搜索

Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets
在无淋巴细胞的小鼠模型中用调节性T细胞和普通T细胞重建外周T细胞亚群   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Transfer of mature T cells into immunodeficient mice results in sub-optimal reconstitution of the peripheral T cell pool. Under lymphopenic conditions, dendritic cells are released from tonic control by regulatory T cells (Tregs), and consequently drive activation and proliferation of low affinity T cells specific for endogenous antigens. This oligoclonal proliferation results in a T cell population dominated by T cells possessing an effector/memory phenotype and a limited TCR repertoire. Oligoclonal expansion can be prevented by selectively reconstituting the Treg compartment prior to T cell transfer (Bolton et al., 2015). Reconstitution of the Treg compartment of lymphopenic mice has been tested in immunodeficient mouse strains such as Rag-1-/-or Rag-2-/- mice, and in immunosufficient mice rendered transiently lymphopaenic by lethal whole body irradiation as conditioning for bone marrow transplantation (BMT). Transfer of purified Tregs into these hosts, combined with treatment with exogenous IL-2 for 7 days, is sufficient to reconstitute the Treg compartment and reduce expression of dendritic cell costimulatory molecules, a critical process in preventing inappropriate expansion of self-reactive T cells. T cells transferred after Treg reconstitution do not undergo rapid spontaneous proliferation, and instead undergo slow homeostatic division to repopulate the T cell pool with naive T cells, thus allowing optimal reconstitution of peripheral T cell pool.

Keywords: Lymphopenia(lymphopenia), Immune reconstitution(免疫重建), T cell(T细胞), Regulatory T cell(调节T细胞), Animal model(动物模型)

Materials and Reagents

  1. 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
  2. 10 ml conical tube (SARSTEDT AG & Co, catalog number: 62.9924.272 )
  3. 70 µm nylon cell strainer (Corning, Falcon®, catalog number: 352350 )
  4. 1 ml syringe (BD, catalog number: 309659 )
  5. 25 gauge needle (BD, catalog number: 305125 )
  6. 1 ml insulin syringe with 29 G needle (BD, catalog number: 326719 )
  7. 225 cm tissue culture flasks (Sigma-Aldrich, catalog number: CLS431082 )
  8. LD columns (Miltenyi Biotec, catalog number: 130-042-901 )
  9. LS columns (Miltenyi Biotec, catalog number: 130-042-401 )
  10. Vitamin K1, 10 mg/ml (International Animal Health Products, Koagulon)
  11. PBS (10x, pH 7.4, without Ca2+ and Mg2+) (Mediatech Inc., catalog number: 46-013-CM )
  12. Fetal bovine serum (FBS), heat-inactivated at 65 °C (Scientifix, catalog number: FBS-500S )
  13. Bovine serum albumin (BSA) Fraction V (Amresco, catalog number: 0332-1 kg )
  14. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: E5134 )
  15. RPMI-1640 (Thermo Fisher Scientific, GibcoTM, catalog number: 11875-093 )
  16. Rat anti-mouse Thy1.2 clone 30H12 hybridoma supernatant (prepared in house)
  17. Rat anti-mouse B220 clone RA3.6B2 hybridoma supernatant (prepared in house)
  18. Rat anti-mouse CD8a clone 53-6.7 hybridoma supernatant (prepared in house)
  19. Rat anti-mouse CD11b clone M1/70 hybridoma supernatant (prepared in house)
  20. Rat anti-mouse erythroid lineage clone Ter119 hybridoma supernatant (prepared in house)
  21. Anti-CD25 PE clone PC61 (BD Biosciences, catalog number: 553866 )
  22. Anti-rat IgG microbeads (Miltenyi Biotec, catalog number: 130-048-501 )
  23. Anti-PE microbeads (Miltenyi Biotec, catalog number: 130-048-801 )
  24. Recombinant mouse IL-2 (Peprotech, catalog number: 212-12 )
  25. Anti-IL-2 mAb clone JES6-1 (WEHI monoclonal antibody facility)
  26. Sulfamethoxazole (400 mg/5 ml) and Trimethoprim (80 mg/5 ml) (Hospira, DBL, catalog number: 618670BAU )
  27. MACS wash buffer (see Recipes)
  28. MACS running buffer (see Recipes)
  29. RPMI + 10% FBS (see Recipes)
  30. IL-2/JES6-1 (see Recipes)
  31. Preparation of hybridoma supernatants (see Recipes)

Equipment

  1. Surgical scissors and forceps
  2. 80 gauge stainless steel mesh sieves
  3. MACS MultiStand (Miltenyi Biotec, catalog number: 130-042-303 )
  4. QuadroMACS separator (Miltenyi Biotec, catalog number: 130-090-976 ) or MidiMACS separator (Miltenyi Biotec, catalog number: 130-042-302 )
  5. 37 °C water bath
  6. Benchtop centrifuge
  7. Gammacell irradiator (Nordion), or equivalent
  8. Heat lamp
  9. Perspex mouse restrainer

