T CELL, PREPARATION METHOD FOR SAME, AND APPLICATIONS THEREOF
20230220339 · 2023-07-13
Assignee
Inventors
Cpc classification
A61P29/00
HUMAN NECESSITIES
C07K14/705
CHEMISTRY; METALLURGY
A61P31/00
HUMAN NECESSITIES
A61K35/17
HUMAN NECESSITIES
C12N2740/16043
CHEMISTRY; METALLURGY
C12N15/11
CHEMISTRY; METALLURGY
C12N15/1138
CHEMISTRY; METALLURGY
International classification
C12N15/11
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
Abstract
Provided are a T cell deleting the Ryr2 gene and a preparation method. Also provided are an Ryr2 antagonist, an Ryr2 overexpressing T cell, a method and applications for regulating Ryr2 expression, regulating basal Ca.sup.2+ oscillation, regulating m-Calpain activity, and increasing bonding strength between T cells and DC cells, applications of a reagent implementing the functions in preparing a medicament for treating an infectious disease, inflammation, or tumor, and a method for transforming Tconv cells into being functionally similar to Treg cells.
Claims
1. A T cell, wherein Ryr2 gene is deleted in the T cell.
2. The T cell according to claim 1, wherein exon 7 of the Ryr2 gene is deleted in the T cell.
3. The T cell according to claim 1, wherein at least a guanine-rich sequence in the Ryr2 gene is deleted in the T cell.
4. (canceled)
5. The T cell according to claim 1, wherein the T cell is a Tconv cell.
6. A method for preparing the T cell according to claim 5, wherein the method is selected from shRNA, siRNA, CRISPR/Cas9, zinc finger nuclease technology, transcription activator-like effector nuclease technology or meganuclease.
7. The method according to claim 6, wherein the shRNA is set forth in SEQ ID NOs: 5 or 6.
8-12. (canceled)
13. A method for treating an infectious disease, an inflammation or a tumor, comprising administering to an individual an effective amount of a reagent for regulating Ryr2 expression, a reagent for regulating basal Ca2+ oscillation, a reagent for regulating m-calpain activity, a reagent for regulating binding strength of a T cell to a DC cell, or the T cell according to claim 1.
14-17. (canceled)
18. The method according to claim 13, wherein the infectious disease is selected from a bacterial infection.
19. The method according to claim 13, wherein the inflammation is selected from systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, scleroderma, asthma, atopic dermatitis, organ-specific inflammatory diseases, allergies, folliculitis, tonsillitis, pneumonia, hepatitis, nephritis, acne, autoimmune diseases, chronic prostatitis, glomerulonephritis, hypersensitivity reactions, colitis, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, transplantation rejection, vasculitis or interstitial cystitis.
20. The method according to claim 13, wherein the tumor is selected from prostate cancer, breast cancer, liver cancer, glioma, intestinal cancer, cervical cancer, non-small cell lung cancer, lung cancer, pancreatic cancer, gastric cancer, bladder cancer, skin cancer, striated muscle cancer, tongue squamous carcinoma, nasopharyngeal cancer, ovarian cancer, placental choriocarcinoma, lymphoma, leukemia, rectal adenocarcinoma, medulloblastoma, meningioma, neurofibroma, ependymoma, schwannoma, astrocytoma, melanoma, mesothelioma, myeloma, chronic granulocytic leukaemia, acute myelogenous leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, epidermoid cancer, colon cancer, thymus cancer, blood cancer, head or neck cancer or oropharyngeal cancer.
21-27. (canceled)
28. The method according to claim 13, wherein the regulation is a reduction or an improvement; the reduction of the Ryr2 expression comprises overexpressing FoxP3 in a T cell or adding an Ryr2 inhibitor or knocking down or knocking out the Ryr2 gene in a T cell, the improvement of the Ryr2 expression comprises reducing FoxP3 expression in a T cell or adding an Ryr2 driving agent; wherein the Ryr2 inhibitor is selected from the Ryr2 antagonist, ryanodine, dantrolene or JTV519, the Rvr2 antagonist targets exon 7 of the Ryr2 gene or a guanine-rich sequence in the Ryr2 gene; wherein the Ryr2 driving agent is selected from nicotinamide adenine dinucleotide phosphate, caffeine, p-chloro-m-cresol, ryanodine, chlorantraniliprole, cyantraniliprole, beta-adrenaline, 4-chloro-3-methylphenol, cyantraniliprole, cyclaniliprole, cyclic adenosine diphosphate ribose, suramin sodium, tetraniliprole or trifluoperazine.
29. The method according to claim 28, wherein the knocking down or knocking out the Ryr2 gene in a T cell comprises a guanine-rich sequence of the Ryr2 gene in the T cell is knocked down or knocked out, the guanine-rich sequence is located within first 200 bp from promoter of the Ryr2 gene, and/or the guanine-rich sequence is located a specific region of 300 bp to 500 bp after the TSS site of promoter of Ryr2, and the guanine-rich sequence comprises GCAGGGG.
30. The method according to claim 28, wherein the knocking down or knocking out the Ryr2 gene in a T cell comprises deleting exon 7 of the Ryr2 gene in a T cell.
31-32. (canceled)
33. A method for transforming a Tconv cell to having similar function to a Treg cell, comprising overexpressing FoxP3 in the Tconv cell, or knocking down or knocking out Ryr2 gene in the Tconv cell.
