Essential Immune Functions Research Articles

Macrophages and autophagy: partners in crime.

Macrophages and autophagy are intricately linked, both playing vital roles in maintaining homeostasis and responding to disease. Macrophages, known for their 'eating' function, rely on a sophisticated digestion system to process a variety of targets, from apoptotic cells to pathogens. The connection between macrophages and autophagy is established early in their development, influencing both differentiation and mature functions. Autophagy regulates essential immune functions, such as inflammation control, pathogen clearance, and antigen presentation, linking innate and adaptive immunity. Moreover, it modulates cytokine production, ensuring a balanced inflammatory response that prevents excessive tissue damage. Autophagy also plays a critical role in macrophage polarization, influencing their shift between pro-inflammatory and anti-inflammatory states. This review explores the role of autophagy in macrophages, emphasizing its impact across various tissues and pathological conditions, and detailing the cellular and molecular mechanisms by which autophagy shapes macrophage function.

300. Disturbance of Gut Microbial Diversity in ICU Patients from Colombia is Driven by Multi-Drug Resistant Organisms (MDRO) Colonization

Abstract Background Gut microbiota plays a critical role in health, with essential functions in physiology, metabolism, immunity, regulation, and protection against pathogens. Critically-ill patients admitted on the ICU experience perturbation in the microbiota, with reduction in the phylogenetic diversity and composition. Here, we aimed to investigate the rectal microbiota in patients admitted on the ICU from Colombia, focused on MDRO colonization. Methods Microbiome analysis and microbiological screening for MDRO and WGS were performed in rectal swabs taken on admission day from 180 patients from 5 ICUs. Microbiome analysis was determined by 16S rRNA sequencing using Illumina Miseq platform. alpha-diversity metrics were calculated by Richness, Simpson, and Shannon indexes, and beta-diversity was determined using Similarity Percentages (SIMPER) analysis. Patients were grouped into 2 cohorts: MDRO-colonized and non-colonized. Clinical data was collected using REDCap. Results MDRO colonization was detected in 13 patients (7%), and Enterobacter cloacae complex was the most prevalent microorganism (38%), followed by K. pneumoniae (23%), and P. aeruginosa (15%). Of these MDRO, 23% and 15% harbored KPC-2 and KPC-3/OXA-9 combination, respectively. Prevotella and Bacteroides were the most abundant genus in the microbiota of all patients. Interestingly, microbiota of MDRO-colonized patients showed lower diversity (Shannon p:0.0025, Simpson p: 0.0031 and richness 0.012) and had less bacterial taxa than the observed in the non-colonized group (n=167), in which Bacteroides, Porphyromonas, Campylobacter, and Oscillospiraceae genus were exclusively detected. Moreover, Parabacteroides was a genus exclusively identified in the MDRO colonized group. Further, 14 genus including Oscillospiraceae, Lactobacillus, and Faecalibacterium related to beneficial functions were lower in abundance from MDRO-colonized patients, whereas some pathobionts were present in high abundance in this group. Most of patients were men (58%); median age was 59 y/o. Charlson comorbidity index 4 (IQR=1-6). Conclusion Loss in microbial diversity, with low abundance of beneficial microorganisms were associated with MDRO gut colonization in ICU patients from Colombia. Disclosures Karen Melissa Ordoñez, n/a, Pfizer: Advisor/Consultant|Pfizer: Honoraria

Abstract 4363: Monocyte-specific epigenetic age acceleration and cardiomyopathy risk among survivors of childhood cancer

