Journal: Cancer Immunology Research
Article Title: PVRL2 Suppresses Antitumor Immunity through PVRIG- and TIGIT-independent Pathways
doi: 10.1158/2326-6066.CIR-23-0722
Figure Lengend Snippet: PVRL2 regulates CD8 T-cell and NK-cell activation. A, Average tumor volume over time following subcutaneous injection of 1 × 10 6 WT and Pvrl2 KO MC38 cells in Rag1 KO mice. Error bars represent SEM. B, AUCs of the MC38 tumors from A and A on day 19. Dots represent individual mice. P values are calculated by unpaired t test. Error bars represent SD. C, Schematic of immunophenotyping experiment: 25 days after subcutaneous injection of 1 × 10 6 MC38 WT and Pvrl2 KO cells in C57BL/6 mice, the tumors were collected. CD45 + cell populations were isolated from the tumor dissociates using CD45 positive magnetic selection kit. Then the isolated cells were subjected to viability dye, CD8, CD4, NK1.1, CD107a, and PD-1 staining followed by flow cytometry analysis. Flow cytometric quantification of the percentage of CD8 + ( D ), CD4 + ( E ), and NK1.1 + CD8 − (NK cells; F ) populations, respectively, in the CD45 + cells isolated from the MC38 WT ( n = 3) and Pvrl2 KO ( n = 4) tumors as indicated in C . P values are calculated by unpaired t test. Line represents mean. Quantification of the percentage of CD107a + ( G ) and PD-1 + ( H ) cells among the CD8 + T cell ( D ) population. P values are calculated by unpaired t test. Line represents mean. I, Quantification of the percentage of CD107a + cells among the NK cell ( F ) population. P value is calculated by unpaired t test. Line represents mean. J, Schematic of experiment design in K – O : After subcutaneous injection of 1 × 10 6 MC38 WT and Pvrl2 KO cells in C57BL/6 WT or Rag1 KO mice, the mice were treated with anti-CD8, CD4, or NK1.1 depleting antibodies or isotype control at the indicated serial doses and schedule. Average tumor volume over time following subcutaneous injection of 1 × 10 6 MC38 WT and Pvrl2 KO cells in WT C57BL/6 mice with anti-CD8 ( K ) and anti-CD4 ( L ) depleting antibodies or isotype control as indicated in J . Error bars represent SEM. M, AUCs of the MC38 tumors from ( K ) and ( L ) on day 17. Dots represent individual mice. P values are calculated by unpaired t test. Error bars represent SD. N, Average tumor volume over time following subcutaneous injection of 1 × 10 6 MC38 WT and Pvrl2 KO cells in Rag1 KO mice with anti-NK1.1 depleting antibody or isotype control as indicated in J . Error bars represent SEM. O, AUCs of the MC38 tumors from N on day 16. Dots represent individual mice. P values are calculated by unpaired t test. Error bars represent SD.
Article Snippet: Flow antibodies: Anti-mouse CD8 Brilliant Violet 605 (53-6.7; BioLegend, catalog no. 100744), anti-mouse CD4 Brilliant Violet 421 (GK1.5; BioLegend, catalog no. 100437), anti-mouse NK1.1 PE (PK136; BioLegend, catalog no. 108707), anti-mouse NK1.1 Brilliant Violet 711 (PK136; BioLegend, catalog no. 108745), anti-mouse CD107a (LAMP-1) Brilliant Violet 711 (1D4B; BioLegend, catalog no. 121631), anti-mouse PD-1 (CD279) APC (J43; BD Biosciences, catalog no. 562671), anti-mouse PVRL2 Brilliant Violet 421 (BD Biosciences, catalog no. 748046), anti-mouse PVR PE (TX56; BioLegend, catalog no. 131507), anti-mouse PVR APC (TX56; BioLegend, catalog no. 131509), anti-mouse TIGIT PE (1G9; BioLegend, catalog no. 142104), anti-mouse CD96 APC (3.3; BioLegend, catalog no. 131711), anti-mouse DNAM-1 Brilliant Violet 785 (TX42.1; BioLegend, catalog no. 133611), anti-mouse CD98 PE (4F2; BioLegend, catalog no. 128207), anti-mouse PD-L1 Super Bright 780 (MIH5; Invitrogen, catalog no. 78-5982-82), Brilliant Violet 605 Rat IgG2a, k Isotype control (RTK2758; BioLegend, catalog no. 400539), Brilliant Violet 421 Rat IgG2b, κ Isotype control (RTK4530; BioLegend, catalog no. 400639), PE Mouse IgG2a, κ Isotype control (MOPC-173; BioLegend, catalog no. 400211), Brilliant Violet 711 Rat IgG2b, κ Isotype control (RTK4530; BioLegend, catalog no. 400653), APC Hamster IgG2, κ Isotype control (B81-3; BD Biosciences, catalog no. 562169).
Techniques: Activation Assay, Injection, Isolation, Selection, Staining, Flow Cytometry