Novel Mouse Models for Cancer Immunology.

Mouse models for the study of cancer immunology provide excellent systems in which to test biological mechanisms of the immune response against cancer. Historically, these models have been designed to have different strengths based on the current major research questions at the time. As such, many mouse models of immunology used today were not originally developed to study questions currently plaguing the relatively new field of cancer immunology, but instead have been adapted for such purposes. In this review, we discuss various mouse model of cancer immunology in a historical context as a means to provide a fuller perspective of each model's strengths. From this outlook, we discuss the current state of the art and strategies for tackling future modeling challenges.

Neoantigen-driven B cell and CD4 T follicular helper cell collaboration promotes anti-tumor CD8 T cell responses.

CD4 T follicular helper (TFH) cells support B cells, which are critical for germinal center (GC) formation, but the importance of TFH-B cell interactions in cancer is unclear. We found enrichment of TFH cell transcriptional signature correlates with GC B cell signature and with prolonged survival in individuals with lung adenocarcinoma (LUAD). We further developed a murine LUAD model in which tumor cells express B cell- and T cell-recognized neoantigens. Interactions between tumor-specific TFH and GC B cells, as well as interleukin (IL)-21 primarily produced by TFH cells, are necessary for tumor control and effector CD8 T cell function. Development of TFH cells requires B cells and B cell-recognized neoantigens. Thus, tumor neoantigens can regulate the fate of tumor-specific CD4 T cells by facilitating their interactions with tumor-specific B cells, which in turn promote anti-tumor immunity by enhancing CD8 T cell effector functions.

A mouse model for the study of anti-tumor T cell responses in Kras-driven lung adenocarcinoma.

Kras-driven lung adenocarcinoma (LUAD) is the most common lung cancer. A significant fraction of patients with Kras-driven LUAD respond to immunotherapy, but mechanistic studies of immune responses against LUAD have been limited because of a lack of immunotherapy-responsive models. We report the development of the immunogenic KP × NINJA (inversion inducible joined neoantigen) (KP-NINJA) LUAD model. This model allows temporal uncoupling of antigen and tumor induction, which allows one to wait until after infection-induced inflammation has subsided to induce neoantigen expression by tumors. Neoantigen expression is restricted to EPCAM+ cells in the lung and expression of neoantigen was more consistent between tumors than when neoantigens were encoded on lentiviruses. Moreover, tumors were infiltrated by tumor-specific CD8 T cells. Finally, LUAD cell lines derived from KP-NINJA mice were immunogenic and responded to immune checkpoint therapy (anti-PD1 and anti-CTLA4), providing means for future studies into the immunobiology of therapeutic responses in LUAD.

A reservoir of stem-like CD8+ T cells in the tumor-draining lymph node preserves the ongoing antitumor immune response.

"Stem-like" TCF1+ CD8+ T (TSL) cells are necessary for long-term maintenance of T cell responses and the efficacy of immunotherapy, but, as tumors contain signals that should drive T cell terminal differentiation, how these cells are maintained in tumors remains unclear. In this study, we found that a small number of TCF1+ tumor-specific CD8+ T cells were present in lung tumors throughout their development. Yet, most intratumoral T cells differentiated as tumors progressed, corresponding with an immunologic shift in the tumor microenvironment (TME) from "hot" (T cell inflamed) to "cold" (non­T cell inflamed). By contrast, most tumor-specific CD8+ T cells in tumor-draining lymph nodes (dLNs) had functions and gene expression signatures similar to TSL from chronic lymphocytic choriomeningitis virus infection, and this population was stable over time despite the changes in the TME. dLN T cells were the developmental precursors of, and were clonally related to, their more differentiated intratumoral counterparts. Our data support the hypothesis that dLN T cells are the developmental precursors of the TCF1+ T cells in tumors that are maintained by continuous migration. Last, CD8+ T cells similar to TSL were also present in LNs from patients with lung adenocarcinoma, suggesting that a similar model may be relevant in human disease. Thus, we propose that the dLN TSL reservoir has a critical function in sustaining antitumor T cells during tumor development and in protecting them from the terminal differentiation that occurs in the TME.

Inducible de novo expression of neoantigens in tumor cells and mice.

Inducible expression of neoantigens in mice would enable the study of endogenous antigen-specific naïve T cell responses in disease and infection, but has been difficult to generate because leaky antigen expression in the thymus results in central T cell tolerance. Here we develop inversion-induced joined neoantigen (NINJA), using RNA splicing, DNA recombination and three levels of regulation to prevent leakiness and allow tight control over neoantigen expression. We apply NINJA to create tumor cell lines with inducible neoantigen expression, which could be used to study antitumor immunity. We also show that the genetic regulation in NINJA mice bypasses central and peripheral tolerance mechanisms and allows for robust endogenous CD8 and CD4 T cell responses on neoantigen induction in peripheral tissues. NINJA will enable studies of how T cells respond to defined neoantigens in the context of peripheral tolerance, transplantation, autoimmune diseases and cancer.

Stereotactic Body Radiation and Interleukin-12 Combination Therapy Eradicates Pancreatic Tumors by Repolarizing the Immune Microenvironment.

