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Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T-cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1– and αCTLA-4–blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R (A1-R) to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T-cell response: Proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1-blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data show a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors. Cancer Immunol Res; 1(2); 123–33. ©2013 AACR.

PDF file - 291K, Figure S1. A1-R SIINF preferentially accumulates in established B16-OVA tumors. Figure S2. A1-R SIINF induces systemic SIINF-specific CD8+ T cell proliferation. Figure S3. Weekly A1-R SIINF treatment leads to rejection of established B16-OVA tumors. Figure S4. The majority of intratumoral SIINF-specific CD8+ T cells still express a high level of PD-1 following A1-R OVA treatment.

PDF file - 122K

Background: The CXCL10/CXCR3 signalling mediates paracrine interactions between tumour and stromal cells that govern leukocyte trafficking and angiogenesis. Emerging data implicate noncanonical CXCL10/CXCR3 signalling in tumourigenesis and metastasis. However, little is known regarding the role for autocrine CXCL10/CXCR3 signalling in regulating the metastatic potential of individual tumour clones. Methods: We performed transcriptomic and cytokine profiling to characterise the functions of CXCL10 and CXCR3 in tumour cells with different metastatic abilities. We modulated the expression of the CXCL10/CXCR3 pathway using shRNA-mediated silencing in both in vitro and in vivo models of B16F1 melanoma. In addition, we examined the expression of CXCL10 and CXCR3 and their associations with clinical outcomes in clinical data sets derived from over 670 patients with melanoma and colon and renal cell carcinomas. Results: We identified a critical role for autocrine CXCL10/CXCR3 signalling in promoting tumour cell growth, motility and metastasis. Analysis of publicly available clinical data sets demonstrated that coexpression of CXCL10 and CXCR3 predicted an increased metastatic potential and was associated with early metastatic disease progression and poor overall survival. Conclusion: These findings support the potential for CXCL10/CXCR3 coexpression as a predictor of metastatic recurrence and point towards a role for targeting of this oncogenic axis in the treatment of metastatic disease.

Summary Ionizing radiation-mediated tumor regression depends on type I interferon (IFN) and the adaptive immune response, but several pathways control I IFN induction. Here, we demonstrate that adaptor protein STING, but not MyD88, is required for type I IFN-dependent antitumor effects of radiation. In dendritic cells (DCs), STING was required for IFN-β induction in response to irradiated-tumor cells. The cytosolic DNA sensor cyclic GMP-AMP (cGAMP) synthase (cGAS) mediated sensing of irradiated-tumor cells in DCs. Moreover, STING was essential for radiation-induced adaptive immune responses, which relied on type I IFN signaling on DCs. Exogenous IFN-β treatment rescued the cross-priming by cGAS or STING-deficient DCs. Accordingly, activation of STING by a second messenger cGAMP administration enhanced antitumor immunity induced by radiation. Thus radiation-mediated antitumor immunity in immunogenic tumors requires a functional cytosolic DNA-sensing pathway and suggests that cGAMP treatment might provide a new strategy to improve radiotherapy.

Radiation therapy is currently one of the most widely utilized treatment strategies in the clinical management of cancer. Classically, radiation therapy was developed as an anticancer treatment on the basis of its capacity to induce DNA double strand breaks in exposed cancer cells, ultimately resulting in tumor cell death. Recently, our understanding of radiation effects has expanded widely in terms of the consequences of radiation-induced tumor cell death and the pertinent cells, signaling pathways, and molecular sensors that modify the tumor response to radiation. It is now well accepted that inflammation plays a complex dual role in promoting or inhibiting tumor growth. The capacity of inflammatory responses to alter the tumor response to radiation therapy, and vice versa, is now the subject of intense scientific and clinical investigation. Herein, we review the concepts regarding the immunostimulatory properties of radiation therapy with particular focus on the effects of radiation therapy on the tumor microenvironment.

Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T-cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1– and αCTLA-4–blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R (A1-R) to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T-cell response: Proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1-blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data show a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors. Cancer Immunol Res; 1(2); 123–33. ©2013 AACR.