Procedure

  1. Transplantation of T cell-depleted bone marrow (optional; if using immunodeficient Rag-KO hosts proceed to step B)
    1. Host mice are typically aged between 8-12 weeks of age at the time of irradiation, and are of mixed sex. Mice aged between 6-8 weeks may also be suitable hosts, although we recommend providing young mice with softened food and avoiding the use of young females with low body weight (<20 g). We use an F1 cross between C57BL/6 and B10.BR mice as host mice for allogeneic bone marrow transplantation experiments, with B10.BR bone marrow/Treg donors. Use of F1 hosts is recommended due to superior Treg reconstitution in this system. We have successfully performed syngeneic Treg reconstitution on non-F1 host mice (e.g., B10.BR), but have observed inferior reconstitution using allogeneic Tregs (C57BL/6), possibly due to rejection by radio-resistant NK cells (Davis et al., 2015).
    2. Inject host mice with 100 μl vitamin K (10 mg/ml solution) via subcutaneous injection in either scruff or flank 1 day prior to irradiation using an insulin syringe. We routinely give lethally irradiated mice this prophylactic treatment to prevent post-irradiation coagulopathy that has occasionally been observed in our mouse colony. Additional injections are given 3 and 7 days after irradiation.
    3. Ear mark and weigh all host mice prior to irradiation.
    4. Transfer host mice into Perspex irradiation chambers and place into an appropriate container, then transfer to irradiator.
    5. Irradiate host mice at 600Rad, then transfer back into cages.
    6. After resting mice for 3 h, perform a further irradiation of 600Rad (repeating steps A4-5).
    7. 1 day after irradiation, harvest femurs and tibias from 6-20 week old allogeneic donor mice and collect into 20 ml RPMI + 10% FBS in a 50 ml conical tube. We recommend using 1 donor mouse for every 5-8 recipients.
    8. Trim the ends off each bone and flush out bone marrow with RPMI + 10% FBS using a 1 ml syringe with a 25 gauge needle.
    9. Resuspend bone marrow cells and filter through 70 μm nylon mesh into a 50 ml conical tube.
    10. Centrifuge cells at 125 x g for 10 min at 4 °C, and resuspend in 1 ml MACS wash and add anti-Thy1.2 hybridoma supernatant at a concentration of 1 x 106 cells/1 μl supernatant. Incubate on ice for 30 min. Alternatively anti-CD3 hybridoma supernatant can be used instead of anti-Thy1.2 to bind T cells.
    11. Spin down cells at 125 x g for 10 min at 4 °C.
    12. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    13. Resuspend pellet in 1 ml MACS wash and add anti-Rat IgG microbeads at a concentration of 1 x 107 cells/5 μl microbead solution and incubate at 4 °C for 15 min.
    14. Spin down cells at 125 x g for 10 min at 4 °C.
    15. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    16. Resuspend cells in 1 ml MACS running buffer per 2.5 x 108 cells.
    17. Add cells to LD column prepared according to manufacturer’s instructions with a pre-separation filter. Collect effluent, and wash column with 1 ml MACS running buffer 2 times. The unbound fraction contains T cell-depleted BM cells.
    18. Centrifuge T cell-depleted BM cells at 125 x g for 10 min at 4 °C.
    19. Resuspend pellet at 16.7 x 106 cells/ml in RPMI for injection. Store cells on ice until ready for use.
      Note: This concentration is suitable for transfer of 5 x 106 cells in a volume of 300 μl. The volume used for intravenous injections may vary depending on the policies of individual institutions. If so, the concentration of cells may be adjusted to allow transfer of 5 x 106 cells in an alternative volume.
    20. Warm host mice (irradiated the previous day) for approximately 10 min under a heat lamp to cause peripheral vasodilation, or for as long as needed to dilate the tail vein. Transfer mice to Perspex mouse restrainer. Using a 1 ml insulin syringe inject 300 μl of T cell-depleted bone marrow cells (or alternative volume containing 5 x 106 cells) into the tail vein of host mice. For a more detailed explanation of intravenous injections refer to Machholz et al. (2012).
    21. The drinking water of all irradiated mice is supplemented with sulfamethoxazole (80 μg/ml) and trimethoprim (160 μg/ml) for three weeks post-irradiation.

