Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing ...macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages.
For complete details on the use and execution of this protocol, please refer to Roehle et al. (2021).1
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•Phagocytosis of live tumor cells by primary mouse or human macrophages•Streamlined protocol for in vitro differentiation of human macrophages from PBMCs•Analysis of phagocytosis in adherent macrophages by flow cytometry•Use of cIAP1/2 antagonism and cytokines to induce phagocytosis
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Although tumor-associated macrophages are generally immunosuppressive, macrophages may also promote tumor clearance via phagocytosis of live tumor cells. Here, we present a protocol for assessing macrophage engulfment of tumor cells in vitro using flow cytometry. We describe steps for cell preparation, reseeding macrophages, and setting up phagocytosis. We then detail procedures for collecting samples, staining macrophages, and flow cytometry. The protocol is applicable to both mouse bone-marrow-derived macrophages and human monocyte-derived macrophages.
Pancreatic cancer is rapidly progressive and notoriously difficult to treat with cytotoxic chemotherapy and targeted agents. Recent demonstration of the efficacy of maintenance PARP inhibition in ...germline
mutated pancreatic cancer has raised hopes that increased understanding of the DNA damage response pathway will lead to new therapies in both homologous recombination (HR) repair-deficient and proficient pancreatic cancer. Here, we review the potential mechanisms of exploiting HR deficiency, replicative stress, and DNA damage-mediated immune activation through targeted inhibition of DNA repair regulatory proteins.
Cytokine-based therapies for cancer have not achieved widespread clinical success because of inherent toxicities. Treatment for pancreatic cancer is limited by the dense stroma that surrounds tumors ...and by an immunosuppressive tumor microenvironment. To overcome these barriers, we developed constructs of single-domain antibodies (VHHs) against PD-L1 fused with IL-2 and IFNγ. Targeting cytokine delivery in this manner reduced pancreatic tumor burden by 50%, whereas cytokines fused to an irrelevant VHH, or blockade of PD-L1 alone, showed little effect. Targeted delivery of IL-2 increased the number of intratumoral CD8
T cells, whereas IFNγ reduced the number of CD11b
cells and skewed intratumoral macrophages toward the display of M1-like characteristics. Imaging of fluorescent VHH-IFNγ constructs, as well as transcriptional profiling, demonstrated targeting of IFNγ to the tumor microenvironment. Many tumors and tumor-infiltrating myeloid cells express PD-L1, rendering them potentially susceptible to this form of targeted immunotherapy.
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XBP‐1, a transcription factor that drives the unfolded protein response (UPR), is activated in B cells when they differentiate to plasma cells. Here, we show that in the B cells, whose capacity to ...secrete IgM has been eliminated, XBP‐1 is induced normally on induction of differentiation, suggesting that activation of XBP‐1 in B cells is a differentiation‐dependent event, but not the result of a UPR caused by the abundant synthesis of secreted IgM. Without XBP‐1, B cells fail to signal effectively through the B‐cell receptor. The signalling defects lead to aberrant expression of the plasma cell transcription factors IRF4 and Blimp‐1, and altered levels of activation‐induced cytidine deaminase and sphingosine‐1‐phosphate receptor. Using XBP‐1‐deficient/Blimp‐1‐GFP transgenic mice, we find that XBP‐1‐deficient B cells form antibody‐secreting plasmablasts in response to initial immunization; however, these plasmablasts respond ineffectively to CXCL12. They fail to colonize the bone marrow and do not sustain antibody production. These findings define the role of XBP‐1 in normal plasma cell development and have implications for management of B‐cell malignancies.
Mitochondrial biogenesis initiates within hours of T cell receptor (TCR) engagement and is critical for T cell activation, function, and survival; yet, how metabolic programs support mitochondrial ...biogenesis during TCR signaling is not fully understood. Here, we performed a multiplexed metabolic chemical screen in CD4+ T lymphocytes to identify modulators of metabolism that impact mitochondrial mass during early T cell activation. Treatment of T cells with pyrvinium pamoate early during their activation blocks an increase in mitochondrial mass and results in reduced proliferation, skewed CD4+ T cell differentiation, and reduced cytokine production. Furthermore, administration of pyrvinium pamoate at the time of induction of experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis in mice, prevented the onset of clinical disease. Thus, modulation of mitochondrial biogenesis may provide a therapeutic strategy for modulating T cell immune responses.
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•High-throughput chemical screen discovers modulators of mitochondrial mass•PP blocks mitochondrial mass increase during T cell activation•PP inhibits T cell function by targeting pyruvate oxidation•PP impairs helper T cell differentiation, preventing the onset of EAE
Kurmi et al. present a high-throughput platform quantifying mitochondrial mass in early T cell activation. Pyrvinium pamoate (PP) emerges as a potent inhibitor of mitochondria, disrupting pyruvate oxidation and impairing T cell function. PP impacts helper T cell differentiation to prevent EAE onset, revealing modulation of mitochondria as a potential target for autoimmunity.
