ABSTRACT
The biological and functional heterogeneity between tumors-both across and within cancer types-poses a challenge for immunotherapy. To understand the factors underlying tumor immune heterogeneity and immunotherapy sensitivity, we established a library of congenic tumor cell clones from an autochthonous mouse model of pancreatic adenocarcinoma. These clones generated tumors that recapitulated T cell-inflamed and non-T-cell-inflamed tumor microenvironments upon implantation in immunocompetent mice, with distinct patterns of infiltration by immune cell subsets. Co-injecting tumor cell clones revealed the non-T-cell-inflamed phenotype is dominant and that both quantitative and qualitative features of intratumoral CD8+ T cells determine response to therapy. Transcriptomic and epigenetic analyses revealed tumor-cell-intrinsic production of the chemokine CXCL1 as a determinant of the non-T-cell-inflamed microenvironment, and ablation of CXCL1 promoted T cell infiltration and sensitivity to a combination immunotherapy regimen. Thus, tumor cell-intrinsic factors shape the tumor immune microenvironment and influence the outcome of immunotherapy.
Subject(s)
Adenocarcinoma/therapy , Immunologic Factors/immunology , Immunotherapy , Lymphocyte Subsets/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Pancreatic Neoplasms/therapy , Tumor Microenvironment/immunology , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Aged , Aged, 80 and over , Animals , CD8-Positive T-Lymphocytes/immunology , Epigenomics , Female , Gene Expression Profiling , Humans , Immunologic Factors/genetics , Male , Mice , Middle Aged , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Primary Cell Culture , Pancreatic NeoplasmsABSTRACT
T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.
Subject(s)
High-Throughput Nucleotide Sequencing , T-Lymphocytes , Base Sequence , Chromosome Mapping , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
Innate immune receptors such as toll-like receptors (TLRs) provide critical molecular links between innate cells and adaptive immune responses. Here, we studied the CD40 pathway as an alternative bridge between dendritic cells (DCs) and adaptive immunity in cancer. Using an experimental design free of chemo- or radiotherapy, we found CD40 activation with agonistic antibodies (âºCD40) produced complete tumor regressions in a therapy-resistant pancreas cancer model, but only when combined with immune checkpoint blockade (ICB). This effect, unachievable with ICB alone, was independent of TLR, STING, or IFNAR pathways. Mechanistically, αCD40/ICB primed durable T cell responses, and efficacy required DCs and host expression of CD40. Moreover, ICB drove optimal generation of polyfunctional T cells in this "cold" tumor model, instead of rescuing T cell exhaustion. Thus, immunostimulation via αCD40 is sufficient to synergize with ICB for priming. Clinically, combination αCD40/ICB may extend efficacy in patients with "cold" and checkpoint-refractory tumors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , CD40 Antigens/agonists , Carcinoma, Pancreatic Ductal/drug therapy , Neoplasms/drug therapy , Pancreatic Neoplasms/drug therapy , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , CD40 Antigens/immunology , CD40 Antigens/metabolism , CTLA-4 Antigen/antagonists & inhibitors , CTLA-4 Antigen/immunology , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/pathology , Cell Line, Tumor , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/metabolism , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/immunology , Drug Screening Assays, Antitumor , Drug Synergism , Female , Humans , Lymphocyte Activation/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/immunology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Programmed Cell Death 1 Receptor/immunology , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
