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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

There is spontaneous release of Th1 and Th2 cytokines from splenic CD4 T cells harvested from mice treated with anti-PD-L1 and anti-CTLA4. The experimental design shown in Figure 3 was used. Mice were treated with anti-PD-L1, or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. T cells were isolated from spleens 21 days after tumor cell injection (14 days after irradiation). CD4 T cells were purified by immunomagnetic cell sorting, then stimulated with MHC class II− 5T33 wild-type cells, 5T33-CIITA MHC class II+ cells or T cells only for 48 hours. Supernatants were collected and cytokine levels from were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD4+ T cells for each group were pooled from 5 individual mice. p < 0.05, **p < 0.01.
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Fig6: There is spontaneous release of Th1 and Th2 cytokines from splenic CD4 T cells harvested from mice treated with anti-PD-L1 and anti-CTLA4. The experimental design shown in Figure 3 was used. Mice were treated with anti-PD-L1, or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. T cells were isolated from spleens 21 days after tumor cell injection (14 days after irradiation). CD4 T cells were purified by immunomagnetic cell sorting, then stimulated with MHC class II− 5T33 wild-type cells, 5T33-CIITA MHC class II+ cells or T cells only for 48 hours. Supernatants were collected and cytokine levels from were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD4+ T cells for each group were pooled from 5 individual mice. p < 0.05, **p < 0.01.

Mentions: CD4 T cells from mice treated with WBI and immune checkpoint blockade were also analyzed for cytokine production in response to tumor cells in vitro. Mice were once again treated according to the schedule in Figure 3A, except they received three antibody doses instead of six. On day 21 after myeloma inoculation, CD4 T cells were harvested from the spleen and purified by immunomagnetic cell sorting. Cells were incubated with 5T33-CIITA tumor cells expressing MHC class II molecules for 48 hours followed by cytokine analysis in multiplex cytokine assays. Spontaneous cytokine release was analyzed by incubating CD4 T cells alone or with wild-type 5T33 cells. CD4 T cells harvested from mice treated with anti-PD-L1 plus anti-CTLA4 spontaneously released IFN-γ, as well as the Th2 cytokines IL-4 and IL-5 (Figure 6). Cytokine release was significantly increased when the T cells were incubated with 5T33-CIITA MHC class II+ tumor. CD4 T cells harvested from mice treated with anti-PD-L1 plus anti-LAG-3 or anti-TIM-3 also released IFN-γ when stimulated with 5T33-CIITA cells, but there was no spontaneous or tumor-induced release of IFN-γ, IL-4 and IL-5.Figure 6


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)

There is spontaneous release of Th1 and Th2 cytokines from splenic CD4 T cells harvested from mice treated with anti-PD-L1 and anti-CTLA4. The experimental design shown in Figure 3 was used. Mice were treated with anti-PD-L1, or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. T cells were isolated from spleens 21 days after tumor cell injection (14 days after irradiation). CD4 T cells were purified by immunomagnetic cell sorting, then stimulated with MHC class II− 5T33 wild-type cells, 5T33-CIITA MHC class II+ cells or T cells only for 48 hours. Supernatants were collected and cytokine levels from were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD4+ T cells for each group were pooled from 5 individual mice. p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: There is spontaneous release of Th1 and Th2 cytokines from splenic CD4 T cells harvested from mice treated with anti-PD-L1 and anti-CTLA4. The experimental design shown in Figure 3 was used. Mice were treated with anti-PD-L1, or the combination of anti-PD-L1 with anti-TIM-3, or anti-LAG-3 or anti-CTLA4. T cells were isolated from spleens 21 days after tumor cell injection (14 days after irradiation). CD4 T cells were purified by immunomagnetic cell sorting, then stimulated with MHC class II− 5T33 wild-type cells, 5T33-CIITA MHC class II+ cells or T cells only for 48 hours. Supernatants were collected and cytokine levels from were determined using a multiplex cytokine assay. The graphs are representative of two independent experiments in which the CD4+ T cells for each group were pooled from 5 individual mice. p < 0.05, **p < 0.01.
Mentions: CD4 T cells from mice treated with WBI and immune checkpoint blockade were also analyzed for cytokine production in response to tumor cells in vitro. Mice were once again treated according to the schedule in Figure 3A, except they received three antibody doses instead of six. On day 21 after myeloma inoculation, CD4 T cells were harvested from the spleen and purified by immunomagnetic cell sorting. Cells were incubated with 5T33-CIITA tumor cells expressing MHC class II molecules for 48 hours followed by cytokine analysis in multiplex cytokine assays. Spontaneous cytokine release was analyzed by incubating CD4 T cells alone or with wild-type 5T33 cells. CD4 T cells harvested from mice treated with anti-PD-L1 plus anti-CTLA4 spontaneously released IFN-γ, as well as the Th2 cytokines IL-4 and IL-5 (Figure 6). Cytokine release was significantly increased when the T cells were incubated with 5T33-CIITA MHC class II+ tumor. CD4 T cells harvested from mice treated with anti-PD-L1 plus anti-LAG-3 or anti-TIM-3 also released IFN-γ when stimulated with 5T33-CIITA cells, but there was no spontaneous or tumor-induced release of IFN-γ, IL-4 and IL-5.Figure 6

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