Abstract Our assessment of epigenetic age acceleration (EAA), calculated with DNA methylation (DNAm) data generated from bulk DNA derived from peripheral blood mononuclear cells (PBMC), supports accelerated aging in childhood cancer survivors (CCS). It is challenging to disentangle variation of DNAm at cell-type specific levels from the effects of age-dependent cell type composition, and bulk measurements may obfuscate the links between EAA and age-related outcomes (e.g., cardiomyopathy). Methylation profiling was generated using Infinium EPIC BeadChips on PBMC-derived DNA from CCS in the St. Jude Lifetime Cohort. Tensor composition analysis was employed to deconvolute bulk DNAm and infer DNAm at each leukocyte subtype level, i.e., a single n (individuals) by m (DNAm sites) matrix of observed DNAm data was deconvoluted into multiple n by m matrices of DNAm data. Epigenetic age (EA, using Levine’s clock) and EAA (residuals from the fit of a simple linear regression of EA on chronological age at blood draw) were calculated for bulk PBMCs and each cell subtype (CD4T, CD8T, B, natural killer, and monocyte). Cardiomyopathy (CMP) was assessed by echocardiography, and severity graded (2 = moderate, 3 = severe/disabling, 4 = life-threatening and 5 = fatal) using a modified version of the National Cancer Institute Common Terminology Criteria for Adverse Events. Cumulative doses of anthracyclines and mean heart radiation doses (heart-RT) calculated through radiation dosimetry were abstracted from medical records. Associations between EAA and CMP were evaluated by multivariable Cox regression. Cell-type specific EA was highly correlated with bulk EA with Pearson r2 between 0.63 (CD8T) and 0.79 (CD4T), but the linear regression lines of cell-type specific EA against bulk EA differed in both intercept and slope, suggesting heterogeneity across leukocyte subtypes. Cell-type specific EAA was moderately correlated with bulk EAA with Pearson r2 between 0.23 (monocyte) and 0.29 (CD8T). Among 2,044 CCS (median age = 33.7 years), 104 (5.1%) developed CMP (≥grade 3). Among bulk EAA and five cell-type specific EAAs, monocyte EAA was the only one significantly associated with CMP (hazard ratio per standard deviation increase in EAA = 1.25, 95% CI = 1.04-1.50, P = 0.018). Cell sorting of PBMC followed by DNAm is currently underway for further validation. We showed an in-depth view of the variability of EAA across leukocyte subtypes, and more importantly, demonstrated that monocyte EAA was associated with CMP risk. Our novel finding is plausible and consistent with the literature implicating monocyte-derived cardiac macrophages in cardiac remodeling, which can be induced by cardiotoxic cancer treatment exposures in CCS. Therapeutic strategies that prevent deleterious effects of monocytes contributing to adverse cardiac remodeling, while sparing their essential immune functions, may prevent or ameliorate CMP among CCS. Citation Format: Cheng Chen, Qian Dong, Qin Na, Nan Song, John Easton, Heather L. Mulder, Emily Walker, Geoffrey Neale, Emily R. Finch, Qian Li, Yutaka Yasui, Daniel A. Mulrooney, Melissa M. Hudson, Kirsten K. Ness, Jinghui Zhang, Xiang Chen, Hui Wang, Leslie L. Robison, Zhaoming Wang. Monocyte-specific epigenetic age acceleration and cardiomyopathy risk among survivors of childhood cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4363.

The diverse roles of C-type lectin-like receptors in immunity.

Our understanding of the C-type lectin-like receptors (CTLRs) and their functions in immunity have continued to expand from their initial roles in pathogen recognition. There are now clear examples of CTLRs acting as scavenger receptors, sensors of cell death and cell transformation, and regulators of immune responses and homeostasis. This range of function reflects an extensive diversity in the expression and signaling activity between individual CTLR members of otherwise highly conserved families. Adding to this diversity is the constant discovery of new receptor binding capabilities and receptor-ligand interactions, distinct cellular expression profiles, and receptor structures and signaling mechanisms which have expanded the defining roles of CTLRs in immunity. The natural killer cell receptors exemplify this functional diversity with growing evidence of their activity in other immune populations and tissues. Here, we broadly review select families of CTLRs encoded in the natural killer cell gene complex (NKC) highlighting key receptors that demonstrate the complex multifunctional capabilities of these proteins. We focus on recent evidence from research on the NKRP1 family of CTLRs and their interaction with the related C-type lectin (CLEC) ligands which together exhibit essential immune functions beyond their defined activity in natural killer (NK) cells. The ever-expanding evidence for the requirement of CTLR in numerous biological processes emphasizes the need to better understand the functional potential of these receptor families in immune defense and pathological conditions.