BACKGROUND: Standard chemotherapy remains inadequate in metastatic pancreatic adenocarcinoma. Combining an agonistic CD40 monoclonal antibody with chemotherapy induces T-cell-dependent tumour regression in mice and improves survival. In this study, we aimed to evaluate the safety of combining APX005M (sotigalimab) with gemcitabine plus nab-paclitaxel, with and without nivolumab, in patients with pancreatic adenocarcinoma to establish the recommended phase 2 dose. METHODS: This non-randomised, open-label, multicentre, four-cohort, phase 1b study was done at seven academic hospitals in the USA. Eligible patients were adults aged 18 years and older with untreated metastatic pancreatic adenocarcinoma, Eastern Cooperative Oncology Group performance status score of 0-1, and measurable disease by Response Evaluation Criteria in Solid Tumors version 1.1. All patients were treated with 1000 mg/m2 intravenous gemcitabine and 125 mg/m2 intravenous nab-paclitaxel. Patients received 0·1 mg/kg intravenous APX005M in cohorts B1 and C1 and 0·3 mg/kg in cohorts B2 and C2. In cohorts C1 and C2, patients also received 240 mg intravenous nivolumab. Primary endpoints comprised incidence of adverse events in all patients who received at least one dose of any study drug, incidence of dose-limiting toxicities (DLTs) in all patients who had a DLT or received at least two doses of gemcitabine plus nab-paclitaxel and one dose of APX005M during cycle 1, and establishing the recommended phase 2 dose of intravenous APX005M. Objective response rate in the DLT-evaluable population was a key secondary endpoint. This trial (PRINCE, PICI0002) is registered with ClinicalTrials.gov, NCT03214250 and is ongoing. FINDINGS: Between Aug 22, 2017, and July 10, 2018, of 42 patients screened, 30 patients were enrolled and received at least one dose of any study drug; 24 were DLT-evaluable with median follow-up 17·8 months (IQR 16·0-19·4; cohort B1 22·0 months [21·4-22·7], cohort B2 18·2 months [17·0-18·9], cohort C1 17·9 months [14·3-19·7], cohort C2 15·9 months [12·7-16·1]). Two DLTs, both febrile neutropenia, were observed, occurring in one patient each for cohorts B2 (grade 3) and C1 (grade 4). The most common grade 3-4 treatment-related adverse events were lymphocyte count decreased (20 [67%]; five in B1, seven in B2, four in C1, four in C2), anaemia (11 [37%]; two in B1, four in B2, four in C1, one in C2), and neutrophil count decreased (nine [30%]; three in B1, three in B2, one in C1, two in C2). 14 (47%) of 30 patients (four each in B1, B2, C1; two in C2) had a treatment-related serious adverse event. The most common serious adverse event was pyrexia (six [20%] of 30; one in B2, three in C1, two in C2). There were two chemotherapy-related deaths due to adverse events: one sepsis in B1 and one septic shock in C1. The recommended phase 2 dose of APX005M was 0·3 mg/kg. Responses were observed in 14 (58%) of 24 DLT-evaluable patients (four each in B1, C1, C2; two in B2). INTERPRETATION: APX005M and gemcitabine plus nab-paclitaxel, with or without nivolumab, is tolerable in metastatic pancreatic adenocarcinoma and shows clinical activity. If confirmed in later phase trials, this treatment regimen could replace chemotherapy-only standard of care in this population. FUNDING: Parker Institute for Cancer Immunotherapy, Cancer Research Institute, and Bristol Myers Squibb.