  2. Selective reconstitution of the Treg compartment of bone marrow transplant recipients or immunodeficient Rag-KO mice
    1. Wild type mice on an immunosufficient background aged between 6-20 weeks are used as donors for transfer into either allogeneic irradiated hosts (described in part A), or syngeneic immunodeficient Rag-KO hosts aged between 8-16 weeks. For transfer into Treg-reconstituted allogeneic bone marrow transplant recipients (part A) allogeneic B10.BR donors of 6-20 weeks of age are used. For transfer into Rag-KO mice several strain combinations have been tested with comparable results, including C57BL/6 donors into C57BL/6 Rag-KO hosts, B10.BR donors into B10.BR Rag-KO hosts, and [C57BL/6 x B10.BR]F1 donors into [C57BL/6 x B10.BR]F1 Rag-KO hosts. Treg reconstitution has been tested in both male and female host mice with success, but use of female host mice is avoided when Tregs are derived from male donors. A ratio of 1 donor mouse for every recipient is recommended.
    2. Remove lymph nodes and spleens from donor mice. Lymph nodes collected include the mandibular, accessory mandibular, superficial parotid, axillary, accessory axillary, subiliac, lumbar aortic, and jejunal (Van den Broeck et al., 2006). Collect into 10 ml RPMI + 10% FBS in a 50 ml conical tube.
    3. Prepare a single cell suspension via gentle mechanical dissociation of organs through stainless steel mesh sieves. Transfer single cell suspension to a 50 ml conical tube.
    4. Centrifuge cells at 125 x g for 10 min at 4 °C. For enrichment of CD4 T cells, resuspend in in 1 ml MACS buffer and add anti-B220, anti-CD8α, anti-CD11b and anti-Ter119 hybridoma supernatants at a concentration of 1 x 106 cells/1 μl of each supernatant. Incubate on ice for 30 min.
    5. Spin down cells at 125 x g for 10 min at 4 °C.
    6. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    7. Resuspend pellet in 1 ml MACS wash and add anti-Rat IgG microbeads at a concentration of 1 x 107 cells/5 μl microbead solution and incubate at 4 °C for 15 min.
    8. Spin down cells at 125 x g for 10 min at 4 °C.
    9. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    10. Resuspend cells in 1 ml MACS running buffer per 2.5 x 108 cells.
    11. Add cells to LD column prepared according to manufacturer’s instructions with a pre-separation filter. Split the sample across multiple LD columns if total cell numbers exceed 5 x 108 cells. Collect effluent in a 10 ml conical tube, and wash column with 1 ml MACS running buffer 2 times. The unbound fraction is enriched for CD4 T cells (approximately 70% pure).
    12. Centrifuge the CD4 T cell enriched fraction at 125 x g for 10 min at 4 °C.
    13. For selection of CD25+ cells from the CD4 T cell enriched fraction, resuspend cells in 1 ml MACS wash with CD25-PE at a concentration of 2 x 106 cells/1 μl antibody solution (i.e., 0.2 μg/2 x 106 cells). Incubate on ice for 30 min.
    14. Spin down cells at 125 x g for 10 min at 4 °C.
    15. Discard supernatant and resuspend pellet in 10 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    16. Resuspend pellet in 1 ml MACS wash and add anti-PE microbeads at a concentration of 1 x 106 cells/1 μl microbead solution and incubate at 4 °C for 15 min.
    17. Spin down cells at 125 x g for 10 min at 4 °C.
    18. Discard supernatant and resuspend pellet in 10 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    19. Resuspend cells in 1 ml MACS running buffer per 2 x 108 cells.
    20. Add cells to LS column prepared according to manufacturer’s instructions with a pre-separation filter. Collect effluent in 10 ml conical tubes, and wash column with 3 ml MACS running buffer 3 times. Elute the CD25-enriched fraction bound to the column in 5 ml MACS running buffer into a 10 ml conical tube.
    21. Spin down cells at 125 x g for 10 min at 4 °C.
    22. Discard supernatant and resuspend pellet at 8.3 x 106 cells/ml in RPMI for injection into either imunnodeficient Rag-KO hosts, or lethally irradiated BMT recipients (as described in part A). If Treg cells are to be injected into irradiated hosts the purified Treg cell suspension can be mixed with bone marrow cells (step A19) to be injected simultaneously.
      Note: This concentration is suitable for transfer of 2.5 x 106 cells in a volume of 300 μl. The volume used for intravenous injections may vary depending on the policies of individual institutions. If so, the concentration of cells may be adjusted to allow transfer of 2.5 x 106 cells in an alternative volume.
    23. Warm host mice under a heat lamp to cause peripheral vasodilation. Transfer mice to Perspex mouse restrainer. Using a 1 ml insulin syringe inject 300 μl of Treg cells (or alternative volume containing 2.5 x 106 cells) into the tail vein of host mice.
    24. All host mice receive a single injection of IL-2/JES6-1 complexes (see Recipes for preparation) in a volume of 100 μl using a 1 ml insulin syringe. Administer a repeat injection 2, 4 and 6 days post-Treg transfer.

  3. T cell transfer and reconstitution
    1. Remove lymph nodes (mandibular, accessory mandibular, superficial parotid, axillary, accessory axillary, subiliac, lumbar aortic, and jejunal) from donor mice and collect into 10 ml RPMI + 10% FBS in a 50 ml conical tube. For transfer of 2.5 x 106 cells, 1 donor is sufficient to reconstitute 8-10 recipient mice. For transfer into Treg-reconstituted allogeneic bone marrow transplant recipients (parts A and B) allogeneic B10.BR donors of 6-20 weeks of age are used. For Treg-reconstituted Rag-KO mice (part B) syngeneic T cell donors of 6-20 weeks of age are used (as described for part B). Either male or female donors may be used, but transfer of male donor cells into female hosts should be avoided.
    2. Prepare a single cell suspension via gentle mechanical dissociation of organs through stainless steel mesh sieves. Transfer into a 50 ml conical tube.
    3. Centrifuge cells at 125 x g for 10 min at 4 °C.
    4. For selection of CD4 T cells, resuspend pellet in 1 ml MACS buffer and add anti-B220, anti-CD8α, anti-CD11b and anti-Ter119 hybridoma supernatants at a concentration of 1 x 106 cells/1 μl of each supernatant. Incubate on ice for 30 min. If a mixed population of CD4 and CD8 T cells is required the anti-CD8α hybridoma supernatant can be omitted.
    5. Spin down cells at 125 x g for 10 min at 4 °C.
    6. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    7. Resuspend pellet in 1 ml MACS wash and add anti-Rat IgG microbeads at a concentration of 5 μl/1 x 107 cells/5 μl microbead solution and incubate at 4 °C for 15 min.
    8. Spin down cells at 125 x g for 10 min at 4 °C.
    9. Discard supernatant and resuspend pellet in 20 ml MACS wash, then spin down cells at 125 x g for 10 min at 4 °C. Repeat.
    10. Resuspend cells in 1 ml MACS running buffer per 2.5 x 108 cells.
    11. Add cells to LD column prepared according to manufacturer’s instructions with a pre-separation filter. Split the sample across multiple LD columns if total cell numbers exceed 5 x 108 cells. Collect effluent, and wash column with 1 ml MACS running buffer 2 times. The unbound fraction is enriched for T cells.
    12. Spin down cells at 125 x g for 10 min at 4 °C. 
    13. Discard supernatant and resuspend pellet at 8.3 x 106 cells/ml in RPMI for injection.
      Note: This concentration is suitable for transfer of 2.5 x 106 cells in a volume of 300 μl. The volume used for intravenous injections may vary depending on the policies of individual institutions. If so, the concentration of cells may be adjusted to allow transfer of 2.5 x 106 cells in an alternative volume.
    14. Warm host mice (from part B) under a heat lamp to cause peripheral vasodilation. Transfer mice to Perspex mouse restrainer.
    15. Using a 1 ml insulin syringe inject 300 μl of purified T cell suspension (or alternative volume containing 2.5 x 106 cells) into the tail vein of Treg-reconstituted mice (part B) at day 7 post-Treg transfer.