Essential immune functions of fibroblasts in innate host defense.

The term fibroblast has been used generally to describe spindle-shaped stromal cells of mesenchymal origin that produce extracellular matrix, establish tissue structure, and form scar. Current evidence has found that cells with this morphology are highly heterogeneous with some fibroblastic cells actively participating in both innate and adaptive immune defense. Detailed analysis of barrier tissues such as skin, gut, and lung now show that some fibroblasts directly sense pathogens and other danger signals to elicit host defense functions including antimicrobial activity, leukocyte recruitment, and production of cytokines and lipid mediators relevant to inflammation and immunosuppression. This review will synthesize current literature focused on the innate immune functions performed by fibroblasts at barrier tissues to highlight the previously unappreciated importance of these cells in immunity.

CD73/NT5E is a Potential Biomarker for Cancer Prognosis and Immunotherapy for Multiple Types of Cancers.

Cluster of Differentiations 73 (CD73)/ecto-5'-nucleotidase (NT5E) is a novel type of immune molecular marker expressed on many tumor cells and involved in regulating the essential immune functions and affecting the prognosis of cancer patients. However, it is not clear how the NT5E is linked to the infiltration levels of the immune cells in pan-cancer patients and their final prognosis. This study explores the role of NT5E in 33 tumor types using GEPIA, TIMER, Oncomine, BioGPS databases, and several bioinformatic tools. The findings reveal that the NT5E is abnormally expressed in a majority of the types of cancers and can be used for determining the prognosis prediction ability of different cancers. Moreover, NT5E is significantly related to the infiltration status of numerous immune cells, immune-activated pathways, and immunoregulator expressions. Last, specific inhibitor molecules, like NORNICOTINE, AS-703026, and FOSTAMATINIB, which inhibit the expression of NT5E in various types of cancers, are screened with the CMap. Thus, it is proposed that NT5E can be utilized as a potential biomarker for predicting the prognosis of cancer patients and determining the infiltration of various immune cells in different types of cancers.

Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection invivo and human cells invitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1β heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

Human Platelets Contain, Translate, and Secrete Azurocidin; A Novel Effect on Hemostasis

Platelets play a significant role in hemostasis and perform essential immune functions, evidenced by the extensive repertoire of antimicrobial molecules. Currently, there is no clear description of the presence of azurocidin in human platelets. Azurocidin is a 37 kDa cationic protein abundant in neutrophils, with microbicidal, opsonizing, and vascular permeability-inducing activity. Therefore, this work aimed to characterize the content, secretion, translation, and functions of azurocidin in platelets. Our results show the presence of azurocidin mRNA and protein in α-granules of platelet and megakaryoblasts, and stimulation with thrombin, ADP, and LPS leads to the secretion of free azurocidin as well as within extracellular vesicles. In addition, platelets can translate azurocidin in a basal or thrombin-induced manner. Finally, we found that the addition of low concentrations of azurocidin prevents platelet aggregation and activation. In conclusion, we demonstrate that platelets contain, secrete, and translate azurocidin, and this protein may have important implications for hemostasis.

Micronutrients early in critical illness, selective or generous, enteral or intravenous?

