The Role And Mechanistic Study Of ER-associated Degradation Adaptor Molecule SEL1L In Regulating CD8~+ T-Cell Homeostasis

Background:CD8+T cells are the key players against pathogenic microbial infections and tumorigenesis.In the absence of antigen stimulation,peripheral CD8+T cells are often quiescent characterized with low metabolic rate,low transcriptional and translational activity,and their survival and homeostasis are tightly regulated and maintained by coordination of tonic TCR,IL-7,and sphingosine 1-phosphate(SIP)signals.The removal of these "homeostatic factors" disrupts CD8+T-cell immune response.ER-resident misfolded proteins and terminal unfolded proteins are mainly eliminated by endoplasmic reticulum(ER)-associated degradation(ERAD)pathway.SEL1L is a key adaptor of the SEL1L/HRD1 branch by stabilizing and recruiting the ER transmembrane protein HRD1 to direct the proteins to be degraded through HRD1 channel into the cytosol for degradation by the proteasome.Recent studies have suggested that SEL1L controls hematopoietic stem cell homeostasis and early stage thymocyte development.However,it remains unclear whether SEL1L is involved in homeostasis maintenance of peripheral CD8+ T cells.In this study,we intend to elucidate the role of SEL1L in maintaining CD8+T homeostasis by investigating the mechanism by which Selll deletion affects CD8+T cell survival,homeostatic proliferation,differentiation and metabolism.Our present study will provide new theoretical support for better understanding of T cell homeostasis maintenance.Methods:The Cre-Loxp conditional gene editing system was used to construct mice(Sel1l-/-)with T cell-specific loss of Sel1l.We first confirmed the specific deletion of Sel1l in T cells by both genomic PCR and Western blot analysis.The proportions of T cells in WT and Sel1l-/-mice were detected by flow cytometry,and the absolute number was calculated.Bone marrow chimeric mice transplanted with WT and Sel1l-/-bone marrow cells were constructed,and the frequencies of the donor T cells were measured by flow cytometry.The recipient mice were irradiated with 5 Gy and administered equal proportions of sorted splenic CFSE-labeled WT and Sel1l-/-CD8+ T cells,and CFSE dilution,the survival and cell cycle phase of adoptive T cells was detected by flow cytometry.To further determine the effect of Selll deficiency on CD8+T-cell ER,the morphology of ER was firstly revealed by transmission electron microscopy and the accumulation of protein aggregates in WT and Sel1l-/-CD8+T cells were detected by flow cytometry.The expression of UPR effector molecules in peripheral CD8+T cells from WT and Sel1l-/-mice was measured by RT-qPCR and Western blot.Subsequently,peripheral CD8+T cells were treated with inhibitors of three UPR branches,and the survival was detected by flow cytometry.Mice with T cell-specific deficiency of both Sel1l and Ern1 were also constructed and the frequency and number of T cells were detected and calculated.The percentage,absolute number and survival of T cells were detected and calculated in Sel1l-/mice treated with GSK2606414,and CFSE dilution of adoptive WT and Sel1l-/-CD8+ T cells in the recipient mice irradiated with 5 Gy and administered equal proportions of adoptive T cells was detected.To explore how SEL1L affects the maintenance of naive CD8+T cells in vivo,single-cell transcriptome sequencing(scRNA-seq)detection and UMAP(uniform manifold approximate projection)dimensionality reduction algorithm were used to analyze the changes in splenic CD4+and CD8+T cell subsets from WT and Sel1l-/-mice.To this end,the percentages of naive CD4+or CD8+T cells were detected by flow cytometry.In addition,the frequency of naive T cell in the recipient mice sub-lethally irradiated and administered with equal proportions of sorted WT and Sel1l-/-na(?)ve CD8+ or CD4+ T cells,was detected by flow cytometry.The expression