34. The method according to claim 33, comprising knocking down or knocking out a guanine-rich sequence of the Ryr2 gene in a Tconv cell.
35. The method according to claim 33, comprising knocking out exon 7 of the Ryr2 gene.
36-41. (canceled)
42. The T cell according to claim 3, wherein the guanine-rich sequence is located within first 200 bp from promoter of the Ryr2 gene, and/or the guanine-rich sequence is located a specific region of 300 bp to 500 bp after the TSS site of promoter of Ryr2.
43. The T cell according to claim 42, wherein the guanine-rich sequence comprises GCAGGGG.
44. The method according to claim 34, wherein the guanine-rich sequence is located within first 200 bp from promoter of the Ryr2 gene, and/or the guanine-rich sequence is located a specific region of 300 bp to 500 bp after the TSS site of promoter of Ryr2.
45. The method according to claim 44, wherein the guanine-rich sequence comprises GCAGGGG.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0088] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:
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DETAILED DESCRIPTION OF THE EMBODIMENTS
[0136] Technical schemes in the examples of the present invention will be described clearly and completely in conjunction with the accompanying drawings. It is apparent that the examples described herein are only some examples of the present invention, but not all of them. Based on the examples of the present invention, all other examples obtained by those of ordinary skill in the art without intensive steps shall fall within the protection scope of the present invention.
[0137] Sources of experimental animals, reagents and devices used in the examples are as follows.
[0138] Mice: All mice were in C57BL/6 background. Ryr2fl/fl mice were produced by GemPharmatech Co., Ltd, and identified the genotype by PCT. OT-II transgenic mice were purchased from Jackson laboratory. Wild-type mice, Foxp3-IRES-GFP mice, Ryr2fl/fl mice, Ryr2 transgenic mice, OT-II transgenic mice, CD4-Cre mice and Foxp3-Cre-YFP mice, female Foxp3+/- mice were bred and housed at Tsinghua University Animal Facilities. All mice were bred in specific pathogen-free (SPF) conditions. All animal experiments met animal welfare guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University.
[0139] Cell lines and primary cells culture: DC2.4 cells were provided by Dr. Ken Rock in UMass Medical School; Vero cells were provided by Dr. Tan Xu in School of Pharmaceutical Science of Tsinghua university; Renca cells were provided by doctor An Guangyu in Beijing Chao-Yang Hospital; HEK293FT cells were provided by Dr. Guo Wei in School of Medicine of Tsinghua University; and MC38 cells were purchased from American Type Culture Collection (ATCC). MC38, Renca, NIH-3T3 cells and Vero cells were cultured in DMEM containing 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. All other cells were grown in RPMI-1640 at the same dose containing 50 .Math.M β-mercaptoethanol. All cell lines were tested for mycoplasma contamination by PCR analysis. Mouse CD4+ CD25+ Treg and CD4+ CD25- Tconv were isolated from the spleen using a mouse CD4+ T cell isolation kit (StemCell, 19852) and a mouse CD25 Treg cell positive selection kit (Stem cell, 18782). Treg and Tconv cells were sometimes selected by FACS from Foxp3-IERS-GFP mouse CD4+ spleen cells. Mouse DCs were isolated from the spleen using mouse CD11c selection kit II (Stem cell, 28007). OT-II T cells were isolated from OT-II spleen cells using mouse CD4+ T cell isolation kit (Stem cell, 19852), and sometimes sorted by FACS with anti-TCRVα2 antibodies.
[0140] Antibodies and reagents: All primary cell isolation kits were from StemCell. Recombinant human IL-2 was from R&D systems. Anti-mouse CD3e monoclonal antibody, and anti-mouse CD28 and anti-mouse TCRVα2-PE antibodies were from eBioscience. Flow antibodies were from eBioscience, including anti-CD4, anti-CD8, anti-GITR, anti-CD25, anti-PD-1, anti-CTLA-4, anti-Tim-3 and anti-LAG3 antibodies. Anti-FoxP3 antibodies were from Invitrogen. The following clones were used: anti-mouse CD4, anti-mouse CD8, anti-mouse GITR, anti-mouse CD25, anti-mouse PD-1, anti-mouse/rat CTLA-4, anti-mouse/rat FoxP3 (FJK-16s), anti-mouse/human Tim-3, anti-mouse LAG3, anti-mouse PD-1 (J43.1), anti-mouse CTLA4 (UC10-4B9) and anti-mouse GITR (DTA-1). The following antibodies were used for western blotting: m-calpain large subunit (M-type) antibodies and anti-mouse IgG-HRP linked antibodies from CST. Anti-talin antibodies (8D4) were purchased from Sigma-Aldrich.
[0141] pLKO.1 vectors purchased from shRNA library platform in the Center of Biomedical Analysis of Tsinghua University were used for all our gene knock down studies. All pre-designed shRNA sequences were synthesized by Ruiboxingke Biotech.
[0142] pLVX-IRES-mcherry vectors were provided by Dr. ShenXiaohua in School of Medicine of Tsinghua University.
