Primary Dendritic Cell Research Articles

On-chip human lymph node stromal network for evaluating dendritic cell and T-cell trafficking

The lymph node paracortex, also known as the T-cell zone, consists of a network of fibroblastic reticular cells (FRCs) that secrete chemokines to induce T-cell and dendritic cell (DC) trafficking into the paracortex. To model the lymph node paracortex, we utilize multi-channel microfluidic devices to engineer a 3D lymph node stromal network from human cultured FRCs embedded in a collagen I-fibrin hydrogel. In the hydrogel, the FRCs self-assemble into an interconnected network, secrete the extracellular matrix proteins entactin, collagen IV, and fibronectin, as well as express an array of immune cell trafficking chemokines. Although the engineered FRC network did not secrete characteristic CCR7-ligand chemokines (i.e. CCL19 and CCL21), human primary TNF-αmatured monocyte-derived DCs, CD45RA+T-cells, and CD45RA-T-cells migrate toward the lymph node stromal network to a greater extent than toward a blank hydrogel. Furthermore, the FRCs co-recruit DCs and antigen-specific T-cells into the lymph node stromal network. This engineered lymph node stromal network may help evaluate how human DCs and T-cells migrate into the lymph node paracortex via CCR7-independent mechanisms.

ADP-heptose attenuates Helicobacter pylori-induced dendritic cell activation

ABSTRACT Sophisticated immune evasion strategies enable Helicobacter pylori (H. pylori) to colonize the gastric mucosa of approximately half of the world’s population. Persistent infection and the resulting chronic inflammation are a major cause of gastric cancer. To understand the intricate interplay between H. pylori and host immunity, spatial profiling was used to monitor immune cells in H. pylori infected gastric tissue. Dendritic cell (DC) and T cell phenotypes were further investigated in gastric organoid/immune cell co-cultures and mechanistic insights were acquired by proteomics of human DCs. Here, we show that ADP-heptose, a bacterial metabolite originally reported to act as a bona fide PAMP, reduces H. pylori-induced DC maturation and subsequent T cell responses. Mechanistically, we report that H. pylori uptake and subsequent DC activation by an ADP-heptose deficient H. pylori strain depends on TLR2. Moreover, ADP-heptose attenuates full-fledged activation of primary human DCs in the context of H. pylori infection by impairing type I IFN signaling. This study reveals that ADP-heptose mitigates host immunity during H. pylori infection.

Primary follicular dendritic cell sarcoma of the kidney – a case report of a rare tumor with emphasis on diagnostic pitfalls

BackgroundFollicular dendritic cell sarcoma (FDCS) is a rare low-grade tumor of the lymph nodes, but roughly one-third of the cases emerge from extranodal sites, posing diagnostic challenges.Case presentationIn this report, we present the case of a 59-year-old lady who complained of renal colic. During investigation, a kidney tumor was discovered. A radical nephrectomy was performed, and histological examination identified the tumor as a sarcomatoid renal cell carcinoma. The case was then referred to a genitourinary pathologist for further evaluation. The tumor cells exhibited positive staining for CD21, CD23, somatostatin receptor 2 A, and MDM2 expression. Additionally, MDM2 gene amplification was confirmed by the FISH study. Ultimately, the tumor was diagnosed as a primary renal FDCS. The patient was placed under active oncological surveillance and did not receive any further therapy. Remarkably, after 91 months of follow-up, she remains tumor-free.ConclusionThis case represents a well-documented primary renal FDCS. Our aim in presenting this extremely rare tumor is to enhance awareness and highlight the importance of considering FDCS in the differential diagnosis.

Fully closed and automated enrichment of primary blood dendritic cells for cancer immunotherapy.

