The lncRNA Snhg1-Vps13D vesicle trafficking system promotes memory CD8 T cell establishment via regulating the dual effects of IL-7 signaling

The efficient induction and long-term persistence of pathogen-specific memory CD8 T cells are pivotal to rapidly curb the reinfection. Recent studies indicated that long-noncoding RNAs expression is highly cell- and stage-specific during T cell development and differentiation, suggesting their potential roles in T cell programs. However, the key lncRNAs playing crucial roles in memory CD8 T cell establishment remain to be clarified. Through CD8 T cell subsets profiling of lncRNAs, this study found a key lncRNA-Snhg1 with the conserved naivehi-effectorlo-memoryhi expression pattern in CD8 T cells of both mice and human, that can promote memory formation while impeding effector CD8 in acute viral infection. Further, Snhg1 was found interacting with the conserved vesicle trafficking protein Vps13D to promote IL-7Rα membrane location specifically. With the deep mechanism probing, the results show Snhg1-Vps13D regulated IL-7 signaling with its dual effects in memory CD8 generation, which not just because of the sustaining role of STAT5-BCL-2 axis for memory survival, but more through the STAT3-TCF1-Blimp1 axis for transcriptional launch program of memory differentiation. Moreover, we performed further study with finding a similar high-low-high expression pattern of human SNHG1/VPS13D/IL7R/TCF7 in CD8 T cell subsets from PBMC samples of the convalescent COVID-19 patients. The central role of Snhg1-Vps13D-IL-7R-TCF1 axis in memory CD8 establishment makes it a potential target for improving the vaccination effects to control the ongoing pandemic.


Adoptive transfer and cell sorting
In each individual experiment that if did not need to quantify cell number after infection, a total of ~2-5×10 4 naï ve CD45.1 + P14 cells with retrovirus transduction (~2-5,000 GFP + cells dependent on transduction efficiency) were adoptively transferred into naï ve wild-type (CD45.2 + ) mice, and infected recipient mice with 2×10 5 PFU LCMV Armstrong i.p. on the following day. For cell number quantification experiments, the cell sorting was performed on a FACSAria Ⅲ (BD Biosciences) with 2000 GFP + cells injected for each mouse, and the purity of all sorted populations was > 95%.

Flow cytometry and antibodies
Flow cytometry data were acquired by FACSCantoII (BD Biosciences) and analyzed with FlowJo software (Tree Star). The antibodies and reagents used for flow cytometry staining are listed in Supplementary Table S1. Surface staining was performed in PBS containing 2% BSA or FBS (wt/vol). Intracellular staining of TCF-1, Eomes and Ki67 were performed with the Foxp3/Transcription Factor Staining Buffer Set (00-5523; eBioscience). Staining of BCL-2 was performed with a Cytofix/Cytoperm Fixation/Permeabilization Kit (554722; BD Biosciences). Annexin V staining were performed with an Annexin V Kit eBioscience) according to the manufacturer's instructions. For detection of cytokine production, splenocytes were first stimulated with the indicated peptide (0.2 µg/ml), Golgi Plug, Golgi Stop, anti-CD107a, and anti-CD107b antibodies (BD Biosciences) at 37 o C for 5 hrs. Following surface staining, intracellular cytokine staining was performed with a Cytofix/Cytoperm Fixation/Permeabilization Kit according to the manufacturer's instruction. For detection of phosphorylated STAT signaling proteins, splenocytes were first stained with surface markers and then were stimulated with recombinant murine IL-7 (2 ng/ml, 217-17; PeproTech) at 37 o C for 1 hr. Then immediately fixed the cells with Phosflow Lyse/ Fix buffer (558049; BD Biosciences), followed by permeabilization with Phosflow Perm buffer I (557885; BD Biosciences) and staining with primary unconjugated antibodies to STAT3 (Tyr705) or STAT5 (Tyr694). Afterwards, primary unconjugated antibodies were detected by secondary staining with anti-rabbit IgG A647 antibody.

