PD-1 maintains CD8 T cell tolerance towards cutaneous neoantigens.

The peripheral T cell repertoire of healthy individuals contains self-reactive T cells1,2. Checkpoint receptors such as PD-1 are thought to enable the induction of peripheral tolerance by deletion or anergy of self-reactive CD8 T cells3-10. However, this model is challenged by the high frequency of immune-related adverse events in patients with cancer who have been treated with checkpoint inhibitors11. Here we developed a mouse model in which skin-specific expression of T cell antigens in the epidermis caused local infiltration of antigen-specific CD8 T cells with an effector gene-expression profile. In this setting, PD-1 enabled the maintenance of skin tolerance by preventing tissue-infiltrating antigen-specific effector CD8 T cells from (1) acquiring a fully functional, pathogenic differentiation state, (2) secreting significant amounts of effector molecules, and (3) gaining access to epidermal antigen-expressing cells. In the absence of PD-1, epidermal antigen-expressing cells were eliminated by antigen-specific CD8 T cells, resulting in local pathology. Transcriptomic analysis of skin biopsies from two patients with cutaneous lichenoid immune-related adverse events showed the presence of clonally expanded effector CD8 T cells in both lesional and non-lesional skin. Thus, our data support a model of peripheral T cell tolerance in which PD-1 allows antigen-specific effector CD8 T cells to co-exist with antigen-expressing cells in tissues without immunopathology.

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 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.

Improving Efficacy and Safety of Agonistic Anti-CD40 Antibody Through Extracellular Matrix Affinity.

CD40 is an immune costimulatory receptor expressed by antigen-presenting cells. Agonistic anti-CD40 antibodies have demonstrated considerable antitumor effects yet can also elicit serious treatment-related adverse events, such as liver toxicity, including in man. We engineered a variant that binds extracellular matrix through a super-affinity peptide derived from placenta growth factor-2 (PlGF-2123-144) to enhance anti-CD40's effects when administered locally. Peritumoral injection of PlGF-2123-144-anti-CD40 antibody showed prolonged tissue retention at the injection site and substantially decreased systemic exposure, resulting in decreased liver toxicity. In four mouse tumor models, PlGF-2123-144-anti-CD40 antibody demonstrated enhanced antitumor efficacy compared with its unmodified form and correlated with activated dendritic cells, B cells, and T cells in the tumor and in the tumor-draining lymph node. Moreover, in a genetically engineered BrafV600E ßCatSTA melanoma model that does not respond to checkpoint inhibitors, PlGF-2123-144-anti-CD40 antibody treatment enhanced T-cell infiltration into the tumors and slowed tumor growth. Together, these results demonstrate the marked therapeutic advantages of engineering matrix-binding domains onto agonistic anti-CD40 antibody as a therapeutic given by tumori-regional injection for cancer immunotherapy.Implications: Extracellular matrix-binding peptide conjugation to agonistic anti-CD40 antibody enhances antitumor efficacy and reduces treatment-related adverse events. Mol Cancer Ther; 17(11); 2399-411. ©2018 AACR.

TLR-3 stimulation improves anti-tumor immunity elicited by dendritic cell exosome-based vaccines in a murine model of melanoma.

Dendritic cell (DC)-derived exosomes (Dexo) contain the machinery necessary to activate potent antigen-specific immune responses. As promising cell-free immunogens, Dexo have been tested in previous clinical trials for cancer vaccine immunotherapy, yet resulted in limited therapeutic benefit. Here, we explore a novel Dexo vaccine formulation composed of Dexo purified from DCs loaded with antigens and matured with either the TLR-3 ligand poly(I:C), the TLR-4 ligand LPS or the TLR-9 ligand CpG-B. When poly(I:C) was used to produce exosomes together with ovalbumin (OVA), the resulting Dexo vaccine strongly stimulated OVA-specific CD8(+) and CD4(+) T cells to proliferate and acquire effector functions. When a B16F10 melanoma cell lysate was used to load DCs with tumor antigens during exosome production together with poly(I:C), we obtained a Dexo vaccine capable of inducing robust activation of melanoma-specific CD8(+) T cells and the recruitment of cytotoxic CD8(+) T cells, NK and NK-T cells to the tumor site, resulting in significantly reduced tumor growth and enhanced survival as compared to a Dexo vaccine formulation similar to the one previously tested on human patients. Our results indicate that poly(I:C) is a particularly favorable TLR agonist for DC maturation during antigen loading and exosome production for cancer immunotherapy.

Site-specific integration and tailoring of cassette design for sustainable gene transfer.

Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.