There are numerous cell types with scarcely understood functions, whose interactions with the immune system are not well characterized. To facilitate their study, we generated a mouse bearing enhanced green fluorescent protein (EGFP)-specific CD8+ T cells. Transfer of the T cells into EGFP reporter animals can be used to kill EGFP-expressing cells, allowing selective depletion of desired cell types, or to interrogate T-cell interactions with specific populations. Using this system, we eliminate a rare EGFP-expressing cell type in the heart and demonstrate its role in cardiac function. We also show that naive T cells are recruited into the mouse brain by antigen-expressing microglia, providing evidence of an immune surveillance pathway in the central nervous system. The just EGFP death-inducing (Jedi) T cells enable visualization of a T-cell antigen. They also make it possible to utilize hundreds of existing EGFP-expressing mice, tumors, pathogens and other tools, to study T-cell interactions with many different cell types, to model disease states and to determine the functions of poorly characterized cell populations.

Understanding how drugs affect the immune system has consequences for treating disease and minimizing unwanted side effects. Here we present an integrative computational approach for predicting interactions between drugs and immune cells in a system-wide manner. The approach matches gene sets between transcriptional signatures to determine their similarity. We apply the method to model the interactions between 1,309 drugs and 221 immune cell types and predict 69,995 interactions. The resulting immune-cell pharmacology map is used to predict how five drugs influence four immune cell types in humans and mice. To validate the predictions, we analyzed patient records and examined cell population changes from in vivo experiments. Our method offers a tool for screening thousands of interactions to identify relationships between drugs and the immune system.

Cancer immunotherapy shows limited efficacy against many solid tumors that originate from epithelial tissues, including triple-negative breast cancer (TNBC). We identify the SOX4 transcription factor as an important resistance mechanism to T cell-mediated cytotoxicity for TNBC cells. Mechanistic studies demonstrate that inactivation of SOX4 in tumor cells increases the expression of genes in a number of innate and adaptive immune pathways important for protective tumor immunity. Expression of SOX4 is regulated by the integrin αvβ6 receptor on the surface of tumor cells, which activates TGFβ from a latent precursor. An integrin αvβ6/8-blocking monoclonal antibody (mAb) inhibits SOX4 expression and sensitizes TNBC cells to cytotoxic T cells. This integrin mAb induces a substantial survival benefit in highly metastatic murine TNBC models poorly responsive to PD-1 blockade. Targeting of the integrin αvβ6-TGFβ-SOX4 pathway therefore provides therapeutic opportunities for TNBC and other highly aggressive human cancers of epithelial origin.

Hepatitis C virus (HCV) replication is dependent on a liver-specific microRNA (miRNA), miR-122. A recent clinical trial reported that transient inhibition of miR-122 reduced viral titres in HCV-infected patients. Here we set out to better understand how miR-122 inhibition influences HCV replication over time. Unexpectedly, we observed the emergence of an HCV variant that is resistant to miR-122 knockdown. Next-generation sequencing revealed that this was due to a single nucleotide change at position 28 (G28A) of the HCV genome, which falls between the two miR-122 seed-binding sites. Naturally occurring HCV isolates encoding G28A are similarly resistant to miR-122 inhibition, indicating that subtle differences in viral sequence, even outside the seed-binding site, greatly influence HCV's miR-122 concentration requirement. In addition, we found that HCV itself reduces miR-122's activity in the cell, possibly through binding and sequestering miR-122. Our study provides insight into the interaction between miR-122 and HCV, including viral adaptation to reduced miR-122 bioavailability, and has implications for the development of anti-miR-122-based HCV drugs.

miR-126 is a microRNA expressed predominately by endothelial cells and controls angiogenesis. We found miR-126 was required for the innate response to pathogen-associated nucleic acids and that miR-126-deficient mice had greater susceptibility to infection with pseudotyped HIV. Profiling of miRNA indicated that miR-126 had high and specific expression by plasmacytoid dendritic cells (pDCs). Moreover, miR-126 controlled the survival and function of pDCs and regulated the expression of genes encoding molecules involved in the innate response, including Tlr7, Tlr9 and Nfkb1, as well as Kdr, which encodes the growth factor receptor VEGFR2. Deletion of Kdr in DCs resulted in reduced production of type I interferon, which supports the proposal of a role for VEGFR2 in miR-126 regulation of pDCs. Our studies identify the miR-126-VEGFR2 axis as an important regulator of the innate response that operates through multiscale control of pDCs.

Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen-presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103(+) dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8(+) T cells. CD103(+) DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PD-L1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the growth factor FLT3L followed by intratumoral poly I:C injections expanded and activated CD103(+) DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103(+) DCs in tumors limits checkpoint-blockade efficacy and combined FLT3L and poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection.

The skin immune system must discriminate between innocuous antigens and pathogens. Antigen applied topically using a Viaskin® patch elicits immune tolerance that can suppress colitis and food allergy. Here we show how topical antigen is acquired and presented by dendritic cells in the skin. Topical antigen is acquired by Langerhans cells (LC) and CD11b+ cDC2s but not cDC1s, and both  LCs and CD11b+ cDC2s reaching the lymph node can prime T cells and expand LAP+ Tregs. However, LCs are neither required nor sufficient for T cell priming, and have no role in tolerance induction. Conversely, IRF-4-dependent cDC2s are required for T cell priming. Acquisition of antigen in the dermis, delivery to the draining lymph node, and generation of tolerance are all absent in hairless mice. These results indicate an important function for hair follicle niche and CD11b+ cDC2s in antigen acquisition, and in generation of primary immune tolerance to topical antigens.