Dendritic cell (DC) vaccination is a promising approach to induce tumor-specific immune responses in cancer patients. Until recently, most DC vaccines were based on in vitro-differentiated monocyte-derived DCs. However, through development of efficient isolation techniques, the use of primary blood dendritic cell subsets has come within reach. Manufacturing of blood-derived DCs has multiple advances over monocytes-derived DCs, including more standardized isolation and culture protocols and shorter production processes. In peripheral blood, multiple DC subsets can be distinguished based on their phenotype and function. Plasmacytoid DC (pDC) and myeloid/conventional DCs (cDC) are the two main DC populations, moreover cDC can be further subdivided into CD141/BDCA3+ DC (cDC1) and CD1c/BDCA1+ DC (cDC2). In three separate clinical DC vaccination studies in melanoma and prostate cancer patients, we manufactured DC vaccines consisting of pDCs only, cDC2s only, or a combination of pDC and cDC2s, which we called natural DCs (nDC). Here, we describe a fully closed and automated GMP-compliant method to enrich naturally circulating DCs and present the results of enrichment of primary blood DCs from aphaeresis products of 8 healthy donors, 21 castrate-resistant prostate cancer patients, and 112 stage III melanoma patients. Although primary blood DCs are relatively scarce in aphaeresis material, our results show that it is feasible to isolate highly pure pDC, cDC2, or nDC with sufficient yield to manufacture DC vaccines for natural DC-based immunotherapy.

An Organotypic Human Lymph Node Model Reveals the Importance of Fibroblastic Reticular Cells for Dendritic Cell Function.

Human lymph node (HuLN) models have emerged with invaluable potential for immunological research and therapeutic application given their fundamental role in human health and disease. While fibroblastic reticular cells (FRCs) are instrumental to HuLN functioning, their inclusion and recognition of importance for organotypic in vitro lymphoid models remain limited. Here, we established an in vitro three-dimensional (3D) model in a collagen-fibrin hydrogel with primary FRCs and a dendritic cell (DC) cell line(MUTZ-3 DC). To study and characterise the cellular interactions seen in this 3D FRC-DC organotypic model compared to the native HuLN; flow cytometry, immunohistochemistry, immunofluorescence and cytokine/chemokine analysis were performed. FRCs were pivotal for survival, proliferation and localisation of MUTZ-3 DCs. Additionally, we found that CD1a expression was absent on MUTZ-3 DCs that developed in the presence of FRCs during cytokine-induced MUTZ-3 DC differentiation, which was also seen with primary monocyte-derived DCs (moDCs). This phenotype resembled HuLN-resident DCs, which we detected in primary HuLNs, and these CD1a- MUTZ-3 DCs induced T cell proliferation within a mixed leukocyte reaction (MLR), indicating a functional DC status. FRCs expressed podoplanin (PDPN), CD90 (Thy-1), CD146 (MCAM) and Gremlin-1, thereby resembling the DC supporting stromal cell subset identified in HuLNs. This 3D FRC-DC organotypic model highlights the influence and importance of FRCs for DC functioning in a more realistic HuLN microenvironment. As such, this work provides a starting point for the development of an in vitro HuLN.

A case of primary pulmonary follicular dendritic cell sarcoma

Here, we report a 66-year-old woman who presented with an irregular and lobulated pulmonary mass in the apical segment of the right superior lobe with an enlarged mediastinal lymph node. Abdominal computed tomography (CT), brain magnetic resonance imaging, and fluorine-18 fluorodeoxyglucose positron emission tomography/CT scans showed no extrapulmonary distant metastases. The patient underwent surgical sleeve resection of the right superior lobe with systematic hilar and mediastinal lymph node dissection. Post-operative histopathology, including hematoxylin and eosin staining, immunohistochemistry, and Epstein–Barr virus in situ hybridization, suggested primary pulmonary follicular dendritic cell sarcoma (FDCS). A follow-up plain chest CT indicated recurrence with multiple metastases of the liver, ribs, and mediastinal lymph nodes 1 year after surgery. The patient declined further therapy and was lost follow-up. In this case, FDCS located in the lung was unusual and rare. Corresponding imaging and histopathological features have enhanced our understanding of this disease. Meanwhile, it is necessary to include this tumor as a differential diagnosis of lung tumors and pay particular attention to the possibility of post-operative recurrence and metastasis. Moreover, considering the heterogeneity of FDCS, it is essential to identify the biological or imaging features that can reflect or predict the high risk of tumor recurrence and metastasis. A corresponding post-operative examination is also necessary to determine if any lesions remain.

