Group 2 innate lymphoid cells (ILC2s) regulate inflammation and immunity in mammalian tissues
. Although ILC2s are found in cancers of these tissues
, their roles in cancer immunity and immunotherapy ...are unclear. Here we show that ILC2s infiltrate pancreatic ductal adenocarcinomas (PDACs) to activate tissue-specific tumour immunity. Interleukin-33 (IL33) activates tumour ILC2s (TILC2s) and CD8
T cells in orthotopic pancreatic tumours but not heterotopic skin tumours in mice to restrict pancreas-specific tumour growth. Resting and activated TILC2s express the inhibitory checkpoint receptor PD-1. Antibody-mediated PD-1 blockade relieves ILC2 cell-intrinsic PD-1 inhibition to expand TILC2s, augment anti-tumour immunity, and enhance tumour control, identifying activated TILC2s as targets of anti-PD-1 immunotherapy. Finally, both PD-1
TILC2s and PD-1
T cells are present in most human PDACs. Our results identify ILC2s as anti-cancer immune cells for PDAC immunotherapy. More broadly, ILC2s emerge as tissue-specific enhancers of cancer immunity that amplify the efficacy of anti-PD-1 immunotherapy. As ILC2s and T cells co-exist in human cancers and share stimulatory and inhibitory pathways, immunotherapeutic strategies to collectively target anti-cancer ILC2s and T cells may be broadly applicable.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the classic cold tumor, with few tumor-infiltrating activated CD8+ T cells, a major barrier to immunotherapy. Strategies to convert cold PDACs to ...hot can enable more effective immunotherapies. To identify targets that can convert cold PDACs to hot, we compared a large cohort of rare long-term PDAC survivors with hot tumors to short-term survivors with cold tumors. Surprisingly, hot PDACs were enriched not only in activated CD8+ T cells, but also in group-2 innate lymphoid cells (ILC2s), a class of largely tissue-resident, cytokine-producing innate lymphocyte that potentiates T cell immunity in local tissues. We identified that tumor ILC2s (TILC2s) infiltrate human and mouse PDACs, and TILC2 frequency and tumor expression of the ILC2-activating ligand interleukin (IL)-33 positively correlate with tumor immune cytolytic activity, and long-term patient survival. Using PDAC mouse models, we discovered that the IL33-ILC2 axis activates pancreatic tissue-specific antitumor T-cell immunity. Host IL33 deficiency attenuated TILC2 expansion and CD8+ T-cell activation, accelerating tumor growth in orthotopic PDACs but not heterotopic skin implanted PDACs where TILC2s lack the IL33 receptor. Acute ILC2 depletion partially phenocopied the tissue-specific phenotype of IL33 deficiency, with impaired rejection of immunogenic orthotopic but not heterotopic skin PDACs. Treatment with recombinant IL33 (rIL33), but not with the ILC1-activating ligand rIL18, increased TILC2 frequencies, activated CD8+ T cells, and abrogated orthotopic but not heterotopic PDAC establishment in >70% of mice. Taken together, these studies identify ILC2s as novel anticancer immune cells for PDAC immunotherapy. Strategies to activate tissue-specific ILC2s may be a promising immunotherapeutic approach for PDAC.
Citation Format: Joanne Leung, Luis A Rojas, Jennifer Ruan, Julia Zhao, Billel Gasmi, Zachary Sethna, Anita Ramnarain, Umesh Bhanot, Gokce Askan, Murali Gururajan, Ela Elyada, Youngkyu Park, David A. Tuveson, Mithat Gonen, Steven D. Leach, Jedd D. Wolchok, Ronald P. DeMatteo, Taha Merghoub, Vinod P. Balachandran, John Alec Moral. Tissue-specific innate lymphoid cells are novel targets for pancreatic cancer immunotherapy 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 B02.
Abstract 217
Laminins are heterotrimeric proteins that are the backbone for all basal laminas. They provide structural support, points for focal adhesion, and biochemical feedback for nearby cells. ...Laminins are found in many tissues, including bone marrow and lymphoid organs, and are detected in the discontinuous basal lamina of sinusoidal blood vessels. Laminin g1 is the predominant gamma subunit found in the bone marrow and lymphoid tissues. Here we show that the continuous expression of laminin in adulthood is necessary for viability of the vascular hematopoietic niche (sinusoidal endothelial cells). In the absence of laminin, this niche fails and hematopoiesis is impaired.
