Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Understanding the role of the tumour microenvironment (TME) in lung cancer is critical to improving patient outcome. We identified four histology-independent archetype TMEs in treatment-naive early-stage lung cancer using imaging mass cytometry in the TRACERx study (n=81 patients/198 samples/2.3million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterised by sparse lymphocytes and high tumour-associated neutrophil (TAN) infiltration, had tumour cells spatially separated from vasculature and exhibited low spatial intratumour heterogeneity. TAN-High LUSC had frequent PIK3CA mutations. TAN-High tumours harboured recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis.

Induction of islet autoimmunity to defective ribosomal product of the insulin gene as neoantigen after anti-cancer immunotherapy leading to autoimmune diabetes.

Introduction: The autoimmune response in type 1 diabetes (T1D), in which the beta cells expressing aberrant or modified proteins are killed, resembles an effective antitumor response. Defective ribosomal protein products in tumors are targets of the anti-tumor immune response that is unleashed by immune checkpoint inhibitor (ICI) treatment in cancer patients. We recently described a defective ribosomal product of the insulin gene (INS-DRiP) that is expressed in stressed beta cells and targeted by diabetogenic T cells. T1D patient-derived INS-DRiP specific T cells can kill beta cells and are present in the insulitic lesion. T cells reactive to INS-DRiP epitopes are part of the normal T cell repertoire and are believed to be kept in check by immune regulation without causing autoimmunity. Method: T cell autoreactivity was tested using a combinatorial HLA multimer technology measuring a range of epitopes of islet autoantigens and neoantigen INS-DRiP. INS-DRiP expression in human pancreas and insulinoma sections was tested by immunohistochemistry. Results: Here we report the induction of islet autoimmunity to INS-DRiP and diabetes after ICI treatment and successful tumor remission. Following ICI treatment, T cells of the cancer patient were primed against INS-DRiP among other diabetogenic antigens, while there was no sign of autoimmunity to this neoantigen before ICI treatment. Next, we demonstrated the expression of INS-DRiP as neoantigen in both pancreatic islets and insulinoma by staining with a monoclonal antibody to INS-DRiP. Discussion: These results bridge cancer and T1D as two sides of the same coin and point to neoantigen expression in normal islets and insulinoma that may serve as target of both islet autoimmunity and tumor-related autoimmunity.

Fcγ receptors and immunomodulatory antibodies in cancer.

The discovery of both cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and programmed cell death protein 1 (PD1) as negative regulators of antitumour immunity led to the development of numerous immunomodulatory antibodies as cancer treatments. Preclinical studies have demonstrated that the efficacy of immunoglobulin G (IgG)-based therapies depends not only on their ability to block or engage their targets but also on the antibody's constant region (Fc) and its interactions with Fcγ receptors (FcγRs). Fc-FcγR interactions are essential for the activity of tumour-targeting antibodies, such as rituximab, trastuzumab and cetuximab, where the killing of tumour cells occurs at least in part due to these mechanisms. However, our understanding of these interactions in the context of immunomodulatory antibodies designed to boost antitumour immunity remains less explored. In this Review, we discuss our current understanding of the contribution of FcγRs to the in vivo activity of immunomodulatory antibodies and the challenges of translating results from preclinical models into the clinic. In addition, we review the impact of genetic variability of human FcγRs on the activity of therapeutic antibodies and how antibody engineering is being utilized to develop the next generation of cancer immunotherapies.

Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer.

Background: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. Methods: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour's native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. Results: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. Conclusion: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.

T cell receptor therapeutics: immunological targeting of the intracellular cancer proteome.

The T cell receptor (TCR) complex is a naturally occurring antigen sensor that detects, amplifies and coordinates cellular immune responses to epitopes derived from cell surface and intracellular proteins. Thus, TCRs enable the targeting of proteins selectively expressed by cancer cells, including neoantigens, cancer germline antigens and viral oncoproteins. As such, TCRs have provided the basis for an emerging class of oncology therapeutics. Herein, we review the current cancer treatment landscape using TCRs and TCR-like molecules. This includes adoptive cell transfer of T cells expressing endogenous or engineered TCRs, TCR bispecific engagers and antibodies specific for human leukocyte antigen (HLA)-bound peptides (TCR mimics). We discuss the unique complexities associated with the clinical development of these therapeutics, such as HLA restriction, TCR retrieval, potency assessment and the potential for cross-reactivity. In addition, we highlight emerging clinical data that establish the antitumour potential of TCR-based therapies, including tumour-infiltrating lymphocytes, for the treatment of diverse human malignancies. Finally, we explore the future of TCR therapeutics, including emerging genome editing methods to safely enhance potency and strategies to streamline patient identification.