Nanochannel Electro-Injection as a Versatile Platform for Efficient RNA/DNA Programming on Dendritic Cells.

The utilization of dendritic cell (DC) vaccines is a promising approach in cancer immunotherapy, and the modification of DCs for the expression of tumor-associated antigens is critical for successful cancer immunotherapy. A safe and efficient method for delivering DNA/RNA into DCs without inducing maturation is beneficial to achieve successful DC transformation for cell vaccine applications, yet remains challenging. This work presents a nanochannel electro-injection (NEI) system for the safe and efficient delivery of a variety of nucleic acid molecules into DCs. The device is based on track-etched nanochannel membrane as key components, where the nano-sized channels localize the electric field on the cell membrane, enabling lower voltage (<30V) for cell electroporation. The pulse conditions of NEI are examined so that the transfection efficiency (>70%) and biosafety (viability >85%) on delivering fluorescent dyes, plasmid DNA, messenger RNA, and circular RNA (circRNA) into DC2.4 are optimized. Primary mouse bone marrow DC can also be transfected with circRNA with 68.3% efficiency, but without remarkably affecting cellular viability or inducing DC maturation. These results suggest that NEI can be a safe and efficient transfection platform for in vitro transformation of DCs and possesses a promising potential for developing DC vaccines against cancer.

Establishment and Comparison of Two Different Animal Models of Graves' Orbitopathy.

The purpose of this study was to construct an animal model of Graves' ophthalmopathy (GO) by comparing recombinant adenovirus expressing human thyrotropin receptor A subunit (Ad-TSHR A) gene immunization and dendritic cell (DC) immunization. We evaluated the animal models that are closer to the pathology of human GO, and laid the foundation for the study of GO. Ad-TSHR A was injected intramuscularly into female BALB/c mice to induce the GO animal model. A GO animal model was constructed using TSHR combined with IFN-γ-modified primary DC immunized female BALB/c. The animal models constructed by the above two methods were evaluated in terms of ocular appearance, serology, pathology, and imaging to assess the modeling rate of the animal models, respectively. Both modeled mice exhibited increased serological indexes of free thyroxine (FT4) and TSH receptor antibodies (TRAbs) levels and decreased TSH (P < 0.01). Thyroid pathology analysis revealed the number of thyroid follicles increases, the size varies, and the follicular epithelial cells proliferate to varying degrees in a cuboidal or tall columnar pattern, with a small amount of lymphocytic infiltration visible. Adipose tissue behind the eyeball was accumulated, the muscle outside the eyeball was broken and fibrotic, and hyaluronic acid (HA) behind the eyeball was increased. The animal model of GO constructed by immunization of TSHR with IFN-γ-modified DC had a modeling rate of 60%, whereas that of Ad-TSHR A gene immunization was 72%. Both gene immunization and cellular immunization can be used to construct GO models, and the modeling rate of gene immunization is higher than that of cellular immunization. In this study, two innovative methods, cellular immunity and gene immunity, were used to establish GO animal models, which improved the success rate to a certain extent. To our knowledge, this study presents the first cellular immunity modeling idea of TSHR combined with IFN-γ for the GO animal model, which provides an animal model basis for understanding the pathogenesis of GO and developing new treatment methods.

Primary blastic plasmacytoid dendritic cell neoplasm: a US population-based study.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and poorly understood hematopoietic malignancy. This study aimed to investigate the clinical characteristics and prognostic factors in patients with primary BPDCN. Patients diagnosed with primary BPDCN from 2001 to 2019 were extracted from the Surveillance, Epidemiology and End Results (SEER) database. Survival outcome was analysed with Kaplan-Meier method. Prognostic factors were evaluated based on the univariate and multivariate accelerated failure time (AFT) regression analysis. A total of 340 primary BPDCN patients were included in this study. The average age was 53.7 ± 19.4 years, with 71.5% being male. The mostly affected sites were lymph nodes (31.8%). Most patients (82.1%) received chemotherapy, while 14.7% received radiation therapy. For all the patients, the 1-year, 3-year, 5-year, and 10-year overall survival (OS) were 68.7%, 49.8%, 43.9%, and 39.2%, respectively, and the corresponding disease-specific survival (DSS) were 73.6%, 56.0%, 50.2%, and 48.1%, respectively. Univariate AFT analysis showed that older age, marital status of divorced, widowed and separated at diagnosis, primary BPDCN only, treatment delay for 3-6 months and without radiation therapy were significantly associated with poor prognosis of primary BPDCN patients. But multivariate AFT analysis indicated that older age was independently associated with worse survival, while second primary malignancies (SPMs) and radiation therapy were independently associated with extended survival. Primary BPDCN is a rare disease with poor prognosis. Advanced age was linked independently to poorer survival, while SPMs and radiation therapy were linked independently to prolonged survival.

