533.
- Susan M M Kaech
- Linda S Cauley
- Linda S Cauley
- Linda S Cauley
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Experiments were performed in accordance with protocol AP-200531-0824 approved by the UCONN Health Institutional Animal Care and Use Committee (IACUC). Every effort was made to minimize suffering.
- Urszula Krzych, Walter Reed Army Institute of Research, United States
- Preprint posted: December 16, 2021 (view preprint)
- Received: December 17, 2021
- Accepted: August 5, 2022
- Accepted Manuscript published: August 9, 2022 (version 1)
- Accepted Manuscript updated: August 11, 2022 (version 2)
- Version of Record published: August 24, 2022 (version 3)
- Version of Record updated: August 30, 2022 (version 4)
© 2022, Chandiran et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Age-associated DNA methylation in blood cells convey information on health status. However, the mechanisms that drive these changes in circulating cells and their relationships to gene regulation are unknown. We identified age-associated DNA methylation sites in six purified blood-borne immune cell types (naive B, naive CD4+ and CD8+ T cells, granulocytes, monocytes, and NK cells) collected from healthy individuals interspersed over a wide age range. Of the thousands of age-associated sites, only 350 sites were differentially methylated in the same direction in all cell types and validated in an independent longitudinal cohort. Genes close to age-associated hypomethylated sites were enriched for collagen biosynthesis and complement cascade pathways, while genes close to hypermethylated sites mapped to neuronal pathways. In silico analyses showed that in most cell types, the age-associated hypo- and hypermethylated sites were enriched for ARNT (HIF1β) and REST transcription factor (TF) motifs, respectively, which are both master regulators of hypoxia response. To conclude, despite spatial heterogeneity, there is a commonality in the putative regulatory role with respect to TF motifs and histone modifications at and around these sites. These features suggest that DNA methylation changes in healthy aging may be adaptive responses to fluctuations of oxygen availability.
Infection with Influenza A virus (IAV) causes the well-known symptoms of the flu, including fever, loss of appetite, and excessive sleepiness. These responses, mediated by the brain, will normally disappear once the virus is cleared from the system, but a severe respiratory virus infection may cause long-lasting neurological disturbances. These include encephalitis lethargica and narcolepsy. The mechanisms behind such long lasting changes are unknown. The hypothalamus is a central regulator of the homeostatic response during a viral challenge. To gain insight into the neuronal and non-neuronal molecular changes during an IAV infection, we intranasally infected mice with an H1N1 virus and extracted the brain at different time points. Using single-nucleus RNA sequencing (snRNA-seq) of the hypothalamus, we identify transcriptional effects in all identified cell populations. The snRNA-seq data showed the most pronounced transcriptional response at 3 days past infection, with a strong downregulation of genes across all cell types. General immune processes were mainly impacted in microglia, the brain resident immune cells, where we found increased numbers of cells expressing pro-inflammatory gene networks. In addition, we found that most neuronal cell populations downregulated genes contributing to the energy homeostasis in mitochondria and protein translation in the cytosol, indicating potential reduced cellular and neuronal activity. This might be a preventive mechanism in neuronal cells to avoid intracellular viral replication and attack by phagocytosing cells. The change of microglia gene activity suggest that this is complemented by a shift in microglia activity to provide increased surveillance of their surroundings.