Micronutrients have essential antioxidant and immune functions, while low blood concentrations are frequently observed in critically ill patients. This has led to the concepts of complementation, repletion, or even pharmacological supplementation. Over the last three decades, many clinical studies have tested the latter strategy, with controversial or negative results. Therefore, this review aims at evaluating micronutrient-related interventions that are mandatory or need to be assessed in future trials or clinical registries in all or specific critically ill patients. In the critically ill, low plasma/serum micronutrient levels not always reflect a true deficiency in the absence of demonstrable losses. Current practices of micronutrient provision and monitoring in critical care, vary substantially across the world. Also, recent clinical trials testing high dose as monotherapy (selenium, thiamine, vitamin C, vitamin D) or in combination have failed to demonstrate clinical benefits in sepsis. However, these studies have not applied a physiological integrative approach of micronutrient action. Micronutrients are essential in nutrition but their administration and monitoring are difficult. So far, different well designed RCTs on intravenous and oral high dose micronutrient supplementation have been conducted. Nevertheless, very high-dose single micronutrients cannot be advocated at this stage in sepsis, or any other critical condition. By contrast, studies using combination of moderate doses of micronutrients in specific diseases, such as burns and trauma have been associated with improved outcomes. Intravenous administration seems to be the most efficient route. Future clinical trials need to integrate the physiology underlying the interconnected micronutrient activity, and choose more specific primary and secondary endpoints.

Gut microbiome: from the reference of the norm to pathology

А systemic assessment of the state of the human intestinal microbiome was carried out in relation to its function in the macroorganism, aimed at providing the nutriome, and the factors that determine the adequate nutritional status. A new concept of "reference gut microbiome of a healthy person" was postulated and the requirements to it were formulated: interaction with the host according to the principle of mutualism, provision of immune balance with the macroorganism due to the correct formation of mucosal immunity, implementation of metabolic and regulatory functions without losses for the nutriome. A set of characteristics and biomarkers reflecting the taxonomic composition and population properties of the microbial community, as well as the state of its essential immune and metabolic functions, was proposed as a criterion for its assessment in healthy adults who consume a diet balanced in nutritive and energy value, appropriate for age and energy spending. The influence of alimentary factors on the formation of the human intestinal microbiome in early ontogenesis, the nature of dysbiotic shifts, including those under common non-infectious alimentary-dependent diseases (obesity, food allergy, urolithiasis), in Russians were studied, the ways of their correction and maintenance of the intestinal microbiota in the process of life were substantiated taking into account modern knowledge.

One too many: the surprising heterogeneity of Drosophila macrophages

Macrophages are motile cells that roam the extracellular spaces within organs or the body cavity and carry out essential functions in organ development and immunity. New work published in The EMBO Journal adds surprising new insights into the heterogeneity of Drosophila macrophages revealing many similarities to their vertebrate counterparts.

Cas3 Protein—A Review of a Multi-Tasking Machine

Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

Olfactomedin 4 marks a subset of neutrophils in mice.

Neutrophils are the most abundant immune cell of the innate immune system and participate in essential immune functions. Heterogeneity within neutrophils has been documented, but it is difficult to distinguish if these are altered activation states of a single population or separate subpopulations of neutrophils determined at the time of differentiation. Several groups have identified a subset of human neutrophils that express olfactomedin 4 (OLFM4) and increased OLFM4+ neutrophils during sepsis is correlated with worse outcome, suggesting these neutrophils or the OLFM4 they secrete may be pathogenic. We tested if mice could be used as a model to study OLFM4+ neutrophils. We found the OLFM4 expressing subset of neutrophils is conserved in mice. Depending on the strain, 7–35% of murine neutrophils express OLFM4 and expression is determined early in neutrophil differentiation. OLFM4+ neutrophils phagocytose and transmigrate with similar efficiency as OLFM4− neutrophils. Here we show that within neutrophil extracellular traps (NETs) OLFM4+ and OLFM4− neutrophils undergo NETosis and OLFM4 colocalizes. Finally, we generated an OLFM4 null mouse and show that these mice are protected from death when challenged with sepsis, providing further evidence that the OLFM4 expressing subpopulation of neutrophils, or the OLFM4 they secrete, may be pathogenic during overwhelming infection.

Resolution of macropinosomes, phagosomes and autolysosomes: Osmotically driven shrinkage enables tubulation and vesiculation.