Immunomodulatory Effects by Photodynamic Treatment of Glioblastoma Cells In Vitro.

Multimodal treatment adding immunotherapy and photodynamic treatment (PDT) to standard therapy might improve the devastating therapeutic outcome of glioblastoma multiforme patients. As a first step, we provide investigations to optimize dendritic cell (DC) vaccination by using PDT and ionizing radiation (IR) to achieve maximal synergistic effects. In vitro experiments were conducted on murine glioblastoma GL261 cells, primary DCs differentiated from bone marrow and T cells, isolated from the spleen. Induction of cell death, reactive oxygen species, and inhibition of proliferation by tetrahydroporphyrin-tetratosylat (THPTS)-PDT and IR were confirmed by WST-1, LDH, ROS, and BrdU assay. Tumor cargo (lysate or cells) for DC load was treated with different combinations of THPTS-PDT, freeze/thaw cycles, and IR and immunogenicity analyzed by induction of T-cell activation. Cellular markers (CD11c, 83, 86, 40, 44, 69, 3, 4, 8, PD-L1) were quantified by flow cytometry. Cytotoxic T-cell response was evaluated by calcein AM assay. Immunogenicity of THPTS-PDT-treated GL261 cells lysate was superior to IR-treated lysate, or treated whole cells proven by increased DC phagocytosis, T-cell adhesion, proliferation, cytolytic activity, and cytokine release. These data strongly support the application of PDT together with IR for optimal immunogenic cell death induction in tumor cell lysate used to pulse DC vaccines.

Primary follicular dendritic cell sarcoma of the pleura: A clinicopathological and immunohistochemical study of two cases

Two cases of primary follicular dendritic cell sarcoma (FDCS) of the pleura are presented. The patients are a woman 76-years-old and a man 64-years-old who presented with nonspecific symptoms including chest pain, dyspnea, and cough. Clinical history did not disclose any pertinent history of previous malignancy. Diagnostic imaging showed the presence of a pleural-based mass in both patients and a thoracotomy with resection of the pleural mass was performed. Both tumors were described as solid, light tan, and with ill-defined borders. Histologically, both tumors showed similar histological features, namely the presence of a spindle cellular proliferation composed of elongated cells with fibrillary cytoplasm, oval nuclei, and conspicuous nucleoli. Mild to moderate cellular atypia was present, while mitotic figures ranged from 3 to 4 per 10 high power fields. Mature lymphocytes and plasma cells were also present dispersed throughout the tumor. Immunohistochemical stains in both cases show positive staining for CD21 and CD35 while focal staining was present for D2-40 and clusterin, while negative for other markers including keratin, desmin, S-100 protein, calretinin, and STAT-6. Clinical follow up shows that both patients have remained alive 12 and 14 months after initial diagnosis. The cases herein described represent an unusual occurrence of FDCS arising in the pleural surface and one that must be kept in mind when dealing with spindle cell tumors of the pleura.

IgG-Complexed Adenoviruses Induce Human Plasmacytoid Dendritic Cell Activation and Apoptosis.

Following repeat exposure to many human adenoviruses (HAdVs), most adults harbour long-lived B- and T-cell responses. Combined, this response typically protects us for years from re-infection by the same HAdV type. In spite of these immune responses, some HAdV types are associated with persistent infections that constitute a life-threatening risk when an individual’s T-cell response is compromised. By contrast, patients with B-cell deficiencies do not appear to be at a greater risk of HAdV disease. This dichotomy begs the question of the secondary role of anti-HAdV antibodies during host defence. In this study, we explored IgG-complexed (IC)-HAdV5 and primary human plasmacytoid dendritic cell (pDC) interactions. We found that IC-HAdV5 are efficiently internalized in pDCs, stimulate their activation through TLR9 signalling, and cause apoptosis. These data may help reconcile the enigma of robust immune response to HAdVs, while concurrently allowing persistence.