Example 1: Preparation of Componentwith Ryr2 Gene Knock Down or FoxP3 Overexpression and Experimental Verification
1. Experimental Procedures for Knocking Down Ryr2 Gene
[0143] Lentivirus-based shRNA was used to knock down Ryr2 gene. Specifically, pre-synthesized shRNA sequences were inserted into pLKO.1 vectors. Plasmids including shRNA and packaging (pMD2.G and psPAX2) were purified from transformed Escherichia coli using the EndoFree plasmid Midi kit (CWBIO, CW2105S). Lentiviral packaging was performed according to the reagent instructions. Briefly, 293FT cells were cultured in 10 cm dishes to a 60% to 80% coverage. The media were replaced 2 h before DNA transfection. 2.5 .Math.g of packaging vectors pMD2.G and 2.5 .Math.g of psPAX2, and 5 .Math.g of pLKO.1 vectors (with inserted shRNA) were transfected into 293FT cells using a Neofect system (Neofect). After 72 h, lentiviruses were harvested and T cells were infected with Polybrene (final concentration of 8 .Math.g/mL). Cells were sorted by Aria cytometer (BD Biosciences) 48 h after viral infection. The knock down efficiency was verified by real-time quantitative PCR. The shRNA sequences are as follows: [0144] control-shRNA: NO: 5) and
TABLE-US-00001 5′-CCGGcaacaagatgaagagcaccaaCTCGAGttggtgctcttcatct tgttgTTTTTG-3′
(SEQ ID NO: 3) and
TABLE-US-00002 5′-AATTCAAAAAcaacaagatgaagagcaccaaCTCGAGttggtgctct tcatcttgttgg-3′
(SEQ ID NO: 4); Ryr2-shRNA:
TABLE-US-00003 5′-CCGGccgctaatgaagccatataaaCTCGAGTTTATATGGCTTCATT AGCGGTTTTTG-3′
(SEQ ID
TABLE-US-00004 5′-AATTCAAAAAccgctaatgaagccatataaaCTCGAGTTTATATGGC TTCATTAGCGG-3′
(SEQ ID NO: 6); Itpr1-shRNA:
TABLE-US-00005 5′-CCGGgcagtaggtaagaagttattaCTCGAGtaataacttcttacct actgcTTTTTG-3′
(SEQ ID NO: 7) and
TABLE-US-00006 5′-AATTCAAAAAgcagtaggtaagaagttattaCTCGAGtaataacttc ttacctactgc-3′
(SEQ ID NO: 8); [0145] Orai1-shRNA:
TABLE-US-00007 5′-CCGGcacaaccaccaactcggtcaaaCTCGAGtttgaccgagttgag gttgtgTTTTTG-3′
(SEQ ID NO: 9) and
TABLE-US-00008 5′-AATTCAAAAAcacaacctcaactcggtcaaaCTCGAGtttgaccgag ttgaggttgtg-3′
(SEQ ID NO: 10); Orai3-shRNA:
TABLE-US-00009 5′-CCGGgcccttgcttatctgtataatCTCGAGattatacagataagca agggcTTTTTTGG-3′
(SEQ ID NO: 11) and
TABLE-US-00010 5′-AATTCAAAAAAgcccttgcttatctgtataatCTCGAGattatacag ataagcaagggc-3′
(SEQ ID NO: 12); Trpm1-shRNA:
TABLE-US-00011 5′-CCGGcggagtgaacatgcagcatttCTCGAGaaatgctgcatgttca ctccgTTTTTG-3′
(SEQ ID NO: 13) and
TABLE-US-00012 5′-AATTCAAAAAcggagtgaacatgcagcatttCTCGAGaaatgctgca tgttcactccg-3′
(SEQ ID NO: 14); Trpm4-shRNA:
TABLE-US-00013 5′-CCGGgcacatcttcacggtgaacaaCTCGAGttgttcaccgtgaaga tgtggTTTTTG-3′
[0148] (SEQ ID NO: 15) and
TABLE-US-00014 5′-AATTCAAAAAgcacatcttcacggtgaacaaCTCGAGttgttcaccg tgaagatgtgc-3′
(SEQ ID NO: 16); Trpv2-shRNA:
TABLE-US-00015 5′-CCGGccaaggaacttgtttctatttCTCGAGaaatagaaacaagttc cttggTTTTTG-3′
(SEQ ID NO: 17) and
TABLE-US-00016 5′-AATTCAAAAAccaaggaacttgtttctatttCTCGAGaaatagaaac aaagagctccttgg-3′
[0149] (SEQ ID NO: 18); Cacnb1-shRNA:
TABLE-US-00017 5′-CCGGtaggaacgcaatggatattaaCTCGAGttaatatccattgcgt tcctaTTTTTG-3′
(SEQ ID NO: 19) and
TABLE-US-00018 5′-AATTCAAAAAtaggaacgcaatggatattaaCTCGAGttaatattcc attgcgttccta-3′
(SEQ ID NO: 20).