Retroviral constructs and transduction
3 The constructs pMKO.1 (IRES-GFP) and pMIT (IRES-Thy1.1) were obtained from Dr. Rafi Ahmed. The shRNA sequences targeting Snhg1 or Vps13D were cloned into vector pMKO.1, and the shRNA used here were listed in Table S3. The Bcl2 and Tcf7 coding sequences (p33 and p45 isoform) were amplified and cloned into the vector pMIT. Retroviruses were packaged by transfection of 293T cells with the retroviral vectors along with plasmid pCLeco. P14 CD8 T cells were activated in vivo by injection of 200μg peptide (LCMV glycoprotein amino acids 33-41) into P14 mice. 16-18 hrs later, activated P14 CD8 T cells were isolated and purified by negative selection with BeaverBeads Mag500 Streptavidin Matrix (22302, Beaver) , and then spin-infected for 120 min at 37 o C with centrifugation (800g) in freshly harvested retrovirus supernatants containing 8 µg/ml polybrene (H9268, Sigma-Aldrich) and 20 ng/ml of Miltenyi Biotec). Then, the transducted P14 cells were transferred into recipient mice, followed by infection of the hosts with LCMV Arm 12-16 hrs later.

Quantitative RT-PCR
For comparison of gene expression in P14 cells transducted with retrovirus expressing empty vector or Snhg1/Vps13D shRNA, the cells were sorted by a FACSAria Ⅲ cell sorter (BD Biosciences) and total RNA was extracted using the RNeasy Plus Mini Kit (74134; QIAGEN), thus reverse transcribed with a RevertAid H Minus First Strand cDNA Synthesis Kit (K1632; Thermo Scientific). The relative expression of various genes was examined using AceQ qPCR SYBR Green Master Mix (Q111; Vazyme) on a CFX96 Touch Real-Time System (Bio-Rad). The primers for the test genes are listed in Table S3.

RNA immunoprecipitation (RIP)
RNA immunoprecipitation was performed using the Magna RIP TM RNA-Binding Protein Immunoprecipitation kit (Millipore). Briefly, the harvested El4 or Jurkat cells were washed with ice-cold PBS and resuspended on ice with RIP lysis buffer. A/G magnetic beads were incubated with normal Rabbit IgG (Millipore), Rabbit anti-Vps13D (ab202285, Abcam) or Rabbit anti-CD127 (Abcam) for 30 min at room temperature. Then the cell lysate was incubated with the antibody-coated magnetic beads for 4 hours or overnight at 4 o C, and the immunoprecipitates were treated with Proteinase K Buffer (RIP Wash Buffer, 10 % SDS and proteinase K ) at 55 o C for 30 minutes to digest the protein. RNA was then extracted and quantitative RT-PCR was performed for further analysis.

RNA pulldown coupled with mass spectrometry
Full-length Snhg1 was cloned into the pGEX-3Z vector from Dr. Liuqing Yang (M.D. Anderson Cancer Centre), and then Snhg1 RNA (sense with antisense control) was transcribed in-vitro using the Biotin RNA labeling mix (Roche, 24552321) and T7 or SP6 RNA polymerase (Ambion) and purified by RNA Clean & Concentrator-5 (Zymo Research). The sorted memory CD8 T cells (~5 million, bulk) were freshly prepared using ProteaPrep Zwitterionic Cell Lysis Kit, Mass Spec Grade (Protea) with Anti-RNase, Protease/Phosphatase Inhibitor Cocktail, Panobinostat, and Methylstat, supplemented in the lysis buffer. The BcMag Monomer Avidin Magnetic Beads (Bioclone) were first prepared in accordance with manufacturer's instructions and then immediately subjected to RNA (20 ug) capture in RNA capture buffer for 30 min at room temperature with agitation. The RNAcaptured beads were washed once with NT2 buffer and incubated with 30 mg cell lysates diluted in NT2 buffer supplemented with 50 U/ml RNase inhibitor, 2 mM dithiothreitol, 30 mM EDTA, and 0.02 mg/ml Heparin for 4 hr at 4oC with rotation. The RNA-binding protein complexes were washed sequentially with NT2 buffer, NT2 high-salt buffer, NT2-KSCN buffer and PBS, thus eluted by 2 mM D-biotin in PBS. The eluted protein complexes were denatured, reduced, alkylated, and digested with immobilized trypsin (Promega), and samples were sent to Shanghai Applied Protein Technology Co. Ltd. for mass spectrometry analysis.