Cancer cell-intrinsic mechanisms driving acquired immune tolerance.

Immune evasion is a hallmark of cancer, enabling tumors to survive contact with the host immune system and evade the cycle of immune recognition and destruction. Here, we review the current understanding of the cancer cell-intrinsic factors driving immune evasion. We focus on T cells as key effectors of anti-cancer immunity and argue that cancer cells evade immune destruction by gaining control over pathways that usually serve to maintain physiological tolerance to self. Using this framework, we place recent mechanistic advances in the understanding of cancer immune evasion into broad categories of control over T cell localization, antigen recognition, and acquisition of optimal effector function. We discuss the redundancy in the pathways involved and identify knowledge gaps that must be overcome to better target immune evasion, including the need for better, routinely available tools that incorporate the growing understanding of evasion mechanisms to stratify patients for therapy and trials.

Updates on radiotherapy-immunotherapy combinations: Proceedings of 6th annual ImmunoRad conference.

Focal radiation therapy (RT) has attracted considerable attention as a combinatorial partner for immunotherapy (IT), largely reflecting a well-defined, predictable safety profile and at least some potential for immunostimulation. However, only a few RT-IT combinations have been tested successfully in patients with cancer, highlighting the urgent need for an improved understanding of the interaction between RT and IT in both preclinical and clinical scenarios. Every year since 2016, ImmunoRad gathers experts working at the interface between RT and IT to provide a forum for education and discussion, with the ultimate goal of fostering progress in the field at both preclinical and clinical levels. Here, we summarize the key concepts and findings presented at the Sixth Annual ImmunoRad conference.

Glioblastoma cell fate is differentially regulated by the microenvironments of the tumor bulk and infiltrative margin.

Glioblastoma (GBM) recurrence originates from invasive margin cells that escape surgical debulking, but to what extent these cells resemble their bulk counterparts remains unclear. Here, we generated three immunocompetent somatic GBM mouse models, driven by subtype-associated mutations, to compare matched bulk and margin cells. We find that, regardless of mutations, tumors converge on common sets of neural-like cellular states. However, bulk and margin have distinct biology. Injury-like programs associated with immune infiltration dominate in the bulk, leading to the generation of lowly proliferative injured neural progenitor-like cells (iNPCs). iNPCs account for a significant proportion of dormant GBM cells and are induced by interferon signaling within T cell niches. In contrast, developmental-like trajectories are favored within the immune-cold margin microenvironment resulting in differentiation toward invasive astrocyte-like cells. These findings suggest that the regional tumor microenvironment dominantly controls GBM cell fate and biological vulnerabilities identified in the bulk may not extend to the margin residuum.

The tumour ecology of quiescence: Niches across scales of complexity.

Quiescence is a state of cell cycle arrest, allowing cancer cells to evade anti-proliferative cancer therapies. Quiescent cancer stem cells are thought to be responsible for treatment resistance in glioblastoma, an aggressive brain cancer with poor patient outcomes. However, the regulation of quiescence in glioblastoma cells involves a myriad of intrinsic and extrinsic mechanisms that are not fully understood. In this review, we synthesise the literature on quiescence regulatory mechanisms in the context of glioblastoma and propose an ecological perspective to stemness-like phenotypes anchored to the contemporary concepts of niche theory. From this perspective, the cell cycle regulation is multiscale and multidimensional, where the niche dimensions extend to extrinsic variables in the tumour microenvironment that shape cell fate. Within this conceptual framework and powered by ecological niche modelling, the discovery of microenvironmental variables related to hypoxia and mechanosignalling that modulate proliferative plasticity and intratumor immune activity may open new avenues for therapeutic targeting of emerging biological vulnerabilities in glioblastoma.