2. Knock Down Efficiency Verification by Real-Time Quantitative PCR
[0150] The specific procedures are as follows:
[0151] Total RNA was extracted from the indicated cells using TRIzol reagent (Invitrogen), and a first strand cDNA was synthesized using reverse transcriptase M-MLV (TaKaRa). Real-time quantitative PCR was performed using SYBR Green Master Mix (No Rox) (Yeasen). GAPDH or 18S RNA was used for normalization control. The primer sequences were as follows:
[0152] mouse GAPDH:
TABLE-US-00019 5′-CATCACTGCCACCCAGAAGACTG-3′
(SEQ ID NO: 21) and
TABLE-US-00020 5′-ATGCCAGTGAGCTTCCCGTTCAG-3′
(SEQ ID NO: 22); mouse 18S RNA:
TABLE-US-00021 5′-CGGACAGGATTGACAGATTG-3′
(SEQ ID NO: 23) and
TABLE-US-00022 5′-CAAATCGCTCCACCAACTAA-3′
(SEQ ID NO: 24); mouse Ryr2:
TABLE-US-00023 5′-ATGGCTTTAAGGCACAGCG-3′
(SEQ ID NO: 25) and
TABLE-US-00024 5′-CAGAGCCCGAATCATCCAGC-3′
(SEQ ID NO: 26); mouse FoxP3:
TABLE-US-00025 5′-CCCATCCCCAGGAGTCTTG-3′
(SEQ ID NO: 27) and
TABLE-US-00026 5′-ACCATGACTAGGGGCACTGTA-3′
(SEQ ID NO: 28); mouse m-calpain:
TABLE-US-00027 5′-GGTCGCATGAGAGAGCCATC-3′
(SEQ ID NO: 49) and
TABLE-US-00028 5′-CCCCGAGTTTTGCTGGAGTA-3′
(SEQ ID NO: 50) ; mouse Itpr1:
TABLE-US-00029 5′-CGTTTTGAGTTTGAAGGCGTTT-3′
(SEQ ID NO: 29) and
TABLE-US-00030 5′-CATCTTGCGCCAATTCCCG-3′
(SEQ ID NO: 30); mouse Orai1:
TABLE-US-00031 5′-GATCGGCCAGAGTTACTCCG-3′
(SEQ ID NO: 31) and
TABLE-US-00032 5′-TGGGTAGTCATGGTCTGTGTC-3′
(SEQ ID NO: 32); mouse Orai3:
TABLE-US-00033 5′-GGCTACCTGGACCTTATGGG-3′
(SEQ ID NO: 33) and
TABLE-US-00034 5′-GCAGGCACTAAATGTGACC-3′
(SEQ ID NO: 34); mouse Trpm1:
TABLE-US-00035 5′-ATCCGAGTCTCCTACGACACC-3′
(SEQ ID NO: 35) and
TABLE-US-00036 5′-CAGTTTGGACTGCATCTCGAA-3′
(SEQ ID NO: 36); mouse Trpm4:
TABLE-US-00037 5′-GGACTGCACACAGGCATTG-3′
(SEQ ID NO: 37) and
TABLE-US-00038 5′-GTACCTTGCGGGGAATGAGC-3′
(SEQ ID NO: 38); mouse Trpv2:
TABLE-US-00039 5′-TGCTGAGGTGAACAAAGGAAAG-3′
(SEQ ID NO: 39) and
TABLE-US-00040 5′-TCAAACCGATTTGGGTCCTGT-3′
(SEQ ID NO: 40); mouse Cacnb1:
TABLE-US-00041 5′-GGCAGCAAGTTATCTCCCAG-3′
(SEQ ID NO: 41) and
TABLE-US-00042 5′-CCACAGGATGATTGGCGTCTT-3′
(SEQ ID NO: 42); HSV-1 gB:
TABLE-US-00043 5′-AACGCGACGCACATCAAG-3′
(SEQ ID NO: 43) and
TABLE-US-00044 5′-CTGGTACGCGATCAGAAAGC-3′
(SEQ ID NO: 44); HSV-1 LAT:
TABLE-US-00045 5′-GGGTGGGCTCGTGTTACAG-3′
(SEQ ID NO: 45) and
TABLE-US-00046 5′-GGACGGGTAAGTAACAGAGTCTCTA-3′
(SEQ ID NO: 46).
3. Experimental Procedures for Overexpressing FoxP3
[0153] Vectors were constructed, cDNA of Treg total RNA was used as template, and FoxP3 was amplified by PCR using the following primers: the forward primer
TABLE-US-00047 5′-ATCGCTCGAGATGTGCACACCTAGGCCA-3′
(SEQ ID NO: 47) and the reverse primer
TABLE-US-00048 5′-ATCGGAATTCTCAAGGGCAGGGATTGGA-3′
(SEQ ID NO: 48). The amplified fragments were purified by a gel, digested (XhoI and EcoRI) and cloned into pLVX-IRES-mcherry plasmids to give a pLVX-FoxP3-IRES-mcherry construct. Lentivirus production of pLVX-FoxP3-IRES-mcherry and cell lines generation of FoxP3 overexpression was performed according to the knock down protocol described above. The FoxP3 expression was verified by RT-PCR.
4. Procedures of Flow Cytometry
[0154] The cells were incubated with Fc blocking agent (CD16/32 antibodies; 2.4G2) for 5 min, and then incubated with surface antibodies (CD4, CD8, GITR, CD25, PD-1, CTLA-4, TIM-3, LAG3) for 15 min at room temperature away from the light. FoxP3 cells were stained, and surface-stained cells were incubated with FoxP3/transcription factor immobilization/permeation buffer (Thermo Fisher) for 30 min, followed by incubation with antibodies for 2 h. Sample were analyzed using FACS Diva software and Fortessa cytometer (BD Biosciences). Data was analyzed by Flowjo software.