The majority of cancer patients respond poorly to either vaccine or checkpoint blockade, and even to the combination of both. They are often resistant to high doses of radiation therapy as well. We examined prognostic markers of immune cell infiltration in pancreatic cancer. Patients with low CD8+ T cell infiltration and high PD-L1 expression (CD8+ TloPD-L1hi) experienced poor outcomes. We developed a mouse tumor fragment model with a trackable model antigen (SIYRYYGL or SIY) to mimic CD8+ TloPD-L1hi cancers. Tumors arising from fragments contained few T cells, even after vaccination. Fragment tumors responded poorly to PD-L1 blockade, SIY vaccination or radiation individually. By contrast, local ionizing radiation coupled with vaccination increased CD8+ T cell infiltration that was associated with upregulation of CXCL10 and CCL5 chemokines in the tumor, but demonstrated modest inhibition of tumor growth. The addition of an anti-PD-L1 antibody enhanced the effector function of tumor-infiltrating T cells, leading to significantly improved tumor regression and increased survival compared to vaccination and radiation. These results indicate that sequential combination of radiation, vaccination and checkpoint blockade converts non-T cell-inflamed cancers to T cell-inflamed cancers, and mediates regression of established pancreatic tumors with an initial CD8+ TloPD-L1hi phenotype. This study has opened a new strategy for shifting "cold" to hot tumors that will respond to immunotherapy.

The most widely held explanation for the efficacy of local radiotherapy (RT) is based on direct cytotoxicity to cancer cells through the induction of lethal DNA damage. Recent studies have shown that local ablative radiation of established tumors can lead to increased T-cell priming and T-cell–dependent tumor regression, but the underlying mechanism remains unclear. Here, we describe an essential role for type I IFN in local RT-mediated tumor control. We show that ablative RT increases intratumoral production of IFN-β and, more surprisingly, the antitumor effect of RT is abolished in type I IFN nonresponsive hosts. Furthermore, the major target of RT-induced type I IFN is the hematopoietic compartment. RT drastically enhances the cross-priming capacity of tumor-infiltrating dendritic cells (TIDC) from wild-type mice but not type I IFN receptor–deficient mice. The enhanced cross-priming ability of TIDCs after RT was dependent on autocrine production of type I IFNs. By using adenoviral-mediated expression of IFN-β, we show that delivery of exogenous IFN-β into the tumor tissue in the absence of RT is also sufficient to selectively expand antigen-specific T cells leading to complete tumor regression. Our study reveals that local high-dose RT can trigger production of type I IFN that initiates a cascading innate and adaptive immune attack on the tumor. Cancer Res; 71(7); 2488–96. ©2011 AACR.

Radiation has been a staple of cancer therapy since the early 20th century and is implemented in nearly half of current cancer treatment plans. Originally, the genotoxic function of radiation led to a focus on damage and repair pathways associated with deoxyribonucleic acid as important therapeutic targets to augment radiation efficacy. However, in recent decades, the participation of endogenous immune responses in modifying radiation effects have been widely documented and exploited in both preclinical and clinical settings. In particular, preclinical studies have highlighted the capacity of hypofractionated–radiation dose schedules to modify endogenous immune responses raising interest in the use of hypofractionation in the clinical setting to harness the indirect immune effects of radiation and improve clinical responses. We review the current literature regarding the immunomodulatory effects of hypofractionated "ablative" radiation with a primary focus on the preclinical literature but also highlight examples from the clinical literature.

High-dose ionizing irradiation (IR) results in direct tumor cell death and augments tumor-specific immunity, which enhances tumor control both locally and distantly. Unfortunately, local relapses often occur following IR treatment, indicating that IR-induced responses are inadequate to maintain antitumor immunity. Therapeutic blockade of the T cell negative regulator programmed death–ligand 1 (PD-L1, also called B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 was upregulated in the tumor microenvironment after IR. Administration of anti–PD-L1 enhanced the efficacy of IR through a cytotoxic T cell–dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti–PD-L1 synergistically reduced the local accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs), which suppress T cells and alter the tumor immune microenvironment. Furthermore, activation of cytotoxic T cells with combination therapy mediated the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-L1/PD-1 axis and establish a basis for the rational design of combination therapy with immune modulators and radiotherapy.