Primary Follicular Dendritic Cell Sarcoma of Urinary Bladder.

Follicular dendritic cell sarcoma is a rare malignant mesenchymal neoplasm composed of follicular dendritic cells. A subset of cases can be found at extranodal sites. Only 2 follicular dendritic cell sarcomas were previously reported in the urinary bladder. We report a follicular dendritic cell sarcoma that involved the urinary bladder of an 81-year-old woman, who presented with painless gross hematuria. The neoplasm was composed of large epithelioid cells with atypical nuclei, admixed with inflammatory cells. On immunohistochemistry, the neoplastic cells were reactive only for CD21, CD35, and vimentin. The tumor was considered unresectable due to its large size and proximity to the iliac vessels and the patient was treated with bortezomib, cyclophosphamide, and dexamethasone. Although a full course of chemotherapy was not completed due to adverse side effects, the tumor was reduced in size. A subsequent involved-field radiotherapy was performed. Three years postdiagnosis and 2 years after the radiotherapy treatment, a recurrent tumor was identified on imaging in the left posterolateral bladder wall. However, at that time the patient was found to have a newly developed advanced colonic adenocarcinoma. Three and a half months later she developed a bowel perforation and died.

The Migration of Human Follicular Dendritic Cell-Like Cell Is Facilitated by Matrix Metalloproteinase 3 Expression That Is Mediated through TNFα-ERK1/2-AP1 Signaling.

Follicular dendritic cells are important stromal components of the germinal center (GC) and have pivotal roles in maintaining the GC microenvironment for high-affinity antibody production. Tumor necrosis factor-α (TNFα) is essential for the development and functions of follicular dendritic cells. Despite the importance of follicular dendritic cells in humoral immunity, their molecular control mechanisms have yet to be fully elucidated due to the lack of an adequate investigation system. Here, we have used a unique human primary follicular dendritic cell-like cell (FDCLC) to demonstrate that the migration of these cells is enhanced by TNFα-mediated metalloproteinase 3 (MMP3) expression. MMP3 was found to be highly expressed in normal human GCs and markedly upregulated in human primary FDCLCs by TNFα. TNFα induced ERK1/2 phosphorylation and the transcription of MMP3 through AP1. TNFα treatment increased FDCLC migration, and a knockdown of MMP3 significantly reduced the TNFα-induced migration of FDCLCs. Overall, we have newly identified a control mechanism for the expression of MMP3 in FDCLCs that modulates their migration and may indicate an important role in GC biology. Since GCs are observed in the lesions of autoimmune diseases and lymphomas, targeting the MMP3/TNFα-mediated migration of stromal cells in the B cell follicle may have great potential as a future therapeutic modality against aberrant GC-associated disorders.

Primary cutaneous blastic plasmacytoid dendritic cell neoplasm in a child: A challenging diagnosis and management

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and aggressive malignancy of the skin and hematopoietic system. There are few pediatric cases reported in the literature. Management of primary cutaneous BPDCN is challenging because, despite an apparently indolent clinical presentation, rapid dissemination with high mortality can occur. We describe a child with isolated cutaneous involvement who had a good response to chemotherapy as first-line treatmentof BPDCN.

