Phenotyping of adoptively transferred and endogenous tumor-infiltrating CD8+ T cells in the B16F10 orthotopic melanoma model

Phenotyping of adoptively transferred and endogenous tumor-infiltrating CD8⁺ T cells in the B16F10 orthotopic melanoma model

Ⅰ. Isolated lymphocytes from the B16F10 orthotopic melanoma-bearing mice

1. B16F10 orthotopic melanoma-bearing mice were euthanized by CO₂ inhalation.

2. Tumors were collected, followed by shredding and digesting with 10 ml collagenase IV for 1 hour at 37℃.

3. The digested tumors were ground through a 70 µm filter, and the red blood cells were removed by incubation with ACK lysis buffer for 5 minutes at 4℃. After washing with ice-cold PBS thrice, cells were resuspended in 1 ml RPMI 1640 medium to obtain a single cell suspension.

4. To prepare the Percoll separation fluid, 2 ml 70% (v:v) Percoll in phosphate-buffered saline (PBS) was added into the 15 ml centrifuge tube as the lower layer, followed by the addition of 2 ml 40% (v:v) Percoll in PBS as the upper layer. 

5. The single cell suspension (1 mL) was slowly added onto the surface of Percoll separation fluid, followed by centrifugation at 800 g for 30 min.

6. The lymphocytes was collected from the middle layer and washed with ice-cold PBS thrice, which were ready for the following studies.

Ⅱ. Phenotyping of adoptively transferred CD8⁺ T cells in tumor

A. The cytokine and granule production of adoptively transferred CD8⁺ T cells in tumor 

1. The obtained lymphocytes were stimulated by incubating with 1 µM ionomycin and 50 ng/ml phorbol 12-myristate-13-acetate (PMA) for 4 hours in the presence of 5 µg/ml brefeldin A at 37℃, 5% CO₂.

2. Cells were collected and washed with ice-cold PBS thrice.

3. Cells were stained with 1 µg/ml phycoerythrin (PE)-Cy7 anti-mouse Thy1.1 at 4℃ for 30 minutes to label the adoptively transferred CD8⁺ T cells, and washed with ice-cold PBS thrice.

4. Next, cells were fixed with 4% Paraformaldehyde (PFA) for 15 minutes at room temperature and permeabilized by 0.2% Triton X-100 for 15 minutes at room    temperature.

5. Then, cells were washed with ice-cold PBS and stained with 1 µg/ml fluorescein isothiocyanate (FITC) anti-mouse granzyme B (GzmB), 0.6 µg/ml allophycocyanin (APC) anti-mouse interferon-gamma (IFNγ) and 0.5 µg/ml PE anti-mouse tumor necrosis factor α (TNFα) for 30 minutes at 4℃, respectively.

6. After that, cells were washed with ice-cold PBS thrice. The cytokine and granule production of adoptively transferred CD8⁺ T cells were analyzed by flow cytometry.

B. Exhaustion of adoptively transferred CD8⁺ T cells in tumor

1. The obtained lymphocytes were stained with 1 µg/ml PE-Cy7 anti-mouse Thy1.1 at 4℃ for 20 minutes to label the adoptively transferred CD8⁺ T cells, and washed with ice-cold PBS thrice.

2. Then, cells were divided into two groups.

3. Cells of first group were stained with 0.4 µg/ml APC anti-mouse programmed cell death protein 1 (PD-1, CD279) and 0.4 µg/ml PE anti-mouse T cell Ig and ITIM domain (TIGIT, Vstm3) for 20 minutes at 4℃.

4. Cells of second group were stained with 0.4 µg/ml APC anti-mouse CD366 (T cell immunoglobulin domain and mucin domain-3, TIM-3), 0.6 µg/ml PE anti-mouse CD152 (Cytotoxic T lymphocyte-associated antigen-4, CTLA-4) and 0.6 µg/ml FITC anti-mouse CD223 (Lymphocyte-activation gene 3, LAG-3) for 20 minutes at 4℃.

5. The two group cells were collected and washed with ice-cold PBS thrice. Then, the exhausted markers of adoptively transferred CD8⁺ T cells were analyzed by flow cytometry.

C. The cytokine and granule production of exhausted adoptively transferred CD8⁺ T cells in tumor

1. The obtained lymphocytes were stimulated by incubating with 1 µM ionomycin and 50 ng/ml PMA for 4 hours in the presence of 5 µg/ml brefeldin A at 37℃, 5% CO₂.

2. Cells were collected and washed with ice-cold PBS thrice.

3. Cells were stained with 1 µg/ml PE-Cy7 anti-mouse Thy1.1 at 4℃ for 30 minutes to label the adoptively transferred CD8⁺ T cells.

4. Then, cells were washed with ice-cold PBS thrice and divided into three groups.

5. Cells of first group were stained with 0.4 µg/ml APC anti-mouse PD-1 (CD279) for 20 minutes at 4℃, and washed twice with ice-cold PBS. Then cells were fixed with 4% PFA for 15 minutes at room temperature and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature. After washing with ice-cold PBS, cells were stained with 1 µg/ml FITC anti-mouse GzmB, 0.6 µg/ml Peridinin-chlorophyll-protein complex (Percp)-Cy5.5 anti-mouse IFNγ and 0.5 µg/ml PE anti-mouse TNFα for 30 minutes at 4℃.