Immunotherapies have yet to demonstrate significant efficacy in the treatment of hormone receptor-positive (HR+) breast cancer. Given that endocrine therapy (ET) is the primary approach for treating HR+ breast cancer, we investigated the effects of ET on the tumor immune microenvironment (TME) in HR+ breast cancer. Spatial proteomics of primary HR+ breast cancer samples obtained at baseline and after ET from patients enrolled in a neoadjuvant clinical trial (NCT02764541) indicated that ET upregulated β2-microglobulin and influenced the TME in a manner that promotes enhanced immunogenicity. To gain a deeper understanding of the underlying mechanisms, the intrinsic effects of ET on cancer cells were explored, which revealed that ET plays a crucial role in facilitating the chromatin binding of RelA, a key component of the NF-κB complex. Consequently, heightened NF-κB signaling enhanced the response to interferon-gamma, leading to the upregulation of β2-microglobulin and other antigen presentation-related genes. Further, modulation of NF-κB signaling using a SMAC mimetic in conjunction with ET augmented T-cell migration and enhanced MHC-I-specific T-cell-mediated cytotoxicity. Remarkably, the combination of ET and SMAC mimetics, which also blocks prosurvival effects of NF-κB signaling through the degradation of inhibitors of apoptosis proteins, elicited tumor regression through cell autonomous mechanisms, providing additional support for their combined use in HR+ breast cancer.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Immunotherapy is a promising treatment for triple-negative breast cancer (TNBC), but patients relapse, highlighting the need to understand the mechanisms of resistance. We discovered that in primary breast cancer, tumor cells that resist T cell attack are quiescent. Quiescent cancer cells (QCCs) form clusters with reduced immune infiltration. They also display superior tumorigenic capacity and higher expression of chemotherapy resistance and stemness genes. We adapted single-cell RNA-sequencing with precise spatial resolution to profile infiltrating cells inside and outside the QCC niche. This transcriptomic analysis revealed hypoxia-induced programs and identified more exhausted T cells, tumor-protective fibroblasts, and dysfunctional dendritic cells inside clusters of QCCs. This uncovered differential phenotypes in infiltrating cells based on their intra-tumor location. Thus, QCCs constitute immunotherapy-resistant reservoirs by orchestrating a local hypoxic immune-suppressive milieu that blocks T cell function. Eliminating QCCs holds the promise to counteract immunotherapy resistance and prevent disease recurrence in TNBC.

Type 2 diabetes (T2D) results from insulin resistance and inadequate insulin secretion. Insulin resistance initially causes compensatory islet hyperplasia that progresses to islet disorganization and altered vascularization, inflammation, and, finally, decreased functional β-cell mass and hyperglycemia. The precise mechanism(s) underlying β-cell failure remain to be elucidated. In this study, we show that in insulin-resistant high-fat diet-fed mice, the enhanced islet vascularization and inflammation was parallel to an increased expression of vascular endothelial growth factor A (VEGF). To elucidate the role of VEGF in these processes, we have genetically engineered β-cells to overexpress VEGF (in transgenic mice or after adeno-associated viral vector-mediated gene transfer). We found that sustained increases in β-cell VEGF levels led to disorganized, hypervascularized, and fibrotic islets, progressive macrophage infiltration, and proinflammatory cytokine production, including tumor necrosis factor-α and interleukin-1β. This resulted in impaired insulin secretion, decreased β-cell mass, and hyperglycemia with age. These results indicate that sustained VEGF upregulation may participate in the initiation of a process leading to β-cell failure and further suggest that compensatory islet hyperplasia and hypervascularization may contribute to progressive inflammation and β-cell mass loss during T2D.

Stem cells are critical for the maintenance of many tissues, but whether their integrity is maintained in the face of immunity is unclear. Here we found that cycling epithelial stem cells, including Lgr5+ intestinal stem cells, as well as ovary and mammary stem cells, were eliminated by activated T cells, but quiescent stem cells in the hair follicle and muscle were resistant to T cell killing. Immune evasion was an intrinsic property of the quiescent stem cells resulting from systemic downregulation of the antigen presentation machinery, including MHC class I and TAP proteins, and is mediated by the transactivator NLRC5. This process was reversed upon stem cell entry into the cell cycle. These studies identify a link between stem cell quiescence, antigen presentation, and immune evasion. As cancer-initiating cells can derive from stem cells, these findings may help explain how the earliest cancer cells evade immune surveillance.

Immune checkpoint inhibitors (ICIs) have minimal therapeutic effect in hormone receptor-positive (HR+ ) breast cancer. We present final overall survival (OS) results (n = 88) from a randomized phase 2 trial of eribulin ± pembrolizumab for patients with metastatic HR+ breast cancer, computationally dissect genomic and/or transcriptomic data from pre-treatment tumors (n = 52) for molecular associations with efficacy, and identify cytokine changes differentiating response and ICI-related toxicity (n = 58). Despite no improvement in OS with combination therapy (hazard ratio 0.95, 95% CI 0.59-1.55, p = 0.84), immune infiltration and antigen presentation distinguished responding tumors, while tumor heterogeneity and estrogen signaling independently associated with resistance. Moreover, patients with ICI-related toxicity had lower levels of immunoregulatory cytokines. Broadly, we establish a framework for ICI response in HR+ breast cancer that warrants diagnostic and therapeutic validation. ClinicalTrials.gov Registration: NCT03051659.