Identification of a phagosomal F-actin structure that evades dendritic cell immunity to Cryptococcus gattii

Abstract Hypervirulent Cryptococcus gattii is a major cause of life-threatening cryptococcosis in immunocompetent individuals and responsible for the ongoing outbreak in the Pacific Northwest. This deadly fungus is known to subvert dendritic cell (DC) maturation and concomitant T cell immunity via immune evasion mechanisms that are poorly understood. Here, we show that primary human DC can phagocytose endemic yeasts but trafficking to the late phagolysosome is blocked by retention of a filamentous actin (F-actin) cage surrounding the phagosomes. Structural studies by super resolution microscopy revealed a novel highly branched F-actin cage that physically interfered with lysosomal fusion. Mechanistically, we demonstrate that C. gattii F-actin cage promotes immune evasion by silencing the canonical RelA signaling of the NF-κB pathway required for DC costimulation and T cell activation. Disruption of the F-actin cage through targeted inhibition or by TNF-α signaling reprogrammed quiescent DC to immunocompetent antigen-presenting cells (APCs) and revealed the existence of a negative feedback loop between periphagosomal F-actin aggregation and pro-inflammatory NF-κB signaling. Collectively, our results have uncovered a unique mechanism of DC immune subversion by organisms such as hypervirulent C. gattii and revealed the potential clinical significance of the immunomodulatory function of phagosomal F-actin in host-pathogen interaction.

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

Efficacy Proof of Concept for Allogeneic CD123 Targeting CAR T-Cells Against Primary Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN): Efficient Control of Tumor Progression in PDX Model and Potential Loss of CD123 Expression in Relapsed Disease

Anti-inflammatory modulation of human myeloid-derived dendritic cell subsets by lenalidomide

Although immunomodulatory drugs (IMiDs) were originally developed as anti-inflammatory drugs, they are effective for multiple myeloma. In order to gain further insights into the immunomodulatory mechanisms of IMiDs for the treatment of inflammatory disorders and myeloma, we investigated the influence of a representative IMiD, lenalidomide, on human primary dendritic cell (DC) subsets: myeloid-derived CD1c+ DCs, CD141+ DCs, and plasmacytoid DCs. Lenalidomide did not affect the viability or expression of costimulatory molecules, but it potently suppressed the production of the key inflammatory cytokines IL-12 and IL-23, and enhanced the production of the anti-inflammatory cytokine IL-10 by CD1c+ DCs. Lenalidomide also suppressed the production of IFN-α by CD141+ DCs but not that by plasmacytoid DCs. Lenalidomide likely targets pathways downstream of the nuclear translocation of the transcription factors nuclear factor κB (NF-κB) and IFN regulatory 5 (IRF5) in CD1c+ DCs. Consistent with the direct immunomodulatory effects on DCs, lenalidomide decreased the capacity of CD1c+ DCs to induce differentiation of naïve CD4+ T cells into effector cells producing immune activating and myeloma-promoting cytokines. This study demonstrated that lenalidomide has anti-inflammatory effects via the modulation of cytokine production by human myeloid-derived DCs. Such effects on DCs may allow for beneficial immunomodulation aiding in the treatment of inflammatory disorders and multiple myeloma.

Dendritic cell-based immunization induces Coccidioides Ag2/PRA-specific immune response

Valley Fever, or coccidioidomycosis, is caused by a soil-borne, highly virulent fungal pathogen, Coccidioides spp. Infection with Coccidioides can be life-threatening. Since an effective treatment is not available and the T cell-mediated immune response is protective, vaccine development is of interest. In this study, a primary dendritic cell (DC)-vaccine was evaluated for its ability to stimulate Coccidioides antigen-specific immune response in an extremely susceptible BALB/c mouse model. The DC-vaccine (Ag2-DC) was prepared by non-virally transfecting the primary bone marrow-derived DCs with a plasmid DNA encoding Ag2/PRA (protective epitope of Coccidioides). Mice were intranasally immunized with Ag2-DC on days 2 and 10. Immunized mice were necropsied on days 8, 32, and 44. Major organs and blood samples were harvested. The most common indicators of injury (protein, lactate, and albumin), Ag/PRA-specific cytokine-secreting cells, and IgG and its isotypes were determined by biochemical and immunologic assays, respectively. No signs of sickness were noted. Similarly, no significant changes were observed in the levels of total lung protein, lactate, and albumin, in immunized mice compared with healthy control mice. Interferon (IFN-γ), and interleukin (IL)-4 and IL-17 cytokine-secreting cells were observed in lung and lymph nodes upon Ag2-DC immunization. Our results showed that the levels of serum IgG and its isotypes were increased in Ag2-DC-immunized mice. This report provides evidence of DC immunization-stimulated Ag2/PRA-specific immune responses.