To determine if sinusoidal laminin expression correlates with bone marrow failure and recovery, we used a well-established model of bone marrow failure. Wild type mice were treated with a sublethal dose of 5-fluoruracil (5-FU), and bone marrow was analyzed for disruption of sinusoid architecture and endothelial cell receptor and laminin expression. As described in other studies, shortly after treatment with 5-FU, bone marrow sinusoids became disorganized and hemorrhagic, and endothelial cells lost expression of VEGFR2. In addition, sinusoids lost their laminin-containing basal lamina. These changes correlated with bone marrow failure and onset of peripheral blood pancytopenia. As the sinusoids regained normal cytoarchitecture and VEGFR2 expression was normalized, sinusoid expression of laminin also returned to basal levels. With 5-FU-induced bone marrow failure and recovery, hematopoiesis correlated with laminin expression in the vascular hematopoietic stem cell niche.
To test whether laminin expression directly affects the quality of the niche and hematopoiesis, a mutant mouse line was generated in which laminin g1 expression could be deleted by brief exposure to tamoxifen (TM). Control mice were littermates that lacked the TM-responsive Cre transgene. Prior to TM treatment, adult mutant and control mice had normal peripheral blood cell counts, and there was no evidence of gene recombination in genomic DNA samples. Adult mutant and control mice were then injected with either TM or vehicle, and the peripheral blood, bone marrow, spleen, and liver were collected after three weeks. Laminin g1 genes were deleted and laminin expression was lost in bone marrow, spleen, and liver sinusoids of only the TM-treated mutant mice. These mice also became thrombocytopenic and leukopenic. Flow cytometry showed early arrest of B-lymphocyte development within the bone marrow, bone marrow sinusoids became hemorrhagic, and the normal cytoarchitecture of the sinusoids and endothelial expression of VEGFR2 were lost. All other mouse groups were unaffected.
These results suggest that continuous laminin expression is necessary for support of sinusoidal endothelial cells and the vascular hematopoietic niche. Disruption of the hematopoietic microenvironment is sufficient to alter blood cell development and release into peripheral circulation. These studies support the critical role of the microenvironment and stroma in supporting hematopoiesis.
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract 2596
Laminins are heterotrimeric proteins composed of an alpha, beta, and gamma subunit, and are expressed in a majority of organ systems, including hematopoietic and lymphoid tissues. ...Specific laminin subunits have been identified in prognostic stromal gene expression profiles from diffuse large B-cell lymphomas (Lenz et al. 2008). Laminin g1 is the gamma-subunit present in a majority of physiologically expressed laminin heterotrimers. Global laminin-g1 deficiency in mice is embryologically lethal at E5.5 and results in part from failure of the primordial basal lamina to form (Smyth et al. 1998). Conditional knockout mouse lines have therefore been developed to examine the role of laminin-g1 in specific tissues. Disruption of Schwann cell-specific expression of laminin-g1 prevents basal lamina formation within peripheral nerves, decreases glial cell proliferation, and increases glial cell apoptosis leading to a severe dysmyelinated phenotype (Chen and Strickland 2003; Yu et al. 2005).
Here we present data that global disruption of laminin- g1 expression in adult animals induces involution of the spleen and suggests a fundamental role for laminins in maintenance of spleen integrity. Mice homozygous for the LoxP flanked laminin-g1 allele (floxed allele) were crossed with mice expressing a global Cre-ERT2 transgene. Mice homozygous or heterozygous for the floxed laminin-g1 allele that carry the inducible Cre transgene are hereafter referred to as homozygous and heterozygous mutant mice, respectively. Adult heterozygous and homozygous mutant animals were treated with either tamoxifen (TM) or corn oil (control), and three weeks afterwards, tissues were harvested for analysis. Treatment of homozygous mutant mice with TM, but not corn oil, induced laminin-g1 gene recombination. There was a significant decrease in spleen size in TM-treated but not control-treated homozygous mutant animals. Neither TM nor control treatment induced changes in the spleen size of heterozygous mutant animals. Similarly, TM treatment did not alter spleen size of animals that expressed Cre-transgene but were wild type with respect to the floxed laminin-g1 gene, or which were homozygous for the floxed laminin-g1 gene but lacked the Cre-transgene (Figure 1).
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This is the first animal model to show that continued expression of laminins are critical for maintenance of the normal spleen and suggests a potential new target for disorders of lymphoid or splenic hyperproliferation.
Chen ZL, Strickland S. 2003. Laminin gamma1 is critical for Schwann cell differentiation, axon myelination, and regeneration in the peripheral nerve. J Cell Biol 163(4):889-99.
Lenz G, Wright G, Dave SS, Xiao W, Powell J, Zhao H, Xu W, Tan B, Goldschmidt N, Iqbal J and others. 2008. Stromal gene signatures in large-B-cell lymphomas. N Engl J Med 359(22):2313–23.