Chromatin immunoprecipitation (ChIP)
The enriched CD8 T cells were stimulated with IL-7 (10ng/ml) at 37 o C for 3hrs. Then the treated cells were conducted with ChIP assay using the SimpleChIP Enzymatic Chromatin IP Kit (Magnetic Beads) (9003; Cell Signaling Technology), according to the manufacturer's instructions. Chromatin fragments were immunoprecipitated by Rabbit anti-p-STAT3 Y705 (9145; Cell Signaling Technology), anti-p-STAT3 S727 (9134; Cell Signaling Technology), anti-p-STAT5 Y694 (9314; Cell Signaling Technology) or the normal rabbit IgG (3900; Cell Signaling Technology) coupled with ChIP Grade Protein G Magnetic Beads (9006; Cell Signaling Technology). After purification of DNA with a PCR purification kit (28104; Qiagen), quantitative PCR was performed with primers (Supplementary Table S3) flanking the putative p-STAT3 or p-STAT5 binding sites.

Immunoprecipitation (IP)-based mass spectrometry
Immunoprecipitation was performed using the Sure BeadsTM Protein G magnetic beads (BioRad,. Briefly, the harvested EL4 cells were lysed thoroughly in RIPA buffer (proteinase inhibitor added) with pipeting using BD ultra-fine needle on ice, thus with supersonics on ice for getting membrane proteins. After centrifugation, the lysate was incubated with rabbit anti-Vps13D (ab202285, Abcam) or normal rabbit IgG (Abcam) for 2 hrs at 4 o C, followed by incubation with the BSA pre-blocked Protein G magnetic beads for 1 hr at 4 o C. Wash the immunoprecipitated samples with RIPA buffer and RIPA high buffer, thus samples were eluted using 200ul/IP SDT buffer at 95 o C for 10min, thus with beads removed and sent to Shanghai Applied Protein Technology Co. Ltd. for mass spectrometry analysis.

Nucleus-cytoplasmic fractionation
Nucleus-cytoplasmic fractionation experiment was conducted using the Cytoplasmic and Nuclear RNA Extraction Kit (NORGEN, 21000), according to the manufacturer's protocol.

Human PBMC isolation and in-vitro culture
Human peripheral blood mononuclear cells (PBMCs) were isolated with Ficoll (SigmaAldrich) gradient separation from peripheral blood of 4 healthy adult donors (Fig. 1a,  2a) or 3 convalescent COVID-19 patients ( Supplementary Fig. S6i). The blood samples of COVID-19 patients were obtained from Chongqing Public Health Medical Center. The study received IRB approvals at Chongqing Public Health Medical Center (2020-023-01-KY). For the acquisition of human effector CD8 T cells, the sorted naï ve CD8 T cells (CCR7 + CD45RA -) were stimulated with Dynabeads™ Human T-Activator CD3/CD28 (11161D, Thermo Fisher Scientific) for three days in RPMI 1640 with 10% FCS. The study was reviewed and approved by the Ethics Committee of Chongqing Public Health Clinical Center, Third Millitary Medical University. Written informed consents were offered to all study participants.

Cytokines and neutralizing Ab treatments
To examine the cytokines' roles on the regulation of gene expression, cells were incubated at 37 o C with indicated concentrate of recombinant murine IL-7 (rmIL-7, PeproTech) or recombinant murine IL-2 (rmIL-2, PeproTech) for 48 hours in vitro, followed by RT-qPCR analysis. For depletion of IL-7, mice were treated on indicated days with 100 μg anti-IL-7 mAbs (M25, Bio X Cell) per day with i.v. injection in 500 μL PBS.

Virus titration 6
The LCMV viral loads in the spleen were quantified by RT-qPCR analysis as described previously (Mccausland and Crotty, 2008).

Statistical analysis
Statistical analysis was conducted with Prism 6.0 (GraphPad). Paired or unpaired two-tailed t-test with 95% confidence interval was used for calculation of P-values.  Fig. 1d, 1g and 1h; (g) is for Fig. 1e, 1f and 1k, and (h) is for Fig. 1i. (i) log2 ratio of CD127 + KLRG1 -% in GFPto that of GFP + P14 cells by using shRNAs to target the candidate lncRNAs for internal analysis on days 8, 15 and 30 p.i.. Data are representative of two or three independent experiments with at least three replicates or four mice per group.  Fig. 1h). Data are representative of two or three independent experiments with at least three replicates or four mice per group (error bars denote s.e.m.). *p < 0.05, **p < 0.01, ****p < 0.0001 (paired or unpaired two-tailed t-test). were injected into CD45.2 mice followed with LCMV infection, the kinetics of viral titers in spleen with infection timeline were shown. Data are representative of two or three independent experiments with at least four mice per group (error bars denote s.e.m.). ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001 (unpaired two-tailed t-test).    . ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001( paired or unpaired two-tailed t-test).