Subject(s)
Adenocarcinoma/drug therapy , Albumins/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , CD40 Antigens/antagonists & inhibitors , Deoxycytidine/analogs & derivatives , Nivolumab/administration & dosage , Paclitaxel/administration & dosage , Pancreatic Neoplasms/drug therapy , Adenocarcinoma/immunology , Adenocarcinoma/secondary , Aged , Albumins/adverse effects , Antineoplastic Combined Chemotherapy Protocols/adverse effects , CD40 Antigens/immunology , Deoxycytidine/administration & dosage , Deoxycytidine/adverse effects , Female , Humans , Male , Middle Aged , Nivolumab/adverse effects , Paclitaxel/adverse effects , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Time Factors , Treatment Outcome , United States , GemcitabineABSTRACT
Cancer immunotherapies are increasingly effective in the clinic, especially immune checkpoint blockade delivered to patients who have T cell-infiltrated tumors. Agonistic CD40 mAb promotes stromal degradation and, in combination with chemotherapy, drives T cell infiltration and de novo responses against tumors, rendering resistant tumors susceptible to current immunotherapies. Partnering anti-CD40 with different treatments is an attractive approach for the next phase of cancer immunotherapies, with a number of clinical trials using anti-CD40 combinations ongoing, but the optimal therapeutic regimens with anti-CD40 are not well understood. Pancreatic ductal adenocarcinoma (PDA) is classically resistant to immunotherapy and lacks baseline T cell infiltration. In this study, we used a tumor cell line derived from a genetically engineered mouse model of PDA to investigate alterations in the sequence of anti-CD40 and chemotherapy as an approach to enhance pharmacological delivery of chemotherapy. Unexpectedly, despite our previous studies showing anti-CD40 treatment after chemotherapy is safe in both mice and patients with PDA, we report in this article that anti-CD40 administration <3 d in advance of chemotherapy is lethal in more than half of treated C57BL/6 mice. Anti-CD40 treatment 2 or 3 d before chemotherapy resulted in significantly increased populations of both activated myeloid cells and macrophages and lethal hepatotoxicity. Liver damage was fully abrogated when macrophage activation was blocked using anti-CSF-1R mAb. These studies highlight the dual nature of CD40 in activating both macrophages and T cell responses, and the need for preclinical investigation of optimal anti-CD40 treatment regimens for safe design of clinical trials.
Subject(s)
Antibodies, Monoclonal/therapeutic use , CD40 Antigens/agonists , Carcinoma, Ductal/therapy , Drug-Related Side Effects and Adverse Reactions/prevention & control , Immunotherapy/methods , Liver Failure/prevention & control , Pancreatic Neoplasms/therapy , Receptor, Macrophage Colony-Stimulating Factor/metabolism , Animals , Carcinoma, Ductal/complications , Carcinoma, Ductal/immunology , Cell Line, Tumor , Clinical Protocols , Drug Interactions , Drug Therapy , Genetic Engineering , Humans , Liver Failure/etiology , Mice , Mice, Inbred C57BL , Neoplasms, Experimental , Pancreatic Neoplasms/complications , Pancreatic Neoplasms/immunology , Receptor, Macrophage Colony-Stimulating Factor/immunologyABSTRACT
Vitiligo is a CD8 T cell-mediated autoimmune disease that has been shown to promote the longevity of memory T cell responses to melanoma. However, mechanisms whereby melanocyte/melanoma Ag-specific T cell responses are perpetuated in the context of vitiligo are not well understood. These studies investigate the possible phenomenon of naive T cell priming in hosts with melanoma-initiated, self-perpetuating, autoimmune vitiligo. Using naive pmel (gp10025-33-specific) transgenic CD8 T cells, we demonstrate that autoimmune melanocyte destruction induces naive T cell proliferation in skin-draining lymph nodes, in an Ag-dependent fashion. These pmel T cells upregulate expression of CD44, P-selectin ligand, and granzyme B. However, they do not downregulate CD62L, nor do they acquire the ability to produce IFN-γ, indicating a lack of functional priming. Accordingly, adult thymectomized mice exhibit no reduction in the severity or kinetics of depigmentation or long-lived protection against melanoma, indicating that the continual priming of naive T cells is not required for vitiligo or its associated antitumor immunity. Despite this, depletion of CD4 T cells during the course of vitiligo rescues the priming of naive pmel T cells that are capable of producing IFN-γ and persisting as memory, suggesting an ongoing and dominant mechanism of suppression by regulatory T cells. This work reveals the complex regulation of self-reactive CD8 T cells in vitiligo and demonstrates the overall poorly immunogenic nature of this autoimmune disease setting.