Notes

  1. We have found that using hybridoma supernatants in magnetic bead separation protocols to be a cost effective and flexible approach for the selection of various cell populations. However, similar results can also be achieved using commercially available antibodies. These may be either unlabeled and detected with anti-rat IgG microbeads as described in this protocol, or fluorochrome labeled and detected with mircobeads specific for the fluorochrome of interest (such as the CD25-PE positive selection described above). Alternatively, commercial kits for magnetic selection of specific cell populations such as Tregs can also be used.

Recipes

  1. MACS wash buffer
    Supplement 950 ml PBS with 10 mM EDTA and 50 ml sterile heat inactivated FBS Filter sterilize and stored at 4 °C
  2. MACS running buffer
    Supplement 1 L PBS with 0.5% BSA and 5 mM EDTA
    Filter sterilize and stored at 4 °C
  3. RPMI + 10% FBS
    Supplement 450 ml RPMI with 50 ml of sterile heat inactivated FBS
    Keep solution sterile and stored at 4 °C
  4. IL-2/JES6-1
    Note: A single dose of IL-2/JES6-1 complexes consists of 1 μg IL-2/5 μg JES6-1 (approximately a 2:1 molar ratio) (Boyman et al., 2006). Stock solutions of IL-2 and JES6-1 are stored frozen at -30 °C at 1 mg/ml.
    1. To prepare 100 doses of complexes, mix 100 μg IL-2 (0.1 ml) and 500 μg JES6-1 (0.5 ml) and incubate together in a 37 °C water bath for 30 min.
    2. Aliquot into appropriate volumes for single use (6 μl per injection) and store frozen at -70 °C.
    3. Dilute to a final working concentration of 100 μl per injection in PBS immediately prior to injection.
  5. Preparation of hybridoma supernatants
    1. Hybridoma cell lines are thawed and cultured in a 225 cm tissue culture flasks in RPMI + 10% FBS until dead cells begin to appear and medium is yellow (typically 7-10 days). Remove contents of flask and transfer into a 50 ml conical tube.
    2. Pellet cells by spinning at 125 x g for 10 min at 4 °C.
    3. Transfer supernatant into 50mL conical flasks, keeping the solution sterile. Stored at 4 °C.
      Note: We do not routinely quantify the concentration of antibody in our hybridoma supernatants. The volumes used in this protocol are in excess of what we have determined to be required using typical batches of supernatants, and are thus considered sufficient even if a particular batch of supernatant has a lower than average yield.

Acknowledgments

The protocols described here were adapted from our previously published work (Bolton et al., 2015). This work was supported by Australian National Health and Medical Research Council Project Grants 1012930, 1012522, 1012524 and 1051854, and Program Grant 427620. HAB was funded by an Australian Postgraduate Award and BFdeStG was funded by an Australian National Health and Medical Research Council Research Fellowship.

References

  1. Bolton, H. A., Zhu, E., Terry, A. M., Guy, T. V., Koh, W. P., Tan, S. Y., Power, C. A., Bertolino, P., Lahl, K., Sparwasser, T., Shklovskaya, E. and Fazekas de St Groth, B. (2015). Selective Treg reconstitution during lymphopenia normalizes DC costimulation and prevents graft-versus-host disease. J Clin Invest 125(9): 3627-3641.
  2. Boyman, O., Surh, C. D. and Sprent, J. (2006). Potential use of IL-2/anti-IL-2 antibody immune complexes for the treatment of cancer and autoimmune disease. Expert Opin Biol Ther 6(12): 1323-1331.
  3. Davis, J. E., Harvey, M., Gherardin, N. A., Koldej, R., Huntington, N., Neeson, P., Trapani, J. A. and Ritchie, D. S. (2015). A radio-resistant perforin-expressing lymphoid population controls allogeneic T cell engraftment, activation, and onset of graft-versus-host disease in mice. Biol Blood Marrow Transplant 21(2): 242-249.
  4. Machholz, E., Mulder, G., Ruiz, C., Corning, B. F. and Pritchett-Corning, K. R. (2012). Manual restraint and common compound administration routes in mice and rats. J Vis Exp(67).
  5. Van den Broeck, W., Derore, A. and Simoens, P. (2006). Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice. J Immunol Methods 312(1-2): 12-19.

