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Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma.

Jing W, Gershan JA, Weber J, Tlomak D, McOlash L, Sabatos-Peyton C, Johnson BD - J Immunother Cancer (2015)

Bottom Line: When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%).The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells.Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology/Transplant, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226 USA.

ABSTRACT

Background: Multiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.

Methods: To test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.

Results: Temporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.

Conclusions: These data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.

No MeSH data available.


Related in: MedlinePlus

Expression of immune checkpoint proteins are increased on T cells in mice treated with sublethal whole body irradiation and anti-PD-L1. Myeloma bearing KaLwRij mice were treated with 500 cGy whole body irradiation 7 days after tumor cell injection. Treatment with anti-PD-L1 or control IgG (200 μg ip) was initiated 5 days later and specifically given 12, 14, and 19 days after tumor injection. Mice were euthanized at day 21, splenocytes were harvested, and the CD8 T cells were analyzed by flow cytometry for immune checkpoint protein expression. (A) Frequency of CD8+TIM-3+, CD8+LAG-3+ and CD8+2B4+ cells in spleens of anti-PD-L1 treated mice as compared with spleens harvested from control antibody (IgG1) treated mice. ***p < 0.001. (B) Expression of TIM-3 and PD-1, LAG-3 and PD-1 or 2B4 and PD-1 on gated CD8+ T cells. Data shown are representative of more than four independent analyses.
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Fig2: Expression of immune checkpoint proteins are increased on T cells in mice treated with sublethal whole body irradiation and anti-PD-L1. Myeloma bearing KaLwRij mice were treated with 500 cGy whole body irradiation 7 days after tumor cell injection. Treatment with anti-PD-L1 or control IgG (200 μg ip) was initiated 5 days later and specifically given 12, 14, and 19 days after tumor injection. Mice were euthanized at day 21, splenocytes were harvested, and the CD8 T cells were analyzed by flow cytometry for immune checkpoint protein expression. (A) Frequency of CD8+TIM-3+, CD8+LAG-3+ and CD8+2B4+ cells in spleens of anti-PD-L1 treated mice as compared with spleens harvested from control antibody (IgG1) treated mice. ***p < 0.001. (B) Expression of TIM-3 and PD-1, LAG-3 and PD-1 or 2B4 and PD-1 on gated CD8+ T cells. Data shown are representative of more than four independent analyses.

Mentions: We previously showed that blocking the PD-1/PD-L1 axis with a PD-L1-specific monoclonal antibody synergized with lymphodepleting whole body irradiation (WBI) to facilitate a T cell-mediated anti-myeloma response [39]. To determine the influence of this treatment on T cell immune checkpoint expression, mice with established myeloma were treated with 500 cGy WBI 7 days after 5T33 inoculation, followed by three treatments with anti–PD-L1 or control IgG on days 5, 7 and 12 after WBI. On day 14 after WBI, CD8 T cells were harvested from bone marrow and analyzed for expression of PD-1, TIM-3, LAG-3 and 2B4 by flow cytometry. There were significant increases in the percentages of CD8 T cells that expressed TIM-3, LAG-3 or 2B4 in mice treated with anti-PD-L1 as compared to controls treated with IgG1 (Figure 2A). Interestingly, the mice treated with anti-PD-L1 also had a marked increase in percentages of PD-1+ CD8 T cells (Figure 2B), and relatively large percentages of the PD-1+ cells co-expressed TIM-3 (52%), LAG-3 (60%) or 2B4 (40%) (Figure 2B).Figure 2


Combined immune checkpoint protein blockade and low dose whole body irradiation as immunotherapy for myeloma.

Jing W, Gershan JA, Weber J, Tlomak D, McOlash L, Sabatos-Peyton C, Johnson BD - J Immunother Cancer (2015)

Expression of immune checkpoint proteins are increased on T cells in mice treated with sublethal whole body irradiation and anti-PD-L1. Myeloma bearing KaLwRij mice were treated with 500 cGy whole body irradiation 7 days after tumor cell injection. Treatment with anti-PD-L1 or control IgG (200 μg ip) was initiated 5 days later and specifically given 12, 14, and 19 days after tumor injection. Mice were euthanized at day 21, splenocytes were harvested, and the CD8 T cells were analyzed by flow cytometry for immune checkpoint protein expression. (A) Frequency of CD8+TIM-3+, CD8+LAG-3+ and CD8+2B4+ cells in spleens of anti-PD-L1 treated mice as compared with spleens harvested from control antibody (IgG1) treated mice. ***p < 0.001. (B) Expression of TIM-3 and PD-1, LAG-3 and PD-1 or 2B4 and PD-1 on gated CD8+ T cells. Data shown are representative of more than four independent analyses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4302511&req=5