of TCRβ,CD3 and CD5 in T cells from WT,Sel1l-/-and Sel1l-/-Ern1-/-mice.To explore the potential reguation of CD8+T cell metabolism by SEL1L,the levels of the mTORC1 downstream molecules and metabolism by Western blot and flow cytometry.Then,the surface expression of IL-15R and the levels of pSTAT5 in splenic CD8+T cells from WT and Sel1l-/-mice were detected by flow cytometry.The levels of pSTAT5 and mTORCl downstream molecules in WT and Sel1l-/-CD8+T cells treated with IL-15R inhibitor detected by flow cytometry.In addition,the surface expression of IL-15R,the levels of pSTAT5 and mTORC1 downstream molecules and metabolism in CD8+T cells from WT,Sel1l-/-and Sel1l-/-Ern1-/-mice were detected by flow cytometry.Results:Selll deficiency caused markedly decrease in peripheral CD8+T-cell frequency and number.Consistently,the percentages of Sel1l-/-donor-derived CD8+T cells in bone marrow chimera mice was significantly less than WT donor-derived cells.The percentage of transferred Sel1l-/-CD8+T cells was significantly less than that WT in recipient mice co-transferred with equal proportions of WT and Sel1l-/-CD8+T cells.The above-mentioned results showed that Selll deletion led to the reduce in CD8+ T-cell number.The reason was that Sel1l deficiency contributed to increased DNA damage and G1/S arrest in CD8+T cells,which in turn inhibited homeostatic proliferation.In addition,Sel1l deificeincy induced CD8+T-cell pyroptosis,apoptosis,and ferroptosis.Sel1l deletion led to ER stress and subsequently fueled intense PERK signaling in CD8+T cells,which thus disrupted T cell survival.Administration with PERK inhibitor rescued the frequency and number of T cells in Sel1l-/-mice.Highly activated IRE1αsignaling was critical for the maintenance of CD4+ and CD8+T cell numbers in Sel1l-/-mice,and IRE1α deficiency further aggravated the reduce in the numbers of CD4+and CD8+T cell in Sel1l-/-mice.In addition,IL-7 and IL-15 treatment in vitro can significantly inhibit ER stress and rescue survival in Sel1l-/-CD8+T cells.Single-cell transcriptome sequencing data and flow cytometry analysis showed that the percentage of splenic naive CD8+T cells in Sel1l-/-mice was significantly less than that of WT mice while the percentage of memory CD8+T cells was significantly more than that of WT mice.In addition,CD44 expression in peripheral CD8+T cells from Sel1l-/-mice was significantly higher than that from WT mice,and the same phenotype was also confirmed in bone marrow chimera mice.The percentage of memory CD8+T cells derived from adoptive Sel1l-/-naive CD8+T cells in spleen was significantly higher than WT counterparts.Peripheral CD8+T cells from Sel1l-/-mice exhibited mTORC1 activation,upregulated IL-15Rα and IL-15Rβ expression and increased pSTAT5 showed by Western blot and flow cytometry results.In vitro IL-15 treatment augmented mTORC1 downstream molecules in Sel1l-/-CD8+T cells,while blocking IL-15R signaling weakened mTORC1 signaling.In addition,amino acid and lipid metabolism in Sel1l-/-CD8+T cells were significantly increased.IREa deletion attenuated IL-15R signaling and mTORC1 activation and rescueed abnormal metabolism in Sel1l-/-CD8+T cell.Conclusions:Sel1l deletion significantly reduces the number of CD8+T cells in vivo by impairing the survival and homeostatic proliferation.Inhibition of PERK signaling can rescue thesurvival and number of CD8+T cells in Sel1l-/-mice by inhibiting apoptosis and pyroptosis.Sel1l deletion promotes differentiation of na(?)ve CD8+T cells to memory CD8+T cells,which is related to the decreased tonic TCR signaling in naive CD8+T cells.Selll deletion upregulates IL-15R expression on the surface of CD8+T cells,thereby triggering mTORC1 activation and abnormal metabolic shift.Knockout of Ernl reduces IL-15Ra expression and thereby attenuates mTORCl signaling and reverses abnormal metabolic shift in Sel1l-/-naive CD8+T cells.