简介

将成熟T细胞转移到免疫缺陷小鼠中导致外周T细胞库的次优重建。在淋巴细胞减少症状下,树突状细胞通过调节性T细胞(Tregs)从强直对照释放,并因此驱动对内源性抗原特异的低亲和力T细胞的激活和增殖。这种寡克隆增殖导致由具有效应/记忆表型和有限TCR库的T细胞支配的T细胞群。可以通过在T细胞转移之前选择性重建Treg区室来防止寡克隆扩增(Bolton等人,2015)。已经在免疫缺陷小鼠品系例如Rag-1 /或 Rag-2 -/- 中测试了淋巴细胞减少小鼠的Treg区室的重建。 小鼠,以及在通过致死性全身照射作为调节骨髓移植(BMT)的瞬时淋巴细胞减少的免疫小鼠中。将纯化的Treg转移到这些宿主中,结合用外源IL-2处理7天,足以重建Treg区室并减少树突细胞共刺激分子的表达,这是防止自身反应性T细胞不适当扩增的关键过程。在Treg重建后转移的T细胞不经历快速的自发增殖,而是进行慢的内稳态分裂以用天然T细胞重新增殖T细胞库,从而允许外周T细胞库的最佳重建。

关键字:lymphopenia, 免疫重建, T细胞, 调节T细胞, 动物模型

材料和试剂

  1. 50ml锥形管(Corning,Falcon ,目录号:352070)
  2. 10ml锥形管(SARSTEDT AG& Co,目录号:62.9924.272)
  3. 70μm尼龙细胞过滤器(Corning,Falcon ,目录号:352350)
  4. 1ml注射器(BD,目录号:309659)
  5. 25号针(BD,目录号:305125)
  6. 1ml带有29G针头的胰岛素注射器(BD,目录号:326719)
  7. 225cm组织培养瓶(Sigma-Aldrich,目录号:CLS431082)
  8. LD柱(Miltenyi Biotec,目录号:130-042-901)
  9. LS柱(Miltenyi Biotec,目录号:130-042-401)
  10. 维生素K1,10mg/ml(International Animal Health Products,Koagulon)
  11. PBS(10x,pH 7.4,不含Ca 2+和Mg 2+ 2)(Mediatech Inc.,目录号:46-013-CM)
  12. 在65℃热灭活的胎牛血清(FBS)(Scientifix,目录号:FBS-500S)
  13. 牛血清白蛋白(BSA)级分V(Amresco,目录号:0332-1kg)
  14. 乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:E5134)
  15. RPMI-1640(Thermo Fisher Scientific,Gibco TM ,目录号:11875-093)
  16. 大鼠抗小鼠Thy1.2克隆30H12杂交瘤上清液(自制)
  17. 大鼠抗小鼠B220克隆RA3.6B2杂交瘤上清液(自制)
  18. 大鼠抗小鼠CD8a克隆53-6.7杂交瘤上清液(室内制备)
  19. 大鼠抗小鼠CD11b克隆M1/70杂交瘤上清液(自制)
  20. 大鼠抗小鼠红细胞谱系克隆Ter119杂交瘤上清液(室内制备)
  21. 抗CD25 PE克隆PC61(BD Biosciences,目录号:553866)
  22. 抗大鼠IgG微珠(Miltenyi Biotec,目录号:130-048-501)
  23. 抗PE微珠(Miltenyi Biotec,目录号:130-048-801)
  24. 重组小鼠IL-2(Peprotech,目录号:212-12)
  25. 抗IL-2mAb克隆JES6-1(WEHI单克隆抗体工具)
  26. 磺胺甲恶唑(400mg/5ml)和甲氧苄啶(80mg/5ml)(Hospira,DBL,目录号:618670BAU)
  27. MACS洗涤缓冲液(参见配方)
  28. MACS运行缓冲区(参见配方)
  29. RPMI + 10%FBS(参见配方)
  30. IL-2/JES6-1(见配方)
  31. 杂交瘤上清液的制备(参见配方)

设备

  1. 外科剪刀和镊子
  2. 80规格不锈钢网筛
  3. MACS MultiStand(Miltenyi Biotec,目录号:130-042-303)
  4. QuadroMACS分离器(Miltenyi Biotec,目录号:130-090-976)或MidiMACS分离器(Miltenyi Biotec,目录号:130-042-302)
  5. 37°C水浴
  6. 台式离心机
  7. Gammacell辐照器(Nordion)或等同物
  8. 加热灯
  9. 有机玻璃鼠标限制器