Smyth N, Vatansever HS, Meyer M, Frie C, Paulsson M, Edgar D. 1998. The targeted deletion of the LAMC1 gene. Ann N Y Acad Sci 857:283–6.
Yu WM, Feltri ML, Wrabetz L, Strickland S, Chen ZL. 2005. Schwann cell-specific ablation of laminin gamma1 causes apoptosis and prevents proliferation. J Neurosci 25(18):4463–72.
No relevant conflicts of interest to declare
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The mechanisms and underlying factors that predispose individuals to development of chemotherapy-induced peripheral neuropathy (CIPN) are not clearly understood. CIPN results in the dose ...attenuation, delay, or discontinuation of drug in patients otherwise responding to chemotherapy. Moderate to severe CIPN can interfere with function and activities of daily living. Microtubule-stabilizing agents, including taxanes and epothilones are strongly associated with development of CIPN. Ixabepilone, the first FDA-approved epothilone, is specifically used in taxane-resistant metastatic breast cancer. Any ixabepilone-induced CIPN may be as high as 71% depending on administration regimen. Specific risk factors for development of this side effect are unknown.
Here we present data using a murine model of sensory hyperalgesia that suggests pretreatment with paclitaxel, currently a labeling indication for use of ixabepilone in metastatic breast cancer, is itself a predisposing factor for development of ixabepilone-induced peripheral neuropathy. Wild type DB/A2 adult mice were treated with intraperitoneal 25 mg/kg paclitaxel, 2.5 mg/kg ixabepilone, or diluent (cremophor: ethanol) every other day for 4 doses. Mice were analyzed by rota-rod to test sensory-motor function and by hot and cold plate to test for temperature hyperalgesia. There was no difference in performance of either of the three treatment groups following the initial treatment cycle. Mice were then treated with a further cycle of chemotherapy, either the same as they received initially, except that half of the paclitaxel-treated group was crossed-over to ixabepilone. Rota-rod and hot plate testing again failed to show a difference between groups. However, cold-plate testing showed a statistically significant increase in cold hyperalgesia in mice treated first with paclitaxel followed by ixabepilone in comparison to all other treatment groups. EM and immunofluorescence analyses of dorsal root ganglia and sciatic nerve sections from mice in all four treatment groups are ongoing.
Although preliminary, these results suggest that prior chemotherapy regimens have a significant effect on development of CIPN in this model. The exact mechanisms of injury are under evaluation. A complementary clinical study is underway in women with breast cancer to understand the effect of ixabepilone on an ultrastructural level and its potential role in the development of peripheral neuropathy.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4184.
Background
We sought to define the clinical and ultrastructure effects of ixabepilone (Ix), a microtubule‐stabilizing chemotherapy agent on cutaneous sensory nerves and to investigate a potential ...mitochondrial toxicity mechanism.
Methods
Ten breast cancer patients receiving Ix underwent total neuropathy score clinical (TNSc) assessment, distal leg skin biopsies at cycle (Cy) 3 (80–90 mg/m2), Cy5 (160–190 mg/m2), and Cy7 (>200 mg/m2) and were compared to 5 controls. Skin blocks were processed for EM and ultrastructural morphometry of Remak axons done.
Results
At baseline, Ix‐treated subjects had higher TNSc values (4.5 ± 0.8 vs. 0.0 ± 0.0), greater percentage of empty (denervated) Schwann cells (29% vs. 12%), altered axonal diameter (422.9 ± 17 vs. 354.9 ± 14.8 nm, P = 0.01), and axon profiles without mitochondria tended to increase compared to control subjects (71% vs. 70%). With increasing cumulative Ix exposure, an increase in TNSc values (Cy3: 5.4 ± 1.2, Cy7: 10 ± 4, P < 0.001), empty Schwann cells (39% by Cy7), and dilated axons (in nm, Cy3: 506.3 ± 22.1, Cy5: 534.8 ± 33, Cy7: 527.8 ± 24.4; P < 0.001) was observed. In addition, axon profiles without mitochondria (Cy3:74%, Cy7:78%) and mitochondria with abnormal morphology (grade 3 or 4) increased from 24% to 79%. Schwann cells with atypical mitochondria and perineuronal macrophage infiltration in dermis were noted.
Interpretation
This study provides functional and structural evidence that Ix exposure induces a dose‐dependent toxicity on small sensory fibers with an increase in TNSc scores and progressive axonal loss. Mitochondria appear to bear the cumulative toxic effect and chemotherapy‐induced toxicity can be monitored through serial skin biopsy‐based analysis.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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