Abstract A104: Matrix metalloproteinase-2 and Toll-Like receptors modulating immune responses in the tumor microenvironment

Abstract Successful antitumor immunotherapies improve survival in many malignancies but many patients still do not respond or relapse after treatment. This may result from an immunosuppressive tumor microenvironment (TME), composed of many of cells and extracellular matrix proteins such as matrix metalloproteinases (MMPs) that contribute towards tumor growth and metastasis while dampening anti-tumor immunity. We have previously shown that MMP2 is produced in the melanoma TME and modulates human dendritic cell (DC) function to skew T-cells towards a deleterious TH2 phenotype. Briefly, MMP-2 inhibits IL-12 and triggers Toll-like receptor-2 (TLR2) stimulation, leading to NFκB activation to promote OX40L expression and inflammatory cytokine production. We aimed to investigate the mechanisms underlying MMP2-TLR2 interaction in the TME and how they promote immune suppression and melanoma tumor growth. Like human cells, mouse primary and immortalized bone marrow (BM) macrophages and DCs respond to MMP2 and secrete proinflammatory cytokines like TNFα and IL-6. In mice, however, MMP-2 signaling depends on TLR2 and TLR4 with TLR2-/-/TLR4-/- and single KO cells demonstrating compromised TNFα signaling. This response requires MyD88. Moreover, mouse primary splenic and lung DCs respond to MMP-2, and the lack of TLR2/4 abrogates the response, indicating TLR-MMP2 signaling is also systemically significant. Accordingly, in vivo MMP2 injections induced cytokine secretion in the serum of WT but not TLR2-/-, TLR4-/- or dKO mice. To study MMP2 in the TME, we used B16 melanoma tumors. We characterized B16 mRNA and protein expression profiles in cellular compartments and compared with that of whole tumors. We detected MMP2 expression in cultured cells and whole tumors. To identify the source of MMP2 in the TME, we sorted tumor cells and stromal cells and confirmed that both express MMP2 mRNA. Regarding TLR signaling, tumor cells lacked TLR2 and CD14 expression and thus, did not signal via TLR2/TLR4. Following subcutaneous injections of B16 tumors in mice, we observed that TLR2-/-/TLR4-/- mice had significant slower tumor growth kinetics and smaller tumors but similar cellular infiltration in the tumor when compared to WT mice. Next, we modulated TLR and MMP2 signaling in the TME. First we assessed the contributions of the hematopoietic or stromal compartment TLRs into MMP2 signaling using BM chimeras. Our results revealed differences in tumor development due to lack of TLR signaling in hematopoietic cells, as WT mice that received TLR2-/-/TLR4-/- BM have significant slower tumor kinetics and smaller tumors than WT BM recipients. Then, in order to modulate the levels of MMP2 in the TME, we knocked out and overexpressed MMP2 in tumor cells and compared tumor growth in WT mice. Our pilot experiments suggest that the lack of MMP2 in tumor cells significantly delays tumor development and size in WT mice when compared with MMP2-sufficient tumor cells. Co-immunoprecipitation studies confirmed that MMP2 binds with both TLR2 and TLR4. Currently, we are working on pinpointing the exact MMP-2 domain responsible for binding TLRs. Preliminary results thus far indicate the SP-Pro domains of MMP2 is responsible for binding to TLR2 and TLR4 as MMP2 that lacks these domains does not bind TLRs and SP-Pro domains alone are enough for binding. Our data suggests that MMP2, expressed in the TME, serves as a strong promoter of tumor growth and metastasis through stimulation of TLR2 and TLR4 on hematopoietic cells in vivo. Previous work targeting TLRs and endogenous alarmins has showed benefits for tumorigenesis control. Therefore, a deeper knowledge of how MMP-2 interacts with its signaling receptors will provide opportunities to specifically target MMP-2 TLR axis for controlling immune suppression in the TME. Citation Format: Luciana Rebiero Muniz-Bongers, Mansi Saxena, Nina Bhardwaj. Matrix metalloproteinase-2 and Toll-Like receptors modulating immune responses in the tumor microenvironment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A104.