程序

  1. 移植T细胞耗尽的骨髓(任选的;如果使用免疫缺陷的Rag-KO宿主进行步骤B)
    1. 宿主小鼠通常在8-12周龄之间 辐射,并且是混合性别。年龄在6-8周之间的小鼠也可以 是适合的主机,虽然我们建议提供年轻小鼠 软化食物,避免使用体重低的年轻女性  (<20g)。我们使用C57BL/6和B10.BR小鼠之间的F1杂交作为宿主 小鼠用于同种异体骨髓移植实验,用B10.BR  骨髓/Treg供体。推荐使用F1主机,因为优越  Treg重建在这个系统中。我们已经成功执行 对非F1宿主小鼠(例如,B10.BR)的同基因Treg重建,但是 使用同种异体Treg(C57BL/6)观察到较差的重建, 可能是由于放射抗性NK细胞的排斥(Davis等人, 2015)。
    2. 注射宿主小鼠与100微升维生素K(10毫克/毫升 溶液)通过皮下注射在侧腹或侧腹1天 然后使用胰岛素注射器进行照射。 我们常规给 致死辐射小鼠这种预防性治疗以预防 后照射凝血病,偶尔在我们的观察   小鼠集落。 在3和7天后给予额外的注射 辐照
    3. 耳标并在所有的主机小鼠照射前称重。
    4. 将宿主小鼠转移到Perspex照射室,并放入适当的容器,然后转移到辐射器
    5. 照射宿主小鼠在600Rad,然后转移回笼子
    6. 休息小鼠3小时后,进行600Rad的进一步照射(重复步骤A4-5)
    7. 照射后1天,从6-20周收获股骨和胫骨 老同种异体供体小鼠,并收集在20ml RPMI + 10%FBS中 ml锥形管。 我们建议每5-8个使用1个供体小鼠 收件人
    8. 修剪每个骨的末端,并使用1ml具有25号针的注射器用RPMI + 10%FBS冲洗出骨髓。
    9. 重悬的骨髓细胞,通过70微米尼龙网过滤到50毫升锥形管
    10. 在4℃下以125×g离心细胞10分钟,并在1℃重悬 ml MACS洗涤并在a中加入抗Thy1.2杂交瘤上清液 浓度为1×10 6个细胞/1μl上清液。 在冰上孵育30 min。 或者,可以使用抗CD3杂交瘤上清液代替   抗Thy1.2以结合T细胞
    11. 在4℃下以125×g离心细胞10分钟。
    12. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    13. 重悬沉淀在1ml MACS洗涤,并加入抗大鼠IgG微珠 浓度为1×10 7个细胞/5μl微珠溶液并孵育   在4℃下15分钟
    14. 在4℃下以125×g离心细胞10分钟。
    15. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    16. 在每2.5×10 8个细胞的1ml MACS运行缓冲液中重悬细胞
    17. 添加细胞到根据制造商准备的LD柱 说明书带有预分离过滤器。 收集流出物,并洗涤 柱用1ml MACS运行缓冲液2次。 未结合部分 包含T细胞耗尽的BM细胞
    18. 在4℃下以125×g离心10分钟离心T细胞耗尽的BM细胞。
    19. 在注射用RPMI中以16.7×10 6个细胞/ml重悬沉淀物。 将细胞储存在冰上,直到准备使用。
      注意:此浓度适用于传输5 x 10 6个   体积300μl。 用于静脉内注射的体积可以变化 取决于个别机构的政策。 如果是这样, 可以调整细胞浓度以允许在替代体积中转移5×10 个细胞。
    20. 温暖的宿主小鼠(辐照 前一天)在加热灯下引发约10分钟 外周血管舒张,或者只要需要扩张尾部 静脉。 转移小鼠到Perspex鼠标限制器。 使用1ml胰岛素 注射300微升T细胞耗尽的骨髓细胞(或 备选体积含有5×10 6个细胞)进入宿主的尾静脉 老鼠。 有关静脉注射的更详细解释,请参阅   Machholz等人(2012)。
    21. 所有照射的饮用水 小鼠补充磺胺甲恶唑(80μg/ml)和甲氧苄氨嘧啶 (160μg/ml)照射三周。