6. Cells of second group were stained with 0.4 µg/ml APC anti-mouse TIGIT for 20 minutes at 4℃, and washed twice with ice-cold PBS. Then, cells were fixed with 4% PFA for 15 minutes at room temperature and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature. After washing with ice-cold PBS, cells were stained with 1 µg/ml FITC anti-mouse GzmB, 0.6 µg/ml Percp-Cy5.5 anti-mouse IFNγ and 0.5 µg/ml PE anti-mouse TNFα for 30 minutes at 4℃.

7. Cells of third group were stained with 0.4 µg/ml APC anti-mouse CD366 (TIM-3) for 20 minutes at 4℃, and washed twice with ice-cold PBS. Then cells were fixed with 4% PFA for 15 minutes at room temperature and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature. After washing with ice-cold PBS, cells were stained with 1 µg/ml FITC anti-mouse GzmB, 0.6 µg/ml Percp-Cy5.5 anti-mouse IFNγ and 0.5 µg/ml PE anti-mouse TNFα for 30 minutes at 4℃.

8. The three group cells were washed with ice-cold PBS thrice. The cytokine and granule production of exhausted adoptively transferred CD8⁺ T cells were analyzed by flow cytometry.

D. Proliferation and the amount of membrane cholesterol of adoptively transferred CD8⁺ T cells in tumor

1. Lymphocytes were stained with 1 µg/ml PE-Cy7 anti-mouse Thy1.1 at 4℃ for 20 minutes to label the adoptively transferred CD8⁺ T cells.

2. Then, cells were washed with ice-cold PBS thrice and fixed with 4% PFA for 15 minutes at room temperature.

3. Cells were washed with ice-cold PBS and stained with 50 µg/ml Filipin III for 30 minutes at 4℃.

4. Cells were washed with ice-cold PBS and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature. Then, cells were washed with ice-cold PBS and stained with 0.6 µg/ml PE anti-mouse Ki67 for 20 minutes at 4℃.

5. Cells were washed with ice-cold PBS thrice and analyzed by flow cytometry.

Ⅲ. Phenotyping of tumor-infiltrating CD8⁺ T cells in the B16F10 orthotopic melanoma-bearing mice

A. The cytokine and granule production of tumor-infiltrating CD8⁺ T cells

1. The obtained lymphocytes were stimulated by incubating with 1 µM ionomycin and 50 ng/ml PMA for 4 hours in the presence of 5 µg/ml brefeldin A at 37℃, 5% CO₂.

2. Cells were collected and washed with ice-cold PBS thrice.

3. Cells were stained with 1 µg/ml APC-Cy7 anti-mouse CD8a at 4℃ for 20 minutes to label the tumor-infiltrating CD8⁺ T cells, and washed with ice-cold PBS thrice.

4. Next, cells were fixed with 4% PFA for 15 minutes at room temperature and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature.

5. After washing with ice-cold PBS thrice, cells were stained with 1 µg/ml FITC anti-mouse GzmB, 0.6 µg/ml APC anti-mouse IFNg and 0.5 µg/ml PE anti-mouse TNFα for 30 minutes at 4℃, respectively.

6. After that, cells were washed with ice-cold PBS thrice. The cytokine and granule production of tumor-infiltrating CD8⁺ T cells were analyzed by flow cytometry.

B. Exhaustion of tumor-infiltrating CD8⁺ T cells

1. The obtained lymphocytes were stained with 1 µg/ml APC-Cy7 anti-mouse CD8a at 4℃ for 20 minutes to label the tumor-infiltrating CD8⁺ T cells, and washed with ice-cold PBS thrice.

2. Then, cells were divided into two groups.

3. Cells of first group were stained with 0.4 µg/ml APC anti-mouse PD-1 (CD279) and 0.4 µg/ml PE anti-mouse TIGIT (Vstm3) antibody for 30 minutes at 4℃.

4. Cells of second group were stained with 0.4 µg/ml APC anti-mouse CD366 (TIM-3), 0.6 µg/ml PE anti-mouse CD152 (CTLA-4) and 0.6 µg/ml FITC anti-mouse CD223 (LAG-3) for 30 minutes at 4℃, respectively.

5. The two group cells were collected and washed with ice-cold PBS thrice. The exhausted markers of tumor-infiltrating CD8⁺ T cells were analyzed by flow cytometry.

C. Proliferation and the amount of membrane cholesterol of tumor-infiltrating CD8⁺ T cells

1. Lymphocytes were stained with 1 µg/ml APC-Cy7 anti-mouse CD8a at 4℃ for 20 minutes to label the tumor-infiltrating CD8⁺ T cells.

2. Then, cells were washed with ice-cold PBS thrice and fixed with 4% PFA for 15 minutes at room temperature.

3. Cells were washed with ice-cold PBS, and stained with 50 µg/ml Filipin III for 30 minutes at 4℃.

4. Cells were washed with ice-cold PBS and permeabilized by 0.2% Triton X-100 for 15 minutes at room temperature. After washing with ice-cold PBS, cells were stained with 0.6 µg/ml PE anti-mouse Ki67 for 20 minutes at 4℃.

5. Cells were washed with ice-cold PBS thrice and analyzed by flow cytometry.

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