Subject(s)
Autoimmune Diseases/immunology , CD8-Positive T-Lymphocytes/immunology , Immunologic Memory/immunology , Melanoma/immunology , Vitiligo/immunology , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/transplantation , Cell Line, Tumor , Cell Proliferation , Disease Progression , Female , Granzymes/biosynthesis , Hyaluronan Receptors/biosynthesis , Hyaluronan Receptors/metabolism , Interferon-gamma/biosynthesis , Interferon-gamma/metabolism , L-Selectin/biosynthesis , L-Selectin/metabolism , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Male , Melanocytes/immunology , Mice , Mice, Inbred C57BL , P-Selectin/biosynthesis , P-Selectin/metabolism , Skin/immunology , Up-Regulation , gp100 Melanoma Antigen/genetics , gp100 Melanoma Antigen/metabolismABSTRACT
Immune recognition of tumors can limit cancer development, but antitumor immune responses are often blocked by tumor-mediated immunosuppression. Because microbes or microbial constituents are powerful adjuvants to stimulate immune responses, we evaluated whether intratumoral administration of a highly immunogenic but attenuated parasite could induce rejection of an established poorly immunogenic tumor. We treated intradermal B16F10 murine melanoma by intratumoral injection of an attenuated strain of Toxoplasma gondii (cps) that cannot replicate in vivo and therefore is not infective. The cps treatment stimulated a strong CD8(+) T cell-mediated antitumor immune response in vivo that regressed established primary melanoma. The cps monotherapy rapidly modified the tumor microenvironment, halting tumor growth, and subsequently, as tumor-reactive T cells expanded, the tumors disappeared and rarely returned. The treatment required live cps that could invade cells and also required CD8(+) T cells and NK cells, but did not require CD4(+) T cells. Furthermore, we demonstrate that IL-12, IFN-γ, and the CXCR3-stimulating cytokines are required for full treatment efficacy. The treatment developed systemic antitumor immune activity as well as antitumor immune memory and therefore might have an impact against human metastatic disease. The approach is not specific for either B16F10 or melanoma. Direct intratumoral injection of cps has efficacy against an inducible genetic melanoma model and transplantable lung and ovarian tumors, demonstrating potential for broad clinical use. The combination of efficacy, systemic antitumor immune response, and complete attenuation with no observed host toxicity demonstrates the potential value of this novel cancer therapy.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Cancer Vaccines/administration & dosage , Melanoma, Experimental/immunology , Skin Neoplasms/immunology , Toxoplasma/immunology , Adjuvants, Immunologic/therapeutic use , Animals , Cancer Vaccines/immunology , Cell Line, Tumor , Injections, Intradermal , Melanoma, Experimental/prevention & control , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Neoplasm Recurrence, Local/immunology , Neoplasm Recurrence, Local/prevention & control , Skin Neoplasms/prevention & control , Tumor Escape/immunology , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunologyABSTRACT
Tumor molecular data sets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning (ML) to analyze a single-cell, spatial, and highly multiplexed proteomic data set from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcomes. We designed a multiplex immunohistochemistry antibody panel to compare T-cell functionality and spatial localization in resected tumors from treatment-naïve patients with localized pancreatic ductal adenocarcinoma (PDAC) with resected tumors from a second cohort of patients treated with neoadjuvant agonistic CD40 (anti-CD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both cohorts were assayed, and over 1,000 tumor microenvironment (TME) features were quantified. We then trained ML models to accurately predict anti-CD40 treatment status and disease-free survival (DFS) following anti-CD40 therapy based on TME features. Through downstream interpretation of the ML models' predictions, we found anti-CD40 therapy reduced canonical aspects of T-cell exhaustion within the TME, as compared with treatment-naïve TMEs. Using automated clustering approaches, we found improved DFS following anti-CD40 therapy correlated with an increased presence of CD44+CD4+ Th1 cells located specifically within cellular neighborhoods characterized by increased T-cell proliferation, antigen experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of ML in molecular cancer immunology applications, highlight the impact of anti-CD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for anti-CD40-treated patients with PDAC.