  2. 骨髓移植受体或免疫缺陷型Rag-KO小鼠的Treg区室的选择性重建
    1. 在6-20周龄的免疫缺陷背景上的野生型小鼠  用作转移到同种异体照射的宿主中的供体  (在部分A中描述)或老化的同基因的免疫缺陷型Rag-KO宿主 8-16周。转移到Treg重建的同种异体骨  骨髓移植受者(A部分)同种异体B10.BR供体6-20 周龄。为了转移到Rag-KO小鼠几个菌株 组合已经用相当的结果测试,包括C57BL/6  供体进入C57BL/6Rag-KO宿主,B10.BR供体进入B10.BR Rag-KO 宿主和[C57BL/6×B10.BR] F1供体转化为[C57BL/6×B10.BR] F1 Rag-KO 主机。已经在男性和女性宿主中测试了Treg重建 小鼠成功,但当使用雌性宿主小鼠时,避免Tregs  来自男性供体。每个受体的1个供体小鼠的比率 建议使用。
    2. 从捐赠小鼠去除淋巴结和脾。 收集的淋巴结包括下颌,附属下颌,浅表腮腺,腋窝,附属腋窝,subiliac,腰椎 主动脉和空肠(Van den Broeck等人,2006)。 收集10毫升 RPMI + 10%FBS在50ml锥形管中
    3. 准备单个单元格 悬浮通过器官的温和机械解离 不锈钢网筛。 转移单细胞悬浮液到50毫升 锥形管
    4. 在4℃下以125×g离心细胞10分钟。 对于   富集CD4 T细胞,重悬于1ml MACS缓冲液中并加入 抗B220,抗CD8α,抗CD11b和抗Ter119杂交瘤上清液 浓度为1×10 6个细胞/1μl的每种上清液。 孵化 在冰上30分钟
    5. 在4℃下以125×g离心细胞10分钟。
    6. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    7. 重悬沉淀在1ml MACS洗涤,并加入抗大鼠IgG微珠 浓度为1×10 7个细胞/5μl微珠溶液并孵育   在4℃下15分钟
    8. 在4℃下以125×g离心细胞10分钟。
    9. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    10. 在每2.5×10 8个细胞的1ml MACS运行缓冲液中重悬细胞
    11. 添加细胞到根据制造商准备的LD柱 说明书带有预分离过滤器。 拆分样品 多个LD柱,如果总细胞数量超过5×10 8个细胞。 搜集 流出物在10ml锥形管中,并且洗涤柱用1ml MACS运行   缓冲液2次。 未结合部分富集CD4T细胞 (约70%纯)
    12. 在4℃下将CD4 + T细胞富集级分在125×g离心10分钟。
    13. 为了从CD4T细胞富集级分中选择CD25 + sup +细胞,   重悬细胞在1ml MACS洗涤用浓度为2x的CD25-PE   10×10 6个细胞/1μl抗体溶液(即0.2μg/2×10 6个细胞)。 孵化 在冰上30分钟。
    14. 在4℃下以125×g离心细胞10分钟。
    15. 弃去上清液并在10ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    16. 重悬在1ml MACS洗涤沉淀和添加抗PE微珠在a 浓度为1×10 6个细胞/1μl微珠溶液,并在4℃下孵育   ℃下15分钟
    17. 在4℃下以125×g离心细胞10分钟。
    18. 弃去上清液并在10ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    19. 在1ml MACS运行缓冲液/2×10 8个细胞中重悬细胞
    20. 添加细胞到根据制造商准备的LS柱说明书带有预分离过滤器。收集流出物在10毫升 锥形管,并用3ml MACS运行缓冲液洗涤柱3次。 洗脱在5ml MACS中结合到柱上的富含CD25的级分 运行缓冲液倒入10ml锥形管中
    21. 在4℃下以125×g离心细胞10分钟。
    22. 弃去上清液并以8.3×10 6个细胞/ml重悬浮沉淀 RPMI用于注射到免疫缺陷型Rag-KO宿主中,或致死  (如A部分所述)。如果Treg细胞 以将经纯化的Treg细胞悬浮液注射到受照射的宿主中 可以与骨髓细胞混合(步骤A19)以进行注射 同时。
      注意:此浓度适合转移 在300μl体积中的2.5×10 6 用于的卷 静脉注射可能会根据个人的政策而有所不同 机构。如果是,可以调节细胞的浓度以允许在替代卷中传输2.5 x 10 6 个单元格。
    23. 暖主机   小鼠在加热灯下引起外周血管舒张。 转移小鼠 到Perspex鼠标限制器。 使用1毫升胰岛素注射器注射300微升 的Treg细胞(或含有2.5×10 6个细胞的替代体积) 宿主小鼠的尾静脉。
    24. 所有宿主小鼠接受单一 注射IL-2/JES6-1复合物(参见制备方法) 体积为100μl,使用1ml胰岛素注射器。 管理重复 注射2,4和6天后Treg转移。

  3. T细胞转移和重建
    1. 切除淋巴结(下颌,下颌,浅表 腮腺,腋窝,附属腋下,下颌,腰主动脉和 空肠),并在50℃下收集到10ml RPMI + 10%FBS中 ml锥形管。 对于2.5×10 6个细胞的转移,1个供体就足够了 重建8-10受体小鼠。 转入 Treg-重建的同种异体骨髓移植受者(A部分   和B)使用6-20周龄的同种异体B10.BR供体。 对于 Treg重建的Rag-KO小鼠(部分B)6-20的同基因T细胞供体 (如对于部分B所述)。 男性或女性 可以使用供体,但将雄性供体细胞转移到雌性宿主中 应避免。
    2. 通过温和制备单细胞悬浮液 通过不锈钢网筛机械解离器官。 转移到50ml锥形管中
    3. 在4℃下以125×g离心细胞10分钟。
    4. 对于CD4T细胞的选择,将沉淀重悬于1ml MACS缓冲液中 并加入抗B220,抗CD8α,抗CD11b和抗Ter119杂交瘤 浓度为1×10 6个细胞/1μl的上清液 上清液。 在冰上孵育30分钟。 如果混合群体的CD4 和CD8 T细胞需要抗CD8α杂交瘤上清液可以 省略。
    5. 在4℃下以125×g离心细胞10分钟。
    6. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    7. 重悬沉淀在1ml MACS洗涤,并加入抗大鼠IgG微珠 浓度为5μl/1×10 7个细胞/5μl微珠溶液, 在4℃孵育15分钟
    8. 在4℃下以125×g离心细胞10分钟。
    9. 弃去上清液并在20ml MACS洗涤液中重悬沉淀,然后在4℃下以125×g离心细胞10分钟。 重复。
    10. 在每2.5×10 8个细胞的1ml MACS运行缓冲液中重悬细胞
    11. 添加细胞到根据制造商准备的LD柱 说明书带有预分离过滤器。 拆分样品 多个LD柱,如果总细胞数量超过5×10 8个细胞。 搜集 流出物和洗涤柱用1ml MACS运行缓冲液2次。 的 未结合部分富集T细胞
    12. 在4℃下以125×g离心细胞10分钟。
    13. 弃去上清液并以8.3×10 6个细胞/ml重悬浮沉淀 RPMI注射。
      注意:此浓度适合转移   2.5×10 6 用于静脉注射的体积 注射可能会根据个人的政策而有所不同 机构。 如果是,可以调节细胞的浓度以允许   在替代卷中传输2.5 x 10 6 个单元格。
    14. 温热的宿主小鼠(来自部分B)在加热灯下引起外周血管舒张。 将鼠标转移到Perspex鼠标限制器。
    15. 使用1ml胰岛素注射器注射300μl纯化的T细胞 悬浮液(或含有2.5×10 6个细胞的替代体积)尾静脉的Treg重建的小鼠(部分B) 转让。

