CD11ahighCD8+ T Cell Phenotype Identifies Tumor Specific Effector Cells Whose Frequency and Function Are Enhanced by RT

Abstract

Purpose/Objective(s)CD11a is a cellular adhesion molecule, and its expression is required for the rejection of tumors. We previously reported that PD-1 blockade enhanced the RT-induced abscopal effect in melanoma and renal cell carcinoma preclinical models; therefore, we characterized CD11ahighCD8+ T cells from the tumor infiltrating lymphocytes (TILs) of our wild type (WT) and PD-1 knockout (KO) melanoma models (positive for PD-L1).Materials/MethodsC57BL/6 WT or PD-1 KO mice were injected subcutaneously with 1×105 B16-OVA cells in the right hind limb (irradiated) on day 0 and in the left flank (non-irradiated) on days 3–4. On day 8, 15 Gy x 1 was administered to the right hind limb tumors. For the in vitro challenge, 5–10×105 TILs were cultured with OVA peptide (1 μl/mL) for 4–5 hours in the presence of 1 μl/mL of Brefadin A, washed and incubated with rat antimouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFN-γ-FITC or control antibodies according to the manufacturer’s instructions. Tumor antigen specific CD8 T cells were identified by OVA-tetramer staining. Cells were analyzed using single laser, analytical flow cytometer and cytometry data analysis software. Statistical analyses were performed using commercial scientific 2D graphing and statistics software. A two-sided, unpaired, or paired Student t-test was used to assess statistical differences in experimental groups. A P-value of < .05 was considered statistically significant.ResultsPD-1 expressed by CD11ahighCD8+ T cells was higher in the irradiated tumors as compared with those of the non-irradiated tumors (P < .05 on day 15). In contrast, CD11ahighCD8+ T cells in the TILs of non-irradiated mice expressed only modest levels of PD-1. After in vitro OVA stimulation, 33–61% of PD-1+ CD11ahighCD8 T cells from the irradiated and non-irradiated tumors produced IFN-g, thereby suggesting that this population is that of the tumor antigen-reactive effector T cells. A higher percentage of CD11ahighCD8+ T cells were noted in the irradiated and non-irradiated tumors of PD-1 KO mice when compared with WT following RT, which correlated with the extent of tumor growth delay in PD-1 KOs. CD11ahigh but not CD11alowCD8+ T cells from both WT and PD-1 KO mice demonstrated tumor antigen specificity by binding to OVA tetramer. CD11ahighCD8+ T cells from the TILs of irradiated PD-1 KO mice produced IFN-g upon OVA peptide stimulation, in contrast to those from the TILs of non-irradiated mice.ConclusionThe CD11ahighCD8 T cell phenotype within TILs identifies tumor-specific effector cells whose frequency and function are enhanced by RT and PD-1 blockade, which in turn correlates with tumor growth delay and the abscopal effect. The combination of RT and PD-1 blockade may be a promising therapeutic approach for managing metastatic cancer patients, and CD11ahighCD8 T cells in the TILs may predict favorable responders to combined RT and anti-PD-1 therapy. Purpose/Objective(s)CD11a is a cellular adhesion molecule, and its expression is required for the rejection of tumors. We previously reported that PD-1 blockade enhanced the RT-induced abscopal effect in melanoma and renal cell carcinoma preclinical models; therefore, we characterized CD11ahighCD8+ T cells from the tumor infiltrating lymphocytes (TILs) of our wild type (WT) and PD-1 knockout (KO) melanoma models (positive for PD-L1). CD11a is a cellular adhesion molecule, and its expression is required for the rejection of tumors. We previously reported that PD-1 blockade enhanced the RT-induced abscopal effect in melanoma and renal cell carcinoma preclinical models; therefore, we characterized CD11ahighCD8+ T cells from the tumor infiltrating lymphocytes (TILs) of our wild type (WT) and PD-1 knockout (KO) melanoma models (positive for PD-L1). Materials/MethodsC57BL/6 WT or PD-1 KO mice were injected subcutaneously with 1×105 B16-OVA cells in the right hind limb (irradiated) on day 0 and in the left flank (non-irradiated) on days 3–4. On day 8, 15 Gy x 1 was administered to the right hind limb tumors. For the in vitro challenge, 5–10×105 TILs were cultured with OVA peptide (1 μl/mL) for 4–5 hours in the presence of 1 μl/mL of Brefadin A, washed and incubated with rat antimouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFN-γ-FITC or control antibodies according to the