Fig2: Expression of immune checkpoint proteins are increased on T cells in mice treated with sublethal whole body irradiation and anti-PD-L1. Myeloma bearing KaLwRij mice were treated with 500 cGy whole body irradiation 7 days after tumor cell injection. Treatment with anti-PD-L1 or control IgG (200 μg ip) was initiated 5 days later and specifically given 12, 14, and 19 days after tumor injection. Mice were euthanized at day 21, splenocytes were harvested, and the CD8 T cells were analyzed by flow cytometry for immune checkpoint protein expression. (A) Frequency of CD8+TIM-3+, CD8+LAG-3+ and CD8+2B4+ cells in spleens of anti-PD-L1 treated mice as compared with spleens harvested from control antibody (IgG1) treated mice. ***p < 0.001. (B) Expression of TIM-3 and PD-1, LAG-3 and PD-1 or 2B4 and PD-1 on gated CD8+ T cells. Data shown are representative of more than four independent analyses.
Mentions: We previously showed that blocking the PD-1/PD-L1 axis with a PD-L1-specific monoclonal antibody synergized with lymphodepleting whole body irradiation (WBI) to facilitate a T cell-mediated anti-myeloma response [39]. To determine the influence of this treatment on T cell immune checkpoint expression, mice with established myeloma were treated with 500 cGy WBI 7 days after 5T33 inoculation, followed by three treatments with anti–PD-L1 or control IgG on days 5, 7 and 12 after WBI. On day 14 after WBI, CD8 T cells were harvested from bone marrow and analyzed for expression of PD-1, TIM-3, LAG-3 and 2B4 by flow cytometry. There were significant increases in the percentages of CD8 T cells that expressed TIM-3, LAG-3 or 2B4 in mice treated with anti-PD-L1 as compared to controls treated with IgG1 (Figure 2A). Interestingly, the mice treated with anti-PD-L1 also had a marked increase in percentages of PD-1+ CD8 T cells (Figure 2B), and relatively large percentages of the PD-1+ cells co-expressed TIM-3 (52%), LAG-3 (60%) or 2B4 (40%) (Figure 2B).Figure 2

Bottom Line: When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%).The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells.Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology/Oncology/Transplant, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI 53226 USA.

ABSTRACT

Background: Multiple myeloma is characterized by the presence of transformed neoplastic plasma cells in the bone marrow and is generally considered to be an incurable disease. Successful treatments will likely require multi-faceted approaches incorporating conventional drug therapies, immunotherapy and other novel treatments. Our lab previously showed that a combination of transient lymphodepletion (sublethal whole body irradiation) and PD-1/PD-L1 blockade generated anti-myeloma T cell reactivity capable of eliminating established disease. We hypothesized that blocking a combination of checkpoint receptors in the context of low-dose, lymphodepleting whole body radiation would boost anti-tumor immunity.

Methods: To test our central hypothesis, we utilized a 5T33 murine multiple myeloma model. Myeloma-bearing mice were treated with a low dose of whole body irradiation and combinations of blocking antibodies to PD-L1, LAG-3, TIM-3, CD48 (the ligand for 2B4) and CTLA4.

Results: Temporal phenotypic analysis of bone marrow from myeloma-bearing mice demonstrated that elevated percentages of PD-1, 2B4, LAG-3 and TIM-3 proteins were expressed on T cells. When PD-L1 blockade was combined with blocking antibodies to LAG-3, TIM-3 or CTLA4, synergistic or additive increases in survival were observed (survival rates improved from ~30% to >80%). The increased survival rates correlated with increased frequencies of tumor-reactive CD8 and CD4 T cells. When stimulated in vitro with myeloma cells, CD8 T cells from treated mice produced elevated levels proinflammatory cytokines. Cytokines were spontaneously released from CD4 T cells isolated from mice treated with PD-L1 plus CTLA4 blocking antibodies.

Conclusions: These data indicate that blocking PD-1/PD-L1 interactions in conjunction with other immune checkpoint proteins provides synergistic anti-tumor efficacy following lymphodepletive doses of whole body irradiation. This strategy is a promising combination strategy for myeloma and other hematologic malignancies.

No MeSH data available.


Related in: MedlinePlus