5. Procedures of ELISA
[0155] In order to detect IL-10 and TGF-β, 10.sup.6 purified Treg, Ryr2+/+ or Ryr2-/- Tconv cells were stimulated with anti-CD3/anti-CD28 antibodies. After 72 h, the supernatant was collected. High-binding 96-well ELISA plates (Nunc) were incubated overnight at 4° C. After drying, the plates were blocked with 2% BSA for 1 h at room temperature. After washing, 100 .Math.L of diluted cell supernatant was added to per well and incubated for 1 h at room temperature. The plates were then washed with PBST (0.05% Tween20, Sigma-Aldrich, in PBS) and incubated with HRP-labeled goat anti-mouse IL-10 and TGF-β detection antibodies for 0.5 h at room temperature. TMB (100 mL/well) was added and the plates were incubated at room temperature for 10 min in the dark, and then H.sub.2SO.sub.4 (50 .Math.L, 1 M) was added per well to stop the reaction. The optical density (OD) was immediately read at 450 nm using an ELISA microplate reader (Bio-Rad).
6. Calcium Imaging
[0156] For non-adherent cells, Treg, Tconv and A20 cells were stained with 2 mM fluo-4 AM (Thermo Fisher) for 1 h at 37° C. After washing, the cells were adhered to poly-L-lysine coated (0.1 mg/mL; Sigma-Aldrich) round glass slides mounted in the sandwich home-made chamber at room temperature. After 15 min, excess non-adherent cells were removed by washing with buffer. For adherent cells, NIH-3T3 and Renca cells grown on glass slides were stained with 2 mM fluo-4 AM (Thermo Fisher) for 1 hour at 37° C. The measurement chamber was then placed on an Olympus IX-73 microscope equipped with a 20-fold (numerical aperture: 0.8) or 40-fold (numerical aperture: 1.2) Olympus objective. Unless otherwise stated, Ca.sup.2+ oscillation within 20 min was recorded at intervals of 6 s. In the inhibition experiments, Fluo-4-labeled cells were treated with JTV519 (Sigma) at room temperature in the dark for 30 min, and then images were acquired. For the stimulation experiments, Ca.sup.2+ oscillation within 5 min was recorded at intervals of 1 s. 50 to 80 s after starting to acquire images, 4-CmC (Sigma) buffer was added to induce the release of intracellular calcium. The experiments were performed in phenol red-free HBSS medium with or without Ca.sup.2+. The emission signal at 468 nm to 550 nm excited by the 488 nm laser was recorded with a charge coupled device camera (ORCA-AG, Hamamatsu). Data collection was controlled by NIS-Elements 3.0 software (Nikon). The mean fluorescence intensity changes over time in individual cells were analyzed using ImageJ and normalized with fluorescence intensity in the first frame (Fluo-4 F / F0). The peak of Ca.sup.2+ oscillation of the individual cells was shown, and the standard deviation of Ca.sup.2+ fluctuation intensity was calculated as Mean ± SEM.
7. Procedure of Western Blot
[0157] Cells were collected, and lysed with RIPA buffer (Beyotime, P0013B). The cell lysate was centrifuged and the supernatant was collected. Total protein was quantified using BCA protein assay kit (Beyotime, P0012). After mixing with 3XSDS loading buffer and being boiled for 5 min, the proteins were loaded onto 7.5% PAGE gels (EpiZyme, PG111) for separation. The proteins were then transferred to NC membranes, and immunoblotted with primary and secondary antibodies. Finally, immunostained bands were detected using Super ECL detection reagent (Yeasen, 36208ES76).
8. Experimental Results
[0158] Ryr2 could be knocked down in CD4-cre Tconv (
Example 2: Verification of Effect of FoxP3 Expression on Ryr2 Gene and Determination Of Target Sites
[0159] In order to determine the specificity of Ryr2 gene regulation, in this case, it will be linked with FoxP3 expression and determined the target sites.
[0160] FoxP3 was first overexpressed in T cells, B cells (A20), 3T3 and Renca tumor cells (using the method in Example 1). In these four cases, FoxP3 overexpression significantly blocked the transcription of Ryr2 gene, indicating that FoxP3 spontaneously targets Ryr2 (
[0161] Then, a genome-wide Chip-seq data set for gene expression analysis was prepared, wherein the ChIP-seq data was downloaded from the GEO data set, and GEO ID was as follows: Foxp3 in Tconv GSM989036, and FoxP3 in Tconv cells was transduced to expression marker-FoxP3 GSM989034. IGV (v2.4.14) facilitated visualization of ChIP-seq data using the mouse reference genome mm8. As shown in the screenshots at specific gene sites, when the data set was concentrated within a 40-kb region around the Ryr2 gene using the IGV program, a strong signal was detected within the 1.5-kb promoter region in the untransfected control group (
[0162] Furthermore, the above regions were cloned, and expression vectors of luciferase reporter genes were constructed under the control of this promoter for expression in 3T3 and Renca cells, including the FoxP3 overexpression group and the non-FoxP3 overexpression group. As can be seen in
[0163] The FoxP3 binding site was finally determined as the two GCAGGGG sequence repeats. When these two sites were mutated, FoxP3 overexpression lost the ability to inhibit luciferase, indicating that it is the binding site for FoxP3 in this promoter (
[0164] Among them, the fold luciferase reporter assay procedures involved in this example is described in the documents “Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3” (Seth et al., Cell 122, 669-682, 2005) and “LFA-1-mediated adhesion is regulated by cytoskeletal restraint and by a Ca2+-dependent protease, calpain” (Stewart et al., J Cell Biol, 140, 699-707, 1998). The synthesized 1500 bp complete promoter sequence and each truncated promoter sequence were subcloned into the luciferase expression vector pGL3 to construct each mouse Ryr2 reporter plasmid, and the plasmids were confirmed correct by sequencing. The 300 ng of Ryr2 reporter gene and ranilla luciferase reporter gene plasmids were co-transfected to 1.25 × 10.sup.5 FoxP3-overexpressed 3T3, FoxP3 overexpressed Renca or FoxP3-overexpressed A20 cells using Neofect (KS2000). 36 h after transfection, cell lysates were prepared and analyzed using the fold luciferase reporter assay system (Promega).
