Mixed bone marrow and mixed T cell adoptive transfer

Mixed bone marrow and T cell transfer

Material and reagents

1. Rag1, B6, IL15Ra, B6.SJL, OT1 and CD4cre mice were from Jackson laboratory.
2. Otub1 mouse was generated in our laboratory and bred with CD4cre to generate T cell conditional knockout mice.
3. 23-gauge needle (BD Biosciences, catalog number: 305145).
4. 70 μm nylon cell strainer (Corning, catalog number: 431751).
5. 3 ml syringe plunger (BD Biosciences, catalog number: 309657).
6. PBS (Corning, catalog number:21-040-CV)
7. RBC lysis buffer (sigma, catalog number: R7767-100ml)
8. BSA (Roche, catalog number: 03 116 964 001)
9. 0.5M sterile EDTA solution (Amresco, catalog number: E177-500ml)
10. MACS buffer (1xPBS with 2mM EDTA and 0.1% BSA)
11. Anti-CD90.2 microbeads (130-121-278), anti-CD8 microbeads (130-117-044) from Miltenyi Biotec  
12. CellTrace^tm CFSE cell proliferation kit (Thermo Fisher, catalog number: C34554)
13. Anti-mouse CD8a PE-Cy7 (eBioscience, catalog number :25-0081-82); anti-m/h CD44 APC-Cy7 (eBioscience, catalog number: 47-0441-82); anti-mouse CD62L APC (eBioscience, catalog number: 17-0621-83)

Equipment:

1. Sorvall legend RT refrigerated benchtop centrifuge 
2. ¹³⁷Cs irradiator
3. FACSAria(BD Bioscience)

Procedure for bone marrow isolation and transfer

1. Isolate bone marrow cell from femur and tibia of Otub1-CD4cre and SJL mice.
2. Pellet the cells by centrifugation at 500x g for 5 minutes at room temperature and decant the supernatant.
3. Lysate red blood cells with 1ml RBS/mouse for 8 minutes at room temperature.
4. Stop the lysis reaction by adding 10ml PBS. 
5. Filter the cell with 70um cell strainer and centrifuge immediately at 500x g for 5 minutes at room temperature, decant the supernatant.
6. Resuspend pellet with MACS buffer and add anti-CD90.2 microbeads to remove T cells from bone marrow cell.
7. Count bone marrow cells and mix the Otub1-CD4cre bone marrow cell with SJL bone marrow cell in 1:1 ratio.
8. Rag1^−/− mice were exposed to 1000 rads total body irradiation and, 6 h later, were intravenously transferred with 2×10⁶ mixed bone marrow cells.
9. After 6 weeks, recipient mice were sacrificed for flow cytometry analysis of Otub1 knockout (CD45.2) and WT (CD45.1) T cells.

Procedure for polyclonal CD8 T cell isolation and transfer

1. Isolate polyclonal CD8 T cells from pooled spleens and lymph nodes of Otub1-CD4cre and SJL mice and lysis red blood cells with RBC buffer.
2. Purify the CD8 T cells with anti-CD8a microbeads.
3. CD8⁺ T cells were further stained with CD8a PE-Cy7, CD44 APC-Cy7 and CD62L APC antibody to sort CD44^-CD62L⁺ naïve T cells.
4. 10x10⁶ naïve CD8 T cells were stained with CFSE at 1nM final concentration.
5. 0.5x10⁶ of Otub1-CD4cre naïve CD8 T cells were mixed with 0.5x10⁶ of SJL naïve CD8 T cells in 1:1 ratio.
6. B6 and IL15Ra mice were exposed to 600 rads irradiation and, 6h later, were intravenously transferred with 1x10⁶ mixed naïve CD8 T cells.
7. After 7 days, recipient mice were sacrificed for flow cytometry analysis of T cell proliferation and homeostasis.

Procedure for monoclonal CD8 T cell isolation and transfer

1. Isolate monoclonal CD8 T cells from pooled spleens and lymph nodes of Otub1-CD4cre-OT1(CD45.2⁺) and SJL/OT1(CD45.1⁺CD45.2⁺) mice and lysis red blood cells with RBC buffer.
2. Purify the CD8-OT1 T cells with anti-CD8a microbeads.
3. CD8⁺ OT1 T cells were further stained with CD8a PE-Cy7, CD44 APC-Cy7 and CD62L APC antibody to sort CD44^-CD62L⁺ naïve T cells.
4. 10x10⁶ naïve CD8 OT1 T cells were stained with CFSE at 1nM final concentration.
5. 1x10⁶ of Otub1-CD4cre-OT1 naïve CD8 T cells were mixed with 1x10⁶ of SJL/OT1 naïve CD8 T cells in 1:1 ratio.
6. B6 and IL15Ra mice were exposed to 600 rads irradiation and, 6h later, were intravenously transferred with 2x10⁶ mixed naïve CD8-OT1 T cells.
7. After 7 days, recipient mice were sacrificed for flow cytometry analysis of T cell proliferation and homeostasis.

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