Anti-CTLA-4 antibodies drive myeloid activation and reprogram the tumor microenvironment through FcγR engagement and type I interferon signaling.

Despite the clinical success of checkpoint inhibitors, a substantial gap still exists in our understanding of their mechanism of action. While antibodies to cytotoxic T lymphocyte-associated protein-4 (CTLA-4) were developed to block inhibitory signals in T cells, several recent studies have demonstrated that Fcγ receptor (FcγR)-dependent depletion of regulatory T cells (Treg) is critical for antitumor activity. Here, using single-cell RNA sequencing, we dissect the impact of anti-CTLA-4-blocking, Treg cell-depleting and FcR-engaging activity on the immune response within tumors. We observed a rapid remodeling of the innate immune landscape as early as 24 h after treatment. Using genetic Treg cell ablation models, we show that immune remodeling was not driven solely by Treg cell depletion or CTLA-4 blockade but mainly through FcγR engagement, downstream activation of type I interferon signaling and reduction of suppressive macrophages. Our findings indicate that FcγR engagement and innate immune remodeling are involved in successful anti-CTLA-4 treatment, supporting the development of optimized immunotherapy agents bearing these features.

Perspectives in Melanoma: meeting report from the Melanoma Bridge (December 2nd - 4th, 2021, Italy).

Advances in immune checkpoint and combination therapy have led to improvement in overall survival for patients with advanced melanoma. Improved understanding of the tumor, tumor microenvironment and tumor immune-evasion mechanisms has resulted in new approaches to targeting and harnessing the host immune response. Combination modalities with other immunotherapy agents, chemotherapy, radiotherapy, electrochemotherapy are also being explored to overcome resistance and to potentiate the immune response. In addition, novel approaches such as adoptive cell therapy, oncogenic viruses, vaccines and different strategies of drug administration including sequential, or combination treatment are being tested. Despite the progress in diagnosis of melanocytic lesions, correct classification of patients, selection of appropriate adjuvant and systemic theràapies, and prediction of response to therapy remain real challenges in melanoma. Improved understanding of the tumor microenvironment, tumor immunity and response to therapy has prompted extensive translational and clinical research in melanoma. There is a growing evidence that genomic and immune features of pre-treatment tumor biopsies may correlate with response in patients with melanoma and other cancers, but they have yet to be fully characterized and implemented clinically. Development of novel biomarker platforms may help to improve diagnostics and predictive accuracy for selection of patients for specific treatment. Overall, the future research efforts in melanoma therapeutics and translational research should focus on several aspects including: (a) developing robust biomarkers to predict efficacy of therapeutic modalities to guide clinical decision-making and optimize treatment regimens, (b) identifying mechanisms of therapeutic resistance to immune checkpoint inhibitors that are potentially actionable, (c) identifying biomarkers to predict therapy-induced adverse events, and (d) studying mechanism of actions of therapeutic agents and developing algorithms to optimize combination treatments. During the Melanoma Bridge meeting (December 2nd-4th, 2021, Naples, Italy) discussions focused on the currently approved systemic and local therapies for advanced melanoma and discussed novel biomarker strategies and advances in precision medicine as well as the impact of COVID-19 pandemic on management of melanoma patients.

A local human Vδ1 T cell population is associated with survival in nonsmall-cell lung cancer.

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA-CD27- effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

High inter-follicular spatial co-localization of CD8+FOXP3+ with CD4+CD8+ cells predicts favorable outcome in follicular lymphoma.