笔记

  1. 我们已经发现,在磁珠分离方案中使用杂交瘤上清液是用于选择各种细胞群体的成本有效和灵活的方法。 然而,使用市售的抗体也可以获得相似的结果。 这些可以是未标记的并且用本方案中所述的抗大鼠IgG微珠检测,或用荧光染料标记并用对感兴趣的荧光染料特异性的mircobeads(例如上述的CD25-PE阳性选择)检测。 或者,也可以使用用于特定细胞群体(例如Treg)的磁性选择的商业试剂盒。

食谱

  1. MACS洗涤缓冲液
    补充含有10mM EDTA和50ml无菌热灭活FBS的950ml PBS过滤灭菌并在4℃下保存
  2. MACS运行缓冲区
    补充1L含0.5%BSA和5mM EDTA的PBS
    过滤灭菌并在4℃下保存
  3. RPMI + 10%FBS
    补充450毫升RPMI与50毫升无菌热灭活FBS
    保持溶液无菌,储存在4°C
  4. IL-2/JES6-1
    注意:单剂量的IL-2/JES6-1复合物由1μgIL-2 /5μgJES6-1(约2:1摩尔比)组成(Boyman等人,2006)。 将IL-2和JES6-1的储备溶液在-30℃下以1mg/ml冷冻储存。
    1. 为了制备100个剂量的复合物,混合100μgIL-2(0.1ml)和500μg JES6-1(0.5ml),并在37℃水浴中一起孵育30分钟。
    2. 等分到适当的体积一次性使用(每次注射6微升),并存储在-70℃冷冻。
    3. 在注射前,立即稀释至每次注射100μl的最终工作浓度。
  5. 制备杂交瘤上清液
    1. 将杂交瘤细胞系解冻并在225cm组织培养物中培养 烧瓶在RPMI + 10%FBS中,直到死细胞开始出现,培养基是 黄色(通常7-10天)。 取出烧瓶中的内容物并转入一个50ml锥形管。
    2. 通过在4℃下以125×g旋转10分钟来沉淀细胞。
    3. 将上清转移到50mL锥形瓶中,保持溶液无菌。 储存于4°C。
      注意:我们不常规量化我们的抗体浓度   杂交瘤上清液。 此协议中使用的卷过量   我们确定需要使用典型的批次 上清液,因此即使是特定的,也被认为是足够的 批次的上清液具有低于平均产量。

致谢

这里描述的协议改编自我们以前发表的工作(Bolton等人。,2015)。这项工作得到澳大利亚国家卫生和医学研究委员会项目拨款1012930,1012522,1012524和1051854以及计划拨款427620的支持.HAB由澳大利亚研究生奖励资助,BFdeStG由澳大利亚国家卫生和医学研究委员会研究奖学金。

参考文献

  1. Bolton,HA,Zhu,E.,Terry,AM,Guy,TV,Koh,WP,Tan,SY,Power,CA,Bertolino,P.,Lahl,K.,Sparwasser,T.,Shklovskaya,E.and Fazekas de St Groth,B。(2015)。 淋巴细胞减少期间的选择性Treg重建使DC共刺激正常化,并预防移植物抗宿主病。 J Clin Invest 125(9):3627-3641。
  2. Boyman,O.,Surh,C.D.and Sprent,J。(2006)。 潜在使用IL-2 /抗IL-2抗体免疫复合物治疗癌症和自身免疫性疾病。 Expert Opin Biol Ther 6(12):1323-1331。
  3. Davis,J.E.,Harvey,M.,Gherardin,N.A.,Koldej,R.,Huntington,N.,Neeson,P.,Trapani,J.A。和Ritchie, 抗辐射穿孔素的淋巴细胞群控制同种异体T细胞植入,活化和移植的发生 - 小鼠中的 - 宿主疾病。生物血液移植 21(2):242-249。
  4. Machholz,E.,Mulder,G.,Ruiz,C.,Corning,B.F.and Pritchett-Corning,K.R。(2012)。 在小鼠和大鼠中手动约束和常见的化合物给药途径。 Exp (67)。
  5. Van den Broeck,W.,Derore,A。和Simoens,P。(2006)。 鼠淋巴结的解剖和命名:BALB/cAnNCrl小鼠中的描述性研究和命名标准化。 a Immunology Methods 312(1-2):12-19。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bolton, H. A. and Groth, B. F. (2016). Reconstitution of Lymphopaenic Mice with Regulatory and Conventional T cell Subsets. Bio-protocol 6(10): e1814. DOI: 10.21769/BioProtoc.1814.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。