Example 3: Effect of Ryr2 Inhibition on Ca.SUP.2+ Levels in T Cells
1. M-Calpain Differences in Tconv and Treg, and Binding Ability to DC
[0165] Since the m-calpain activity is low in Treg, this example is to determine whether the expression level is low or not. Meanwhile, m-calpain is regulated by intracellular Ca.sup.2+ availability. Therefore, Ca.sup.2+ signals of Tconv and Treg in the resting state were detected.
[0166] The results are shown in
2. Determination of Factors Affecting Ca.SUP.2+ Levels
[0167] Upon T cell activation, Ca.sup.2+ signals were from the IP3R channel as the result of an expansion of the immunotyrosine-based signaling cascade to a secondary Ca.sup.2+ amplification event. However, this activation-induced signal was absent in resting cells. In this example, the data of several factors affecting Ca.sup.2+ signal regulation involved in the database GEO DataSets (see “A mechanism for expansion of regulatory T-cell repertoire and its role in self-tolerance” (Feng et al., Nature, 2015)) were used, and the details are shown in
[0168] The GEO database analysis procedure comprised the following steps: proteins were classified as per expression level according to the three RNA-Seq studies published in NCBI GEO database (GSE71162). Calcium-associated proteins were sorted by GO analysis. The functions of these proteins were annotated based on UniProt (http://www.uniprot.org/). The proteins were sorted by the degree of expression difference between Treg and Tconv.
[0169] Ryr receptors are located on the ER membrane (Ryr1, 2 and 3). In human PBMC, Ryr1 is expressed in the CD19+ population and Ryr2 is expressed in the CD3+ fraction. In order to confirm that Ryr2 is expressed in Treg in low amounts, the digestion rate of CMAC (calpain substrate) was observed using the stimulatory factor 4-CMC. The results are shown in
3. Inhibiting Ryr2 in Tconv Cells Can Reduce Ca.SUP.2+ Levels and Basal Oscillations
[0170] This example further verified the change in Ca.sup.2+ signal after Ryr2 inhibition, and the results are as shown in
[0171] In summary, the lower Ca.sup.2+ levels in the T cell subpopulation were essentially controlled by the inhibition of Ryr2 transcription.
Example 4: Blocking Ryr2 Can Tightly Bind T Cells to DCs
1. Ryr2 Knock Down in Tconv Can Reduce Ca.SUP.2+ Levels
[0172] In order to genetically confirm that Ryr2 inhibition is responsible for the reduction of Ca.sup.2+ levels in Tconv, shRNA interference for knock down of Ryr2 was performed in this example. Transcriptional mRNA levels were compared between group with Ryr2-specific knock down by shRNA and group without Ryr2 knock down (
[0173] Wherein calpain activity measurement procedure: To measure calpain activity, 10.sup.5 T cells were incubated in 200 .Math.L of medium containing 20 .Math.M calpain substrate CMAC (t-BOC-Leu-Met, Thermo Fisher) in the dark at room temperature. After 5 min of incubation, the reaction was terminated with 1 % PFA (Biosharp) and the cells were immediately placed on ice. The fluorescent signal emitted from the digested substrate was measured as calpain activity by Fortessa flow cytometer through the Hoechst blue channel. In all experiments, the inhibitor JTV519 was added 30 min prior to the experiments, and activator 4-CmC and CMAC were added at the same time.
2. Effect of Ryr2 Deletion in Tconv Cells on T Cells Binding to DCs
[0174] To verify the effect of the reduction in Ryr2 and loss of m-calpain activity on T cells binding to DCs, in this example, shRNA and JTV519 were used to block Ryr2 in Tconv. The results showed that both treatments induced strong contact with dendritic cells, and the intensities were even stronger than that of Treg (
[0175] Furthermore, it was confirmed that improved binding of treated Tconv to DC resulted in the failure stable binding of antigen-specific Tconv to the same DC, and a triple cell binding force analysis was conducted (see: “Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy” (Chen et al., The Journal of experimental medicine, 2017)). The results showed that Treg and knock-down Ryr2 Tconv can significantly inhibit the binding strength between OTII and OVA-loaded DC (
[0176] Experimental procedures of inhibition in vitro. Purified OTII T cells were stained with CellTrace CFSE (Thermo Fisher Scientifc). 10.sup.4 purified DCs from spleen cells, 2 × 10.sup.4 OTII T cells, 2 × 10.sup.-4 Treg or Ryr2-/- Tconv or Ryr2-knock down Tconv and 2 .Math.M OVA.sub.323-339 peptides were mixed and cultured in a 96-well U-bottom plate, and the dilution of CFSE was detected using Fortessa flow cytometer to evaluate proliferation of OTII T cell. Inhibition percentage was calculated by (1 - proliferation%), and then the data was normalized by taking the Treg-free group as 0% inhibition and the Treg group as 100% inhibition.