The spatial architecture of the lymphoid tissue in follicular lymphoma (FL) presents unique challenges to studying its immune microenvironment. We investigated the spatial interplay of T cells, macrophages, myeloid cells and natural killer T cells using multispectral immunofluorescence images of diagnostic biopsies of 32 patients. A deep learning-based image analysis pipeline was tailored to the needs of follicular lymphoma spatial histology research, enabling the identification of different immune cells within and outside neoplastic follicles. We analyzed the density and spatial co-localization of immune cells in the inter-follicular and intra-follicular regions of follicular lymphoma. Low inter-follicular density of CD8+FOXP3+ cells and co-localization of CD8+FOXP3+ with CD4+CD8+ cells were significantly associated with relapse (p = 0.0057 and p = 0.0019, respectively) and shorter time to progression after first-line treatment (Logrank p = 0.0097 and log-rank p = 0.0093, respectively). A low inter-follicular density of CD8+FOXP3+ cells is associated with increased risk of relapse independent of follicular lymphoma international prognostic index (FLIPI) (p = 0.038, Hazard ratio (HR) = 0.42 [0.19, 0.95], but not independent of co-localization of CD8+FOXP3+ with CD4+CD8+ cells (p = 0.43). Co-localization of CD8+FOXP3+ with CD4+CD8+ cells is predictors of time to relapse independent of the FLIPI score and density of CD8+FOXP3+ cells (p = 0.027, HR = 0.0019 [7.19 × 10-6 , 0.49], This suggests a potential role of inter-follicular CD8+FOXP3+ and CD4+CD8+ cells in the disease progression of FL, warranting further validation on larger patient cohorts.

Systematic Evaluation of the Immune Environment of Small Intestinal Neuroendocrine Tumors.

PURPOSE: The immune tumor microenvironment and the potential therapeutic opportunities for immunotherapy in small intestinal neuroendocrine tumors (siNET) have not been fully defined. EXPERIMENTAL DESIGN: Herein, we studied 40 patients with primary and synchronous metastatic siNETs, and matched blood and normal tissue obtained during surgery. We interrogated the immune checkpoint landscape using multi-parametric flow cytometry. In addition, matched FFPE tissue was obtained for multi-parametric IHC to determine the relative abundance and distribution of T-cell infiltrate. Tumor mutational burden (TMB) was also assessed and correlated with immune infiltration. RESULTS: Effector tumor-infiltrating lymphocytes (TIL) had a higher expression of PD-1 in the tumor microenvironment compared with the periphery. In addition, CD8+ TILs had a significantly higher co-expression of PD-1/ICOS and PD-1/CTLA-4 (cytotoxic T lymphocyte antigen-4) and higher levels of PD-1 expression compared with normal tissue. IHC revealed that the majority of cases have ≤10% intra-tumoral T cells but a higher number of peri-tumoral T cells, demonstrating an "exclusion" phenotype. Finally, we confirmed that siNETs have a low TMB compared with other tumor types in the TCGA database but did not find a correlation between TMB and CD8/Treg ratio. CONCLUSIONS: Taken together, these results suggest that a combination therapy approach will be required to enhance the immune response, using PD-1 as a checkpoint immunomodulator backbone in combination with other checkpoint targeting molecules (CTLA-4 or ICOS), or with drugs targeting other pathways to recruit "excluded" T cells into the tumor microenvironment to treat patients with siNETs.

Tumor-educated Tregs drive organ-specific metastasis in breast cancer by impairing NK cells in the lymph node niche.

Breast cancer is accompanied by systemic immunosuppression, which facilitates metastasis formation, but how this shapes organotropism of metastasis is poorly understood. Here, we investigate the impact of mammary tumorigenesis on regulatory T cells (Tregs) in distant organs and how this affects multi-organ metastatic disease. Using a preclinical mouse mammary tumor model that recapitulates human metastatic breast cancer, we observe systemic accumulation of activated, highly immunosuppressive Tregs during primary tumor growth. Tumor-educated Tregs show tissue-specific transcriptional rewiring in response to mammary tumorigenesis. This has functional consequences for organotropism of metastasis, as Treg depletion reduces metastasis to tumor-draining lymph nodes, but not to lungs. Mechanistically, we find that Tregs control natural killer (NK) cell activation in lymph nodes, thereby facilitating lymph node metastasis. In line, an increased Treg/NK cell ratio is observed in sentinel lymph nodes of breast cancer patients compared with healthy controls. This study highlights that immune regulation of metastatic disease is highly organ dependent.

Allergens of the urushiol family promote mitochondrial dysfunction by inhibiting the electron transport at the level of cytochromes b and chemically modify cytochrome c1.

BACKGROUND: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. RESULTS: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). CONCLUSION: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.