Prostate cancers are considered to be immunologically 'cold' tumors given the very few patients who respond to checkpoint inhibitor (CPI) therapy. Recently, enrichment of interferon-stimulated genes ...(ISGs) predicted a favorable response to CPI across various disease sites. The enhancer of zeste homolog-2 (EZH2) is overexpressed in prostate cancer and known to negatively regulate ISGs. In the present study, we demonstrate that EZH2 inhibition in prostate cancer models activates a double-stranded RNA-STING-ISG stress response upregulating genes involved in antigen presentation, Th1 chemokine signaling and interferon response, including programmed cell death protein 1 (PD-L1) that is dependent on STING activation. EZH2 inhibition substantially increased intratumoral trafficking of activated CD8
T cells and increased M1 tumor-associated macrophages, overall reversing resistance to PD-1 CPI. Our study identifies EZH2 as a potent inhibitor of antitumor immunity and responsiveness to CPI. These data suggest EZH2 inhibition as a therapeutic direction to enhance prostate cancer response to PD-1 CPI.
Here, we describe a one‐step, in vivo CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in mice. We produced knock‐in (KI) mice wherein a 1.9‐kb DNA fragment bearing a pre‐arranged human ...B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. Our methodology relies on Cas9 nuclease‐induced double‐stranded breaks directed by two sgRNAs to occur within the specific target locus of fertilized oocytes. These double‐stranded breaks are subsequently repaired via homology‐directed repair by a plasmid‐borne template containing the pre‐arranged human immunoglobulin heavy chain. To validate our knock‐in mouse model, we examined the expression of the KI immunoglobulin heavy chains by following B‐cell development and performing single B‐cell receptor sequencing. We optimized this strategy to generate immunoglobulin KI mice in a short amount of time with a high frequency of homologous recombination (30–50%). In the future, we envision that such knock‐in mice will provide much needed vaccination models to evaluate immunoresponses against immunogens specific for various infectious diseases.
Synopsis
We describe an in vivo, CRISPR/Cas9 nuclease‐mediated strategy to generate knock‐in (KI) mice. A large 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus. We optimized this strategy to generate two independent heavy chain KI mouse models in about 3 weeks with a high frequency of homologous recombination (30–50%).
Via a CRISPR/Cas9‐nuclease‐mediated strategy, a 1.9‐kb DNA fragment bearing a pre‐arranged human B‐cell receptor heavy chain was recombined into the native murine immunoglobulin locus.
The frequency of recombination is 30–50%.
This strategy enabled us to generate knock‐in mice in about 3 weeks.
Direct recombination of pre‐arranged human B‐cell receptor into the native immunoglobulin locus in fertilized mouse oocytes provides a facile strategy for obtaining vaccination models for infectious disease.
Selinexor (KPT-330) is a first-in-class nuclear transport inhibitor currently in clinical trials as an anticancer agent. To determine how selinexor might affect antitumor immunity, we analyzed immune ...homeostasis in mice treated with selinexor and found disruptions in T-cell development, a progressive loss of CD8 T cells, and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by selinexor, suggesting that normal immune homeostasis could recover. We found that a high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week did not restore immune homeostasis. Both naïve and effector CD8 T cells cultured
showed impaired activation in the presence of selinexor. These experiments suggest that nuclear exportins are required for T-cell development and function. We determined the minimum concentration of selinexor required to block T-cell activation and showed that T-cell-inhibitory effects of selinexor occur at levels above 100 nmol/L, corresponding to the first 24 hours post-oral dosing. In a model of implantable melanoma, selinexor treatment at 10 mg/kg with a 4-day drug holiday led to intratumoral IFNγ
, granzyme B
cytotoxic CD8 T cells that were comparable with vehicle-treated mice. Overall, selinexor treatment leads to transient inhibition of T-cell activation, but clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T-cell functioning and development of antitumor immunity.
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Abstract
Like virally infected cells, tumors can evade CD8 T-cell recognition by downregulation of MHC class I. Loss of MHC class I and IFNg sensing are major causes of primary and acquired ...resistance to checkpoint blockade immunotherapy. The cellular (c)-IAPs regulate classical and alternative NF-kB signaling, and induction of noncanonical NF-kB signaling using IAP antagonists mimics costimulatory signaling, augmenting antitumor immunity. We now show that induction of noncanonical NF-kB signaling promotes antitumor immunity across a range of orthotopic and spontaneous pancreatic cancer models, and induces T cell-dependent immune responses even in b2m-/- tumors, demonstrating that direct CD8 T-cell recognition of tumor cell expressed MHC class I is not required. Here we show that MHC class I negative tumors can be controlled in a T-cell dependent fashion by repolarized intratumoral phagocytes. In wild-type mice, but not mice incapable of mounting antigen-specific T cell responses, treatment with IAP antagonists increases phagocytosis of tumor cells, as determined by flow cytometry, imaging flow cytometry, and quantitative immunofluorescence. This mechanism of action inspired combination therapy with CD47 blockade, a strategy that induced curative responses in otherwise refractory preclinical models. To our knowledge, this is the first example of antigen specific immunotherapy for tumors that are refractory to checkpoint blockade due to loss of MHC class I or IFNg sensing.
Citation Format: Stephanie K. Dougan. Tumor-specific T cells reprogram mononuclear phagocytes for destruction of checkpoint blockade-resistant pancreatic cancer abstract. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I09.
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