Macropinosomes, phagosomes and autolysosomes are comparatively large, quasi-spherical organelles that play essential functions in immunity and homeostasis. These vacuolar organelles are relatively short-lived, promptly fragmenting into smaller structures. Vacuolar resolution is mediated by tubulation and vesiculation, processes orchestrated by protein complexes that are recruited to highly curved membranes. Importantly, the surface-to-volume ratios of the tubules and vesicles generated during the resolution process are considerably larger than that of the parental vacuole. Because membranes under high hydrostatic tension resist deformation, an active, concomitant loss of volume is required to sustain the resolution process and may even initiate tubulation and vesiculation. Despite its fundamental role in membrane remodeling, the mechanisms that account for organellar volume loss are poorly understood, but are likely to involve the export of solutes followed by osmotically obliged water. In this review, we describe the principles and possible mechanisms underlying the resolution of organelles, with particular attention paid to the osmolytes they contain and the pathways mediating their exit.

The perivascular origin of pathological fibroblasts.

The ability to repair tissues is essential for the survival of organisms. In chronic settings, the failure of the repair process to terminate results in overproduction of collagen, a pathology known as fibrosis, which compromises organ recovery and impairs function. The origin of the collagen-overproducing cell has been debated for years. Here we review recent insights gained from the use of lineage tracing approaches in several organs. The resulting evidence points toward specific subsets of tissue-resident mesenchymal cells, mainly localized in a perivascular position, as the major source for collagen-producing cells after injury. We discuss these findings in view of the functional heterogeneity of mesenchymal cells of the perivascular niche, which have essential vascular, immune, and regenerative functions that need to be preserved for efficient repair.

RETRACTED ARTICLE: Inhibition of airway inflammation in a cockroach allergen model of asthma by agonists of miRNA-33b

MicroRNAs (miRNAs) play powerful roles in immune function by regulating target genes that mediate cell behavior. It is well known that mast cells have essential effector and immune regulatory functions in IgE-associated allergic disorders and in innate and adaptive immune responses. However, the role of miRNAs in mediating mast cell functions and the relevant mechanisms require further exploration. The roles of miR-33b in airway inflammation and mast cell functions are still unknown. To examine the role of miR-33b in mouse mast cells in cockroach allergen-induced asthma, we developed a lentiviral system for miRNA-33b overexpression to examine whether miRNA-33b mediates airway inflammation by regulating mast cell function and to evaluate the underlying mechanism. The results showed that miR-33b inhibited cockroach allergen-induced asthma in vivo: in particular, it inhibited TH2 cytokine production. In addition, we found that in cells in which miRNA-33b had been transfected, mast cell degranulation was inhibited through suppression of the calcium release and IgE/FcεRI pathway. Our study provides new insight into the roles of miR-33b in asthma and mast cell biology and identifies novel mechanisms that may contribute to mast cell-related pathological conditions in airway inflammation.

The evolution of the natural killer complex; a comparison between mammals using new high-quality genome assemblies and targeted annotation

Natural killer (NK) cells are a diverse population of lymphocytes with a range of biological roles including essential immune functions. NK cell diversity is in part created by the differential expression of cell surface receptors which modulate activation and function, including multiple subfamilies of C-type lectin receptors encoded within the NK complex (NKC). Little is known about the gene content of the NKC beyond rodent and primate lineages, other than it appears to be extremely variable between mammalian groups. We compared the NKC structure between mammalian species using new high-quality draft genome assemblies for cattle and goat; re-annotated sheep, pig, and horse genome assemblies; and the published human, rat, and mouse lemur NKC. The major NKC genes are largely in the equivalent positions in all eight species, with significant independent expansions and deletions between species, allowing us to propose a model for NKC evolution during mammalian radiation. The ruminant species, cattle and goats, have independently evolved a second KLRC locus flanked by KLRA and KLRJ, and a novel KLRH-like gene has acquired an activating tail. This novel gene has duplicated several times within cattle, while other activating receptor genes have been selectively disrupted. Targeted genome enrichment in cattle identified varying levels of allelic polymorphism between the NKC genes concentrated in the predicted extracellular ligand-binding domains. This novel recombination and allelic polymorphism is consistent with NKC evolution under balancing selection, suggesting that this diversity influences individual immune responses and may impact on differential outcomes of pathogen infection and vaccination.