3. The Confirmation of Binding to DCs in the Deletion of Ryr2 Using Force Spectrum
[0177] Similarly, the force spectrum also showed improved binding to DCs when Ryr2 was deleted (
[0178] The atomic force microscope-based single cell force spectrum procedures were performed using JPK CellHesion device as described above (see “Alum interaction with dendritic cell membrane lipids is essential for its adjuvanticity” (Flach et al., Nat Med 17, 479-487, 2011) and “Strong adhesion by regulatory T cells induces dendritic cell cytoskeletal polarization and contact-dependent lethargy” (Chen et al., The Journal of experimental medicine, 2017)). Briefly, to measure T-DC adhesion in a two-cell system, DC2.4 cells were cultured on untreated glass plates. T cells were treated with 200 U/mL human IL-2 overnight. The glass plates were moved into an AFM compatible chamber and mounted on a machine table. Clean cantilevers were coated with CellTak (BD), and then stick an individual T cell on thefront end of the cantilever. The AFM cantilever carrying the individual T cell was lowered to allow T cell contact with a single DC and to interact for 15 s, and then moved upward until the two cells were completely separated. A force spectrum curve was obtained. The process was then repeated. For the triple cell system, DC2.4 cells cultured on glass plates were treated with 100 .Math.g/mL soluble OVA protein for 4 h prior to the experiment. IL-2 treated Treg/Tconv cells were stained with 10 .Math.M CFSE, then DC2.4 cells were incubated with these fluorescently labeled Treg or Tconv cells for about 20 min, followed by addition of unlabeled OT-II T cells. The Treg/Tconv cell-DC pair identified with the UV flash lamp was then approached by a cantilever tip carrying OT-II T cells. Treg/Tconv contact-mediated inhibition of OT-II-DC adhesion was analyzed. In each cycle, the AFM cantilever carrying an individual T cell was lowered in 0.5 .Math.m to 2 .Math.m increments until the first force curve was generated. The T cells on the cantilever were then allowed to interact with the DCs for 15 s, and then moved upward until the two cells were completely separated. The incubator containing the device was set at 37° C. and 5% CO.sub.2. In all experiments, at least 14 force curves were collected for further analysis. The force curves were processed using JPK image processing software. Only round and plump cells were selected for AFM testing. For each SCFS experiment, a T-DC pair was used to generate force readings for each up-down cycle during a period of several minutes. At least three pairs of data were used for each condition.
Example 5: Effect of Ryr2-/- T Cells in HSV-1 Infection
1. Preparation of Herpes Infection Model and Experimental Procedures
[0179] Herpes infection was induced by 10.sup.6pfu HSV-1 (strain F) in 20 .Math.L PBS into hinder footpads. Cells were adoptively transferred into the footpadat 3 dpi with 2 × 10.sup.5 cells/20 .Math.L PBS, Treg, or Ryr2-/- Tconv or Ryr2+/+ Tconv. The homogenized footpad tissue was tested for viral titer at 7 dpi. Each group contained 4-6 mice and the experiment was repeated 3 times.
[0180] For DTH, the right footpad was re-challenged with UV-inactivated HSV-1 (10.sup.6pfu/20 .Math.L PBS) at 6 dpi, and the footpad swelling was then measured at 7 dpi with the left footpad as a control. Each group contained 16-21 mice.
2. Experimental Results
[0181] In the herpes infection model, injection of Treg or Ryr2-/- Tconv promoted HSV-1 amplification. The pfu of HSV- 1 was not increased by Ryr2+/+ Tconv infusion compared with viral inoculation alone (PBS) (see
Example 6: Effect of Ryr2-/- T Cells in a Mouse Model With Asthma
1. Preparation of Asthma Model and Experimental Procedures
[0182] Ovalbumin (OVA) can induce airway inflammation. Mice were sensitized on day 0 and day 14 by two i.p. injections of OVA with alum adjuvant (100 .Math.g + 4 mg). Intra-tracheal OVA re-challenge were repeatedly given at back of the tongue on day 21, day 23 and day 25. 10.sup.6 Treg, Ryr2-/- Tconv or Ryr2+/+ Tconv, or PBS were adoptively transferred by i.v. On the 32nd day, the euthanized mice were used to analyze the immune cell infiltration and lung tissue pathology in broncho-alveolar lavage fluid (BALF).
2. H&E Stain Procedures
[0183] Lung tissue were immobilized in 4% formalin at 4° C. The samples were then embedded in paraffin. Cut into five to six micrometer thick slices, and staine with hematoxylin and eosin.