Subject(s)
Carcinoma, Pancreatic Ductal , Immunotherapy , Machine Learning , Neoadjuvant Therapy , Pancreatic Neoplasms , Tumor Microenvironment , Humans , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/therapy , Pancreatic Neoplasms/pathology , Tumor Microenvironment/immunology , Immunotherapy/methods , Carcinoma, Pancreatic Ductal/immunology , Carcinoma, Pancreatic Ductal/therapy , Carcinoma, Pancreatic Ductal/pathology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , CD40 Antigens/metabolism , Treatment Outcome , Female , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , MaleABSTRACT
T cell receptor repertoires can be profiled using next generation sequencing (NGS) to measure and monitor adaptive dynamical changes in response to disease and other perturbations. Genomic DNA-based bulk sequencing is cost-effective but necessitates multiplex target amplification using multiple primer pairs with highly variable amplification efficiencies. Here, we utilize an equimolar primer mixture and propose a single statistical normalization step that efficiently corrects for amplification bias post sequencing. Using samples analyzed by both our open protocol and a commercial solution, we show high concordance between bulk clonality metrics. This approach is an inexpensive and open-source alternative to commercial solutions.
ABSTRACT
Tumor molecular datasets are becoming increasingly complex, making it nearly impossible for humans alone to effectively analyze them. Here, we demonstrate the power of using machine learning to analyze a single-cell, spatial, and highly multiplexed proteomic dataset from human pancreatic cancer and reveal underlying biological mechanisms that may contribute to clinical outcome. A novel multiplex immunohistochemistry antibody panel was used to audit T cell functionality and spatial localization in resected tumors from treatment-naive patients with localized pancreatic ductal adenocarcinoma (PDAC) compared to a second cohort of patients treated with neoadjuvant agonistic CD40 (αCD40) monoclonal antibody therapy. In total, nearly 2.5 million cells from 306 tissue regions collected from 29 patients across both treatment cohorts were assayed, and more than 1,000 tumor microenvironment (TME) features were quantified. We then trained machine learning models to accurately predict αCD40 treatment status and disease-free survival (DFS) following αCD40 therapy based upon TME features. Through downstream interpretation of the machine learning models' predictions, we found αCD40 therapy to reduce canonical aspects of T cell exhaustion within the TME, as compared to treatment-naive TMEs. Using automated clustering approaches, we found improved DFS following αCD40 therapy to correlate with the increased presence of CD44+ CD4+ Th1 cells located specifically within cellular spatial neighborhoods characterized by increased T cell proliferation, antigen-experience, and cytotoxicity in immune aggregates. Overall, our results demonstrate the utility of machine learning in molecular cancer immunology applications, highlight the impact of αCD40 therapy on T cells within the TME, and identify potential candidate biomarkers of DFS for αCD40-treated patients with PDAC.
ABSTRACT
Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC ( NCT03214250 ). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Albumins , Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Carcinoma, Pancreatic Ductal/drug therapy , Carcinoma, Pancreatic Ductal/pathology , Humans , Nivolumab/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Tumor Microenvironment , Pancreatic NeoplasmsABSTRACT
Polymorphisms in the transcription factor Stat4 gene have been implicated as risk factors for systemic lupus erythematosus. Although some polymorphisms have a strong association with autoantibodies and nephritis, their impact on pathophysiology is still unknown. To explore this further we used signal transducers and activators of transcription 4 (Stat4) knockout MRL/MpJ-Fas(lpr)/Fas(lpr) (MRL-Fas(lpr)) mice and found that they did not differ in survival or renal function from Stat4-intact MRL-Fas(lpr) mice. Circulating interleukin (IL)-18 levels, however, were elevated in Stat4-deficient compared to Stat4-intact mice, suggesting that this interleukin might contribute to the progression of lupus nephritis independent of Stat4. In a second approach, Stat4 antisense or missense oligonucleotides or vehicle were given to MRL-Fas(lpr) mice with advanced nephritis. Each of these treatments temporarily ameliorated disease, although IL-18 was increased in each setting. Based on these findings, studies using gene transfer to overexpress IL-18 in MRL-Fas(lpr) and IL-12p40/IL-23 knockout MRL-Fas(lpr) mice reveal a critical role for IL-18 in mediating disease. Thus, the Stat4 and IL-12 (an activator of Stat4)-independent factor, IL-18, can drive autoimmune lupus nephritis in MRL-Fas(lpr) mice. Temporarily blocking Stat4 during advanced nephritis ameliorates disease, suggesting a time-dependent compensatory proinflammatory mechanism.