Local failures following radiation therapy are multifactorial, and the contributions of the tumor and the host are complex. Current models of tumor equilibrium suggest that a balance exists between cell birth and cell death due to insufficient angiogenesis, immune effects, or intrinsic cellular factors. We investigated whether host immune responses contribute to radiation-induced tumor equilibrium in animal models. We report an essential role for immune cells and their cytokines in suppressing tumor cell regrowth in two experimental animal model systems. Depletion of T cells or neutralization of IFN-γ reversed radiation-induced equilibrium, leading to tumor regrowth. We also demonstrate that PD-L1 blockade augments T cell responses, leading to rejection of tumors in radiation-induced equilibrium. We identify an active interplay between tumor cells and immune cells that occurs in radiation-induced tumor equilibrium and suggest a potential role for disruption of the PD-L1/PD-1 axis in increasing local tumor control.

Pancreatic cancer patients often fail to respond to immunotherapy, such as vaccines or checkpoint inhibitors. It is unknown if an immune phenotype predicts the efficacy of immunotherapy for pancreatic cancer and what standard modalities are required to facilitate the response of pancreatic cancer to immunotherapy. We examined prognostic markers of immune cell infiltration in pancreatic cancer. Pancreatic cancer patients with low CD8+ T cell infiltration and high PD-L1 expression (CD8+ TloPD-L1hi) experienced worse outcomes compared to patients whose tumors demonstrate CD8+ TloPD-L1lo, CD8+ ThiPD-L1hi or CD8+ ThiPD-L1lo profiles. To understand how to improve tumor control in CD8+ TloPD-L1hi pancreatic cancers, we developed a mouse tumor fragment model with a trackable model antigen (SIYRYYGL or SIY) to mimic CD8+ TloPD-L1hi pancreatic cancers. Similar to pancreatic cancers, tumors arising from fragments contained few T cells, even after vaccination. Fragment tumors responded poorly to PD-L1 blockade, SIY vaccination or radiation as individual treatments. By contrast, local ionizing radiation coupled with vaccination increased CD8+ T cell infiltration that was associated with upregulation of CXCL10 and CCL5 chemokines in the tumor, but demonstrated modest inhibition of tumor growth. The addition of an anti-PD-L1 antibody enhanced the effector function of tumor-infiltrating T cells, leading to significantly improved tumor regression and increased survival compared to vaccination and radiation. These results indicate that combination of radiation, vaccination and checkpoint blockade could convert non-T cell-inflamed cancers to T cell-inflamed cancers, and thus effectively treat established pancreatic tumors with an initial CD8+ TloPD-L1hi phenotype. This suggests a novel immunostimulatory role for radiotherapy in the treatment of pancreatic cancer. Citation Format: Wenxin Zheng, Kinga B. Skowron, Jukes P. Namm, Byron Burnette, Christian Fernandez, Ainhoa Arina, Hua Liang, Michael T. Spiotto, Mitchell C. Posner, Yang-Xin Fu, Ralph R. Weichselbaum. Radiotherapy sensitizes pancreatic cancer to immunotherapy by promoting T cell infiltration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-251.

Radiotherapy has been considered a local modality and outcomes have emphasized local and regional control of tumors. Recent data suggests that radiotherapy may activate the immune system and the combination of radiation therapy and immune therapies may have the potential to improve both local and distant control of tumor deposits. Below we review principals underlying the concepts of combining both modalities.

Patients with locally advanced cancer or distant metastasis frequently receive prolonged treatment with chemotherapy and/or fractionated radiotherapy (RT). Despite the initial clinical response, treatment resistance frequently develops and cure in these patients is uncommon. Developments in RT technology allow for the use of high-dose (or ablative) RT to target local tumors, with limited damage to the surrounding normal tissue. We report that reduction of tumor burden after ablative RT depends largely on T-cell responses. Ablative RT dramatically increases T-cell priming in draining lymphoid tissues, leading to reduction/eradication of the primary tumor or distant metastasis in a CD8+ T cell–dependent fashion. We further demonstrate that ablative RT-initiated immune responses and tumor reduction are abrogated by conventional fractionated RT or adjuvant chemotherapy but greatly amplified by local immunotherapy. Our study challenges the rationale for current RT/chemotherapy strategies and highlights the importance of immune activation in preventing tumor relapse. Our findings emphasize the need for new strategies that not only reduce tumor burden but also enhance the role of antitumor immunity.

