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 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.
Phage immunoprecipitation sequencing (PhIP-seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-seq for autoantigen discovery, including our previous work (Vazquez et al., 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki disease (KD), multisystem inflammatory syndrome in children (MIS-C), and finally, mild and severe forms of COVID-19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as prodynorphin (PDYN) in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in two patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID-19, including the endosomal protein EEA1. Together, scaled PhIP-seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.
Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here, we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2, and M1 and M4 helices of p22 are involved in the heterodimer formation. Dehydrogenase (DH) domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain, leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the DH in a productive docked conformation.