Subject(s)
Interleukin-18/metabolism , Lupus Nephritis/etiology , STAT4 Transcription Factor/deficiency , Animals , Base Sequence , DNA Primers/genetics , Female , Gene Knockout Techniques , Gene Transfer Techniques , Interferon-gamma/biosynthesis , Interleukin-12/metabolism , Interleukin-18/antagonists & inhibitors , Interleukin-18/genetics , Interleukin-23/metabolism , Kidney/metabolism , Kidney/pathology , Lupus Nephritis/genetics , Lupus Nephritis/metabolism , Lupus Nephritis/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred MRL lpr , Mice, Knockout , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , STAT4 Transcription Factor/antagonists & inhibitors , STAT4 Transcription Factor/geneticsABSTRACT
Immunologically-cold tumors including glioblastoma (GBM) are refractory to checkpoint blockade therapy, largely due to extensive infiltration of immunosuppressive macrophages (MÏs). Consistent with a pro-tumor role of IL-6 in alternative MÏs polarization, we here show that targeting IL-6 by genetic ablation or pharmacological inhibition moderately improves T-cell infiltration into GBM and enhances mouse survival; however, IL-6 inhibition does not synergize PD-1 and CTLA-4 checkpoint blockade. Interestingly, anti-IL-6 therapy reduces CD40 expression in GBM-associated MÏs. We identify a Stat3/HIF-1α-mediated axis, through which IL-6 executes an anti-tumor role to induce CD40 expression in MÏs. Combination of IL-6 inhibition with CD40 stimulation reverses MÏ-mediated tumor immunosuppression, sensitizes tumors to checkpoint blockade, and extends animal survival in two syngeneic GBM models, particularly inducing complete regression of GL261 tumors after checkpoint blockade. Thus, antibody cocktail-based immunotherapy that combines checkpoint blockade with dual-targeting of IL-6 and CD40 may offer exciting opportunities for GBM and other solid tumors.
Subject(s)
Brain Neoplasms/immunology , Brain Neoplasms/therapy , CD40 Antigens/metabolism , Glioblastoma/immunology , Glioblastoma/therapy , Immunotherapy , Interleukin-6/metabolism , Animals , Brain Neoplasms/drug therapy , Gene Deletion , Glioblastoma/drug therapy , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immune Checkpoint Inhibitors/pharmacology , Immune Checkpoint Inhibitors/therapeutic use , Immunosuppression Therapy , Lymphocyte Activation/drug effects , Lymphocytes, Tumor-Infiltrating/drug effects , Lymphocytes, Tumor-Infiltrating/immunology , Macrophages/drug effects , Macrophages/metabolism , Mice, Inbred C57BL , Neutralization Tests , STAT3 Transcription Factor/metabolism , Survival AnalysisABSTRACT
Loss of major histocompatibility complex (MHC) class I and interferon-γ (IFN-γ) sensing are major causes of primary and acquired resistance to checkpoint blockade immunotherapy. Thus, additional treatment options are needed for tumors that lose expression of MHC class I. The cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) regulate classical and alternative nuclear factor κB (NF-κB) signaling. Induction of noncanonical NF-κB signaling with cIAP1/2 antagonists mimics costimulatory signaling, augmenting antitumor immunity. We show that induction of noncanonical NF-κB signaling induces T cell-dependent immune responses, even in ß2-microglobulin (ß2M)-deficient tumors, demonstrating that direct CD8 T cell recognition of tumor cell-expressed MHC class I is not required. Instead, T cell-produced lymphotoxin reprograms both mouse and human macrophages to be tumoricidal. In wild-type mice, but not mice incapable of antigen-specific T cell responses, cIAP1/2 antagonism reduces tumor burden by increasing phagocytosis of live tumor cells. Efficacy is augmented by combination with CD47 blockade. Thus, activation of noncanonical NF-κB stimulates a T cell-macrophage axis that curtails growth of tumors that are resistant to checkpoint blockade because of loss of MHC class I or IFN-γ sensing. These findings provide a potential mechanism for controlling checkpoint blockade refractory tumors.