Background & Aims: The ability of the liver to regenerate hepatic mass is essential to withstanding liver injury. The process of liver regeneration is tightly regulated by distinct signaling cascades involving components of the innate immune system, cytokines, and growth factors. However, the role of the adaptive immune system in regulation of liver regeneration is not well-defined. The role of adaptive immune system in liver regeneration was investigated in lymphocyte-deficient mice and in conditional lymphotoxin-deficient mice. Methods: A model of liver regeneration after 70% partial hepatectomy was used, followed by examination of liver pathology, survival, DNA synthesis, and cytokine expression. Results: We found that mice deficient in T cells show a reduced capacity for liver regeneration following partial hepatectomy. Furthermore, surface lymphotoxin, provided by T cells, is critical for liver regeneration. Mice specifically deficient in T-cell lymphotoxin had increased liver damage and a reduced capacity to initiate DNA synthesis after partial hepatectomy. Transfer of splenocytes from wild-type but not lymphotoxin-deficient mice improved liver regeneration in T cell-deficient mice. We found that an agonistic antibody against the lymphotoxin receptor was able to facilitate liver regeneration by reducing liver injury, increasing interleukin-6 production, hepatocyte DNA synthesis, and survival of lymphocyte-deficient (Rag) mice after partial hepatectomy. Conclusions: The adaptive immune system directly regulates liver regeneration via a T cell-derived lymphotoxin axis, and pharmacological stimulation of lymphotoxin receptor might represent a novel therapeutic approach to improve liver regeneration.

High dose ionizing irradiation (IR) results in direct tumor cell death and has been demonstrated to augment tumor-specific immunity that enhance tumor control both locally and distantly. However, local relapses often occur in patients and in established experimental tumors after treatment with IR suggesting that IR-induced immune responses are inadequate. Therapeutic blockade of the T cell negative regulator Programmed Death Ligand 1 (PD-L1, also named B7-H1) can enhance T cell effector function when PD-L1 is expressed in chronically inflamed tissues and tumors. Here, we demonstrate that PD-L1 is up-regulated in the tumor microenvironment after IR. Administration of anti-PD-L1 enhanced the efficacy of IR through a CD8+ T cell dependent mechanism. Concomitant with IR-mediated tumor regression, we observed that IR and anti-PD-L1 synergistically reduced the local accumulation of tumor-infiltrating Myeloid Derived Suppressor Cells (MDSCs), thus altering the tumor immune microenvironment. We demonstrate that CD8+ T cells, activated by combination therapy, mediate the reduction of MDSCs in tumors through the cytotoxic actions of TNF. Our data provide evidence for a close interaction between IR, T cells, and the PD-1/PD-L1 axis and establish the basis for the rational design of combination therapy with immune modulators and radiotherapy in the clinical setting. Note: This abstract was not presented at the meeting. Citation Format: Liufu Deng, Hua Liang, Byron Burnette, Micheal Beckett, Thomas Darga, Ralph Weichselbaum, Yang-Xin Fu. Irradiation and anti-B7-H1 provide synergistic treatment effects in murine cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5019. doi:10.1158/1538-7445.AM2014-5019

Tumor relapse after radiotherapy may be due to the upregulation of programmed cell death ligand 1 (PD-L1). We demonstrated that anti-PD-L1 antibody synergizes with radiation to control local and distal tumors. CD8+T cells mediated antitumor effects of the combination therapy by the reduction of myeloid-derived suppressor cells (MDSCs) via tumor-necrosis factor (TNF)-mediated signaling. Our study provides insight into immune- and radiation-based combinational therapies.

Despite its efficacy and widespread use, radiation therapy is often associated with local or distal tumor relapse. We have recently found that CD8+ T cells and their cytokines are essential for maintaining irradiated neoplasms under control. In line with this notion, enhancing T-cell functions by means of immune checkpoint inhibitors can tilt the balance toward tumor rejection.