Over 80% of pancreatic ductal adenocarcinoma (PDA) patients are diagnosed with non-resectable late-stage disease that lacks effective neoadjuvant therapies. Stereotactic body radiation therapy (SBRT) has shown promise as an emerging neoadjuvant approach for treating PDA, and here, we report that its combination with local interleukin-12 (IL-12) microsphere (MS) immunotherapy results in marked tumor reduction and cures in multiple preclinical mouse models of PDA. Our findings demonstrate an increase of intratumoral interferon gamma (IFNγ) production following SBRT/IL-12 MS administration that initiates suppressor cell reprogramming and a subsequent increase in CD8 T cell activation. Furthermore, SBRT/IL-12 MS therapy results in the generation of systemic tumor immunity that is capable of eliminating established liver metastases, providing a rationale for follow-up studies in advanced metastatic disease.

Lineage Specifiers in Lung Cancer Are Ahead of Their TIME.

The factors that shape the distinctive tumor-immune landscapes of various types and subtypes of cancer remain poorly understood. In this issue of Immunity, Mollaoglu et al. (2018) reveal a mechanistic link between the function of lineage specifiers SOX2 and NKX2-1 and the presence of neutrophils in the tumor-immune microenvironment of lung cancer.

Investigating Tumor-Associated Tertiary Lymphoid Structures in Murine Lung Adenocarcinoma.

Genetically engineered mouse models (GEMMs), in which autochthonous tumors develop into advanced-stage disease in the presence of a functional immune system, have contributed significantly to the understanding of most types of cancer. Using a GEMM of lung adenocarcinoma, we have found that immune cells are present in complex, highly organized, lymph node (LN)-like structures known as the tumor-associated tertiary lymphoid structures (TA-TLS). TA-TLS have been characterized in human lung cancer patients, but not in animal tumor models, and hence remain untapped targets for therapeutic interventions. We have shown that TA-TLS emerge as a result of tumor growth and that therapeutically depleting regulatory T cells (Tregs) from TA-TLS results in tumor elimination. Hence, a strong antitumor immune response exists but is suppressed in TA-TLS. Here, we describe a high-throughput immunofluorescence (IF) analysis pipeline for visualization and quantification of TA-TLS. Imaging the relatively small size of TA-TLS within tumor-bearing lung lobes using confocal microscopy is a labor-intensive process that can take up to 1 month. We have optimized this process and reduced the time required per lung lobe to 1-2 weeks using automated microscopy methods. Combining IF with multicolor fluorescence-activated cell sorting (FACS), we are able to interrogate not only the size and location of TA-TLS but also the activation status of immune cells within these structures. Using these techniques, investigation of TLS in lung adenocarcinoma combines cutting-edge technological tools in cancer biology and immunology to interrogate a fundamental, but poorly understood, tumor-associated immune structure.

Increasing the efficacy of radiotherapy by modulating the CCR2/CCR5 chemokine axes.

Although radiotherapy (RT) is widely used to control tumor growth across many cancer types, there is a relatively high incidence of RT failure exhibited by tumor recurrence, therefore a clear need exists to achieve improved effectiveness of RT. The RT-elicited immune response largely impacts the efficacy of RT and includes immune cells that kill tumor cells, but also immunosuppressive cells, which dampen anti-tumor immunity. Using murine models in which syngeneic tumor cell lines (Colon38, Glioma261, Line1) are grown intramuscularly and treated with 15 Gy local RT, we assessed the effects of RT on both the systemic and intratumoral immune response. Here we demonstrate that RT stimulates increased production of two chemokines, CCL2 and CCL5, at the tumor site. Further, that this leads to increased CCR2+ CCR5+ monocytes in circulation and subsequently alters the intratumoral immune infiltrate favoring the largely immunosuppressive CCR2+ CCR5+ monocytes. Importantly, a CCR2/CCR5 antagonist administered daily (15 mg/kg subcutaneously) starting two days prior to RT reduces both circulating and intratumoral monocytes resulting in increased efficacy of RT in radioresponsive tumors. Overall, these data have important implications for the mechanism of RT and present a means to improve RT efficacy across many cancer types.

Radio-responsive tumors exhibit greater intratumoral immune activity than nonresponsive tumors.

Radiation therapy (RT) continues to be a cornerstone in the treatment for many cancers. Unfortunately, not all individuals respond effectively to RT resulting clinically in two groups consisting of nonresponders (progressive disease) and responders (tumor control/cure). The mechanisms that govern the outcome of radiotherapy are poorly understood. Interestingly, a new paradigm has emerged demonstrating that the immune system mediates many of the antitumor effects of RT. Therefore, we hypothesized that the immune response following RT may dictate the efficacy of treatment. To examine this, we developed a tumor model that mirrors this clinically relevant phenomenon in which mice bearing Colon38, a colon adenocarcinoma, were treated locally with 15Gy RT resulting in both nonresponders and responders. More importantly, we were able to distinguish responders from nonresponders as early as 4 days post-RT allowing for the unique opportunity to identify critical events that ultimately determined the effectiveness of therapy. Intratumoral immune cells and interferon-gamma were increased in responsive tumors and licensed CD8 T cells to exhibit lytic activity against tumor cells, a response that was diminished in tumors refractory to RT. Combinatorial treatment with RT and the immunomodulatory cytokine IL-12 resulted in complete remission of cancer in 100% of cases compared to a cure rate of only 12% with RT alone. Similar data were obtained when IL-12 was delivered by microspheres. Therefore, the efficacy of RT may depend on the strength of the immune response induced after radiotherapy. Additionally, immunotherapy that further stimulates the immune cells may enhance the effectiveness of RT.