Subject(s)
Cellular Reprogramming , Histocompatibility Antigens Class I , Immunotherapy , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Neoplasms/therapy , Phagocytes , T-Lymphocytes/immunology , Animals , Humans , Interferon-gamma , Macrophages , Mice , NF-kappa B , Neoplasms/immunology , Signal TransductionABSTRACT
PURPOSE: CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood. PATIENTS AND METHODS: Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) before surgery followed by adjuvant chemotherapy and CD40 mAb. RESULTS: The toxicity profile was acceptable, and overall survival was 23.4 months (95% confidence interval, 18.0-28.8 months). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared with resection specimens from treatment-naïve PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T-cell enriched, compared with 37% of untreated tumors (P = 0.004) and 23% of chemotherapy/chemoradiation-treated tumors (P = 0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines/sec CXCL10 and CCL22 increased systemically after selicrelumab. CONCLUSIONS: This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal/therapeutic use , Pancreatic Neoplasms/drug therapy , Tumor Microenvironment/drug effects , Adult , Aged , Antibodies, Monoclonal/pharmacology , CD40 Antigens/immunology , Female , Humans , Male , Middle Aged , Neoadjuvant Therapy , Pancreatic Neoplasms/surgeryABSTRACT
Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø)-mediated mechanism in MRL-Fas(lpr) mice. By constructing mutant MRL-Fas(lpr) strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Fas(lpr) mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in Møs, apoptosis in the skin, and CLE in MRL-Fas(lpr), but not in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Møs that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Fas(lpr) but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.
Subject(s)
Lupus Erythematosus, Systemic/etiology , Lupus Erythematosus, Systemic/pathology , Macrophage Colony-Stimulating Factor/genetics , Skin Diseases/pathology , Sunlight/adverse effects , Adoptive Transfer , Animals , Enzyme-Linked Immunosorbent Assay , Fibroblasts/metabolism , Fibroblasts/pathology , Flow Cytometry , Fluorescent Antibody Technique , Gene Expression , Immunohistochemistry , Keratinocytes/metabolism , Keratinocytes/pathology , Lupus Erythematosus, Systemic/immunology , Macrophage Colony-Stimulating Factor/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred MRL lpr , Mice, Transgenic , Skin Diseases/etiology , Skin Diseases/immunologyABSTRACT
Macrophages mediate kidney disease and are prominent in a mouse model (MRL-Fas(lpr)) of lupus nephritis. Colony stimulating factor-1 (CSF-1) is the primary growth factor for macrophages, and CSF-1 deficiency protects MRL-Fas(lpr) mice from kidney disease and systemic illness. Whether this renoprotection derives from a reduction of macrophages and whether systemic CSF-1, as opposed to intrarenal CSF-1, promotes macrophage-dependent lupus nephritis remain unclear. Here, we found that increasing systemic CSF-1 hastened the onset of lupus nephritis in MRL-Fas(lpr) mice. Using mutant MRL-Fas(lpr) strains that express high, moderate, or no systemic CSF-1, we detected a much higher tempo of kidney disease in mice with the highest level of CSF-1. Furthermore, we uncovered a multistep CSF-1-dependent systemic mechanism central to lupus nephritis. CSF-1 heightened monocyte proliferation in the bone marrow (SSC(low)CD11b(+)), and these monocytes subsequently seeded the circulation. Systemic CSF-1 skewed the frequency of monocytes toward "inflammatory" (SSC(low)CD11b(+)Ly6C(high)) and activated populations that homed to sites of inflammation, resulting in a more rapid accumulation of intrarenal macrophages (CD11b(+)CSF-1R(+) or CD68(+)) that induced apoptosis of tubular epithelial cells, damaging the kidney. In humans, we found increased levels of CSF-1 in the serum, urine, and kidneys of patients with lupus compared with healthy controls. Furthermore, serum and urine CSF-1 levels correlated with lupus activity, and intrarenal CSF-1 expression correlated with the histopathology activity index of lupus nephritis. Taken together, circulating CSF-1 is a potential therapeutic target for lupus nephritis.