manufacturer’s instructions. Tumor antigen specific CD8 T cells were identified by OVA-tetramer staining. Cells were analyzed using single laser, analytical flow cytometer and cytometry data analysis software. Statistical analyses were performed using commercial scientific 2D graphing and statistics software. A two-sided, unpaired, or paired Student t-test was used to assess statistical differences in experimental groups. A P-value of < .05 was considered statistically significant. C57BL/6 WT or PD-1 KO mice were injected subcutaneously with 1×105 B16-OVA cells in the right hind limb (irradiated) on day 0 and in the left flank (non-irradiated) on days 3–4. On day 8, 15 Gy x 1 was administered to the right hind limb tumors. For the in vitro challenge, 5–10×105 TILs were cultured with OVA peptide (1 μl/mL) for 4–5 hours in the presence of 1 μl/mL of Brefadin A, washed and incubated with rat antimouse CD16/CD32 mAb (2.4G2) to block nonspecific binding, and then stained with CD8-PE-Cy5 and IFN-γ-FITC or control antibodies according to the manufacturer’s instructions. Tumor antigen specific CD8 T cells were identified by OVA-tetramer staining. Cells were analyzed using single laser, analytical flow cytometer and cytometry data analysis software. Statistical analyses were performed using commercial scientific 2D graphing and statistics software. A two-sided, unpaired, or paired Student t-test was used to assess statistical differences in experimental groups. A P-value of < .05 was considered statistically significant. ResultsPD-1 expressed by CD11ahighCD8+ T cells was higher in the irradiated tumors as compared with those of the non-irradiated tumors (P < .05 on day 15). In contrast, CD11ahighCD8+ T cells in the TILs of non-irradiated mice expressed only modest levels of PD-1. After in vitro OVA stimulation, 33–61% of PD-1+ CD11ahighCD8 T cells from the irradiated and non-irradiated tumors produced IFN-g, thereby suggesting that this population is that of the tumor antigen-reactive effector T cells. A higher percentage of CD11ahighCD8+ T cells were noted in the irradiated and non-irradiated tumors of PD-1 KO mice when compared with WT following RT, which correlated with the extent of tumor growth delay in PD-1 KOs. CD11ahigh but not CD11alowCD8+ T cells from both WT and PD-1 KO mice demonstrated tumor antigen specificity by binding to OVA tetramer. CD11ahighCD8+ T cells from the TILs of irradiated PD-1 KO mice produced IFN-g upon OVA peptide stimulation, in contrast to those from the TILs of non-irradiated mice. PD-1 expressed by CD11ahighCD8+ T cells was higher in the irradiated tumors as compared with those of the non-irradiated tumors (P < .05 on day 15). In contrast, CD11ahighCD8+ T cells in the TILs of non-irradiated mice expressed only modest levels of PD-1. After in vitro OVA stimulation, 33–61% of PD-1+ CD11ahighCD8 T cells from the irradiated and non-irradiated tumors produced IFN-g, thereby suggesting that this population is that of the tumor antigen-reactive effector T cells. A higher percentage of CD11ahighCD8+ T cells were noted in the irradiated and non-irradiated tumors of PD-1 KO mice when compared with WT following RT, which correlated with the extent of tumor growth delay in PD-1 KOs. CD11ahigh but not CD11alowCD8+ T cells from both WT and PD-1 KO mice demonstrated tumor antigen specificity by binding to OVA tetramer. CD11ahighCD8+ T cells from the TILs of irradiated PD-1 KO mice produced IFN-g upon OVA peptide stimulation, in contrast to those from the TILs of non-irradiated mice. ConclusionThe CD11ahighCD8 T cell phenotype within TILs identifies tumor-specific effector cells whose frequency and function are enhanced by RT and PD-1 blockade, which in turn correlates with tumor growth delay and the abscopal effect. The combination of RT and PD-1 blockade may be a promising therapeutic approach for managing metastatic cancer patients, and CD11ahighCD8 T cells in the TILs may predict favorable responders to combined RT and anti-PD-1 therapy. The CD11ahighCD8 T cell phenotype within TILs identifies tumor-specific effector cells whose frequency and function are enhanced by RT and PD-1 blockade, which in turn correlates with tumor growth delay and the abscopal effect. The combination of RT and PD-1 blockade may be a promising therapeutic approach for managing metastatic cancer patients, and CD11ahighCD8 T cells in the TILs may predict favorable responders to combined RT and anti-PD-1 therapy.