3. Experimental Results
[0184] Infusion of Treg and Ryr2-/- Tconv was equally effective in limiting the number of BALF cells in an OVA-sensitized asthma model generated according to the sensitization schedule shown in
Example 7: Effect of Ryr2-/- T Cells in a Mouse Model With Inflammatory Bowel Disease
1. Preparation of DSS-Induced Inflammatory Bowel Disease (IBD) Model and Experimental Procedures
[0185] The DSS-induced mouse experimental inflammatory bowel disease model can be seen in the documents: “Inhibition of Dectin-1 Signaling Ameliorates Colitis by Inducing Lactobacillus-Mediated Regulatory T Cell Expansion in the Intestine” (Tang et al., Cell Host Microbe 18, 183-197, 2015) and “Myeloid-Derived Suppressor Cells Are Controlled by Regulatory T Cells via TGF-beta during Murine Colitis” (Lee et al., Cell 17, 3219-3232, 2016). Briefly, wild-type Tconv or Treg were transferred into 6-week-old male C57BL/6 mice by i.v. adoptive transfer of 3 × 10.sup.6 Ryr2-/- Tconv. The next day, colitis was induced by 4% DSS water (w/v) (Yeason, MW = 36,000 Da to 50,000 Da) for 7 days, and then the mice was fed with ordinary drinking water. Normal control mice were treated with PBS and given normal drinking water. Mice were sacrificed on day 10, the colon was dissected and the colon length was measured. The colon was then immobilized in 4% PFA at 4° C. for 48 h for subsequent H&E staining. The colon sections were observed by 3DHISTECH Pannoramic SCAN (3DHISTECH). Each group contained 11-18 mice.
2. H&E Histological Procedures
[0186] Colonic tissue were immobilized in 4% formalin at 4° C. The samples were then embedded in paraffin. Cut into five to six micrometer thick slices, staine with hematoxylin and eosin.
3. Experimental Results
[0187] In the colitis model generated according to the induction schedule shown in
Example 8: Effect of Ryr2-/- T Cells in a Mouse Colon Cancer Model
1. Preparation of Colon Cancer Model and Experimental Procedures
[0188] The mouse model with the transplanted tumor could be established by reference to the “Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor” (Maj et al., Nat Immunol 18, 1332-1341, 2017). Briefly, MC38 colon cancer cells were washed twice in PBS and then 5 × 10.sup.5 MC38 cells and 10.sup.6 T cells were mixed and injected subcutaneously into the abdomen of 6-week-old male C57BL/6 mice. Tumor size was monitored using a vernier caliper every 4-5 days from day 7 after tumor inoculation. The volume was calculated as (length × width × width) / 2. Group 1: MC38 + PBS; group 2: MC38 + Ryr2+/+ Tconv; group 3: MC38 + Treg; group 4: MC38 + Ryr2-/- Tconv. Each group contained ten mice.
2. Experimental Results
[0189] In the MC38 tumor model, Treg and Ryr2-/- Tconv injection promoted the tumor growth, while no significant effect was seen in PBS and Ryr2+/+ Tconv groups (
Example 9: Immune Homeostasis Restoring Effect of Ryr2-/- T Cells in a Mouse Psoriasis Model
[0190] Ryr2-/- Tconv cells play the most important role in general inflammation caused by multi-organ autoimmunity. Therefore, an Foxp3-deficient mouse model was constructed in this example to verify immune homeostasis restoring under the regulation of Ryr2-/- Tconv cells in FoxP3-deficient immunodeficiency mice.
1. Psoriasis Mouse (Scurfy) Model
[0191] The Foxp3-deficient psoriasis mouse model was established with reference to the “A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity” (Haribhai et al., Immunity 35, 109-122, 2011) and “TGF-beta-induced Foxp3+ regulatory T cells rescue scurfy mice” (Huter et al., Eur J Immunol 38, 1814-1821, 2008). 5 × 10.sup.6 T cells were purified, washed twice in PBS and resuspended in 50 .Math.L PBS for intraperitoneal injection. The injection was performed on day 2 or day 3 after birth of newborn homologous psoriasis mice. Adoptive transfer of T cells was performed every three or four days in the first two weeks, and then performed every two weeks. At adoptive transfer, body weights of the psoriasis model mice and male WT littermates were recorded and the survival rates were monitored. All mice were sampled on the day of birth and genotyped for the sf mutant gene by PCR, and their genotypes were confirmed by sequencing. FoxP3 PCR primers were 5′-CATCCCACTGTGACGAGATG-3′ (SEQ ID NO: 1) and 5′-ACTTGGAGCACAGGGGTCT-3′ (SEQ ID NO: 2). For histology, mice receiving PBS were analyzed at week 3, and mice adoptively transferred Treg and Ryr2-/-Tconv were analyzed at weeks 8-12.
2. H&E Histological Procedures
[0192] Skin, ear, liver and other tissues were all immobilized in 4% formalin at 4° C. The samples were then embedded in paraffin. Cut into five to six micrometer thick slices, staine with hematoxylin and eosin.
3. Experimental Results
[0193] 2-3 days after birth, psoriasis mice (FoxP3-/-, C57BL/6) were injected with PBS, Treg, Ryr2+/+ Tconv or Ryr2-/- Tconv, respectively. All mice receiving PBS or Ryr2+/+ Tconv died within 2-4 weeks after birth as expected. In contrast, all mice receiving Treg or Ryr2-/- Tconv survived for at least 20 weeks (
[0194] The histological results are shown in
[0195] In conclusion, Ryr2-/- T and Treg cells all showed functional equivalence in the mouse model with herpes infection, the asthma model, the colitis model and the colon cancer model, and can also restore the immune function of psoriasis mouse . That is, Ryr2-/- T may all play an equivalent role in some diseases where Treg are known to have immunoregulation effects.
[0196] The preferred embodiments of the present invention are described in detail above, which, however, are not intended to limit the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, all of which will fall within the protection scope of the present invention. In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, such combinations will not be illustrated separately.