Subject(s)
Lupus Nephritis/etiology , Macrophage Colony-Stimulating Factor/blood , Macrophages/physiology , Monocytes/physiology , Animals , Cell Proliferation , Disease Models, Animal , Female , Humans , Inflammation/etiology , Inflammation/pathology , Inflammation/physiopathology , Kidney/pathology , Kidney/physiopathology , Lupus Nephritis/blood , Lupus Nephritis/pathology , Lupus Nephritis/physiopathology , Macrophage Colony-Stimulating Factor/genetics , Macrophage Colony-Stimulating Factor/physiology , Macrophage Colony-Stimulating Factor/urine , Macrophages/classification , Macrophages/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Monocytes/classification , Monocytes/pathology , PhenotypeABSTRACT
Cancer immunotherapy has revolutionized the treatment landscape in medical oncology, but its efficacy has been variable across patients. Biomarkers to predict such differential response to immunotherapy include cytotoxic T lymphocyte infiltration, tumor mutational burden, and microsatellite instability. A growing number of studies also suggest that baseline tumor burden, or tumor size, predicts response to immunotherapy. In this review, we discuss the changes in immune profile and therapeutic responses that occur with increasing tumor size. We also overview therapeutic approaches to reduce tumor burden and favorably modulate the immune microenvironment of larger tumors.
Subject(s)
Immunotherapy , Neoplasms , Tumor Burden/immunology , Humans , Neoplasms/immunology , Neoplasms/pathology , Neoplasms/therapyABSTRACT
The role CD4+ T cells play in tumor immunity is less well appreciated than the cytotoxic role of CD8+ T cells. Despite clear evidence for CD4+ T cell dependency across multiple immunotherapies, the mechanisms by which CD4+ T cells infiltrate tumors remain poorly understood. Prior studies by our group have shown in a mouse model of pancreatic cancer that systemic activation of the cell surface TNF superfamily member CD40 drives T cell infiltration into tumors and, in combination with immune checkpoint blockade, leads to durable tumor regressions and cures that depend on both CD8+ and CD4+ T cells. Here, we used single-cell transcriptomics to examine the tumor microenvironment following treatment with agonist CD40 antibody with or without immune checkpoint blockade. We show that intratumor myeloid cells produce the chemokine CCL5 in response to CD40 agonist and that CCL5 mediates an influx of CD4+ T cells into the tumor microenvironment. Disruption of CCL5 genetically or pharmacologically mitigates the influx of CD4+ but not CD8+ T cells into tumors and blunts the therapeutic efficacy of immunotherapy. These findings highlight a previously unappreciated role for CCL5 in selectively mediating CD4+ T cell tumor infiltration in response to effective immunotherapy.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD40 Antigens/immunology , Chemokine CCL5/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms, Experimental/immunology , Animals , CD4-Positive T-Lymphocytes/pathology , CD40 Antigens/genetics , Chemokine CCL5/genetics , Immunotherapy , Lymphocytes, Tumor-Infiltrating/pathology , Mice , Mice, Knockout , Neoplasms, Experimental/genetics , Neoplasms, Experimental/pathology , Neoplasms, Experimental/therapyABSTRACT
The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.