Analysis of a Nonlocal and Nonlinear Fokker--Planck Model for Cell Crawling Migration

Cell movement has essential functions in development, immunity and cancer. Various cell migration patterns have been reported and a general rule has recently emerged, the so-called UCSP (Universal Coupling between cell Speed and cell Persistence), [30]. This rule says that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to migration speed. In [30], the advection of polarity cues by a dynamic actin cytoskeleton undergoing flows at the cellular scale was proposed as a first explanation of this universal coupling. Here, following ideas proposed in [30], we present and study a simple model to describe motility initiation in crawling cells. It consists of a non-linear and non-local Fokker-Planck equation, with a coupling involving the trace value on the boundary. In the one-dimensional case we characterize the following behaviours: solutions are global if the mass is below the critical mass, and they can blow-up in finite time above the critical mass. In addition, we prove a quantitative convergence result using relative entropy techniques.

Autophagy-Related Proteins Target Ubiquitin-Free Mycobacterial Compartment to Promote Killing in Macrophages.

Autophagy is a lysosomal degradative process that plays essential functions in innate immunity, particularly, in the clearance of intracellular bacteria such as Mycobacterium tuberculosis. The molecular mechanisms involved in autophagy activation and targeting of mycobacteria, in innate immune responses of macrophages, are only partially characterized. Autophagy targets pathogenic M. tuberculosis via a cytosolic DNA recognition- and an ubiquitin-dependent pathway. In this report, we show that non-pathogenic M. smegmatis induces a robust autophagic response in THP-1 macrophages with an up regulation of several autophagy-related genes. Autophagy activation relies in part on recognition of mycobacteria by Toll-like receptor 2 (TLR2). Notably, LC3 targeting of M. smegmatis does not rely on membrane damage, ubiquitination, or autophagy receptor recruitment. Lastly, M. smegmatis promotes recruitment of several autophagy proteins, which are required for mycobacterial killing. In conclusion, our study uncovered an alternative autophagic pathway triggered by mycobacteria which involves cell surface recognition but not bacterial ubiquitination.

Abstract A47: Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer

Abstract The median survival rate for women with metastatic breast cancer is 1-2 years, and the 5-year survival rate is only 15 percent. Early detection of metastatic disease is often ineffective, since once the cancer recurs at a distant site, it may be refractory to conventional treatments. A promising strategy for targeting metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. Immunotherapeutic approaches are effective treatments for many types of cancer, such as melanoma. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. Moreover, immunotherapy can cause adverse side effects such as colitis. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to induce a tumor-specific immune response, which can eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) that in turn activate tumor-specific cytotoxic T cells. While not well-understood, ICD may also sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs, such as anthracyclines, used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of mice with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) which is essential for the folding of actin and tubulin and other critical proteins into their native forms. We found CCT to be overexpressed in breast cancer cells susceptible to the peptide. Inhibition of CCT activity by CT20p, indicated by decreased F-actin, disrupted the cytoskeleton, causing loss of cell motility and adhesion that led to breast cancer cell death. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that can generate the danger signals associate with ICD. To this end, we observed that breast cancer cells treated with CT20p displayed alterations in PERK (protein kinase RNA-like ER kinase), one of the key mediators of the unfolded protein response (UPR). As a result, CT20p-treated breast cancer cells were more readily phagocytosed. This also suggests that peptide treatment could potentially have an impact on NK cell cytotoxicity by inducing the expression of activating ligands on cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p treatment due reduced levels and activity of CCT. These studies indicate that CT20p, by targeting CCT, can be used to induce ICD and stimulate essential innate immune functions like NK cell cytotoxic activity and leukocyte phagocytosis that are necessary for promoting a robust anti-cancer adaptive immune response in order to mitigate the development of lethal metastatic disease. Citation Format: Arati Limaye, Rania Bassiouni, Jeremiah Oyer, Robert W. Igarashi, Orielyz Flores, J. Manuel Perez, Alijca Copik, Annette R. Khaled. Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A47.