Abstract P043: Extracellular matrix modulates T cell clearance of malignant cells in vitro

Author(s):  
Claire Robertson ◽  
Aimy Sebastian ◽  
Aubree Hinckley ◽  
Naiomy Rios-Arce ◽  
William Hynes ◽  
...  
2017 ◽  
Vol 3 (1) ◽  
Author(s):  
Jessica MS Jutzy ◽  
Nathan R Wall

The tumor microenvironment is an area of intense interaction between normal and malignant cells. Factors and cell types within this environment can play a crucial role in the progression or regression of the tumor. Of primary interest are tumor-infiltrating T lymphocytes, which have been shown to have a key role in modifying the dynamics of the tumor microenvironment to promote or prevent tumor growth. While there is much in vitro and in vivo evidence for a modification of the tumor infiltrating T cell population toward a pro-tumor environment, what induces these changes within the tumor microenvironment has remained elusive. Our lab previously identified a role for the Inhibitor of Apoptosis protein Survivin as a secreted protein in the extracellular milieu, where it is capable of entering malignant cells and inducing a more aggressive phenotype. We hypothesized that tumorsecreted Survivin could be responsible for modulation of T lymphocytes in the tumor microenvironment. We first isolated tumor released Survivin and confirmed its ability to be taken up by T cells as it is by malignant cells by confocal microscopy, flow cytometry and Western blotting. Subsequently, we evaluated Survivin’s affect on T cell proliferation and found that tumor-released Survivin impairs T cell expansion, but does not alter its activation after exposure to appropriate stimuli. Assessment of phenotypic changes within the cytotoxic and helper T lymphocyte populations showed an increase in anti-inflammatory type 2 T cells and a reduction in type 1 T cells, whichcorrelates to what has been observed in cancer patients. Although often modified in the tumor microenvironment in patients, we did not observe any changes in regulatory T cells or Th17 cells in the presence of Survivin. The results of this study provide evidence of Survivin’s role as an extracellular mediator of the tumor microenvironment, specifically its role in inducing a pro-tumor type 2 T cell population.


Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Maciej Szydlowski ◽  
Michał Pawlak ◽  
Filip Garbicz ◽  
Patryk Górniak ◽  
Justyna Żurańska ◽  
...  

Increased frequency of tumor-associated macrophages (TAMs) predicts shortened survival of patients with classical Hodgkin lymphoma (cHL), suggesting their protumoral role in the disease. In vitro, malignant cells of cHL (Reed-Sternberg cells, RS) produce humoral factors that convert macrophages into TAMs capable of suppressing T-cell proliferation and exhibiting matrix remodeling activity that fosters lymphoma cell dissemination. Despite these features, phenotype and tumor-supportive activities of cHL-TAMs still remain to be elucidated. Herein, using an in vitro model of cHL-TAMs (RS-conditioned macrophages; RS-M), we provide more comprehensive insights into the biology of macrophages in cHL. To confirm the ability of RS cells to polarize macrophages, THP1 cells and donor-derived CD14+ monocytes were first differentiated into M0 macrophages(Mϕ-0) using PMA and CSF-1, respectively. Obtained Mϕ-0 were nest cocultured for five days with L428 or L1236 RS cells under conditions prohibiting direct contacts (transwell system) and the expression of established M1/M2 polarization markers was assessed by flow cytometry. In comparison to control Mϕ-0, RS-M (L428- and L1236-conditioned: L428-M and L1236-M) exhibited increased surface levels of PD-L1 and M2-associated markers: CD163, CD206 and CD209, but failed to induce M1-specific expression of HLA-DR, indicating that RS cells alter macrophage phenotype in a paracrine manner. Next, using phospho-protein kinase arrays, we studied RS cell-triggered intracellular signaling in conditioned macrophages (L1236-M). In comparison to Mϕ-0, L1236-M exhibited increased phosphorylation levels of M2-specific transcription factors (TFs) and signaling intermediates (CREB1, AKT, STAT-3/6), M1/M2 TF cJun, but also exhibited elevated activity of the M1-specific STAT1 TF. To determine whether M1/M2 states exist within RS-M, we profiled transcriptomes of THP1 and CD14+-derived Mϕ-0 and RS-M by RNAseq, and performed Gene Set Enrichment Analysis (GSEA) using known M1/M2-macrophage signatures. Surprisingly, RS-M showed enrichment for both M1- and M2-signatures, indicating that RS-M phenotype is more complex and cannot be unequivocally classified as M1 or M2 state. Consistent with the transcriptional profiles of RS-M, THP1-derived L1236-M exhibited both enhanced glycolysis (typical for M1 macrophages) and oxidative phosphorylation (typical for M2). To look for genes and processes overrepresented in RS-M, we performed Gene Ontology Enrichment analysis, and found that THP1- and CD14+-derived RS-M similarly induced expression of genes involved in chemotaxis/immunomodulation (CCLs: 2, 5, 7, 8, 13, 17, 18 and 24), extracellular matrix organization (TGM2 and MMP-1, -7, -9 and -12), T cell repression (PD-L1) and angiogenesis (VEGFA, PDGFB, TIMP1, CHI3L1 and -2). Consistently, THP-1-derived L1236-M secreted higher levels of CCL-2, -5, -7 and -17, VEGF and CHI3L1 than Mϕ-0, as determined using cytokine arrays. Furthermore, L1236-M produced additional pro-angiogenic factors, Angiogenin and IL8, suggesting that cHL-TAMs support tumor growth by fostering angiogenesis. To verify this hypothesis, we incubated HUVEC endothelial cells in medium supplemented with VEGFA (10ng/ml), or THP1-derived Mϕ-0- or L1236-M-conditioned media and assessed blood vessel formation in a matrigel assay. In comparison to HUVEC cells grown in Mϕ-0-conditioned medium, addition of VEGFA (10ng/ml) increased number of master junctions by 169%, whereas L1236-M-conditioned medium by 187%, indicating that RS cells induce pro-angiogenic function in macrophages. Together, our data indicate that RS cells determine TAM phenotype in a paracrine manner. Proteomic, transcriptional and metabolic profiles of the in vitro-generated cHL-conditioned TAMs indicate that these macrophages cannot be categorized into one of the two extreme M1/M2 polarization states. On the contrary, our data identify unique and disease-specific phenotype of cHL-TAMs, characterized by elevated expression of molecules involved in the recruitment and modulation of immune cell function, T-cell suppression, extracellular matrix remodeling and angiogenesis. Study supported by National Centre of Science Poland grants: 2017/26/D/NZ5/00561, 2016/22/M/NZ5/00668 and 2018/31/N/NZ5/03214 Disclosures Zaucha: Cellgene: Other: travel, accomodations, expenses; Abbvie: Honoraria; Sandoz: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Other: travel, accomodations, expenses; Takeda: Consultancy, Honoraria, Other: travel, accomodations, expenses; BMS: Consultancy; Novartis: Consultancy. Juszczynski:Ryvu Therapeutics: Other: member of advisory board.


1990 ◽  
Vol 171 (1) ◽  
pp. 211-219
Author(s):  
Y Sharabi ◽  
D H Sachs

The effects of in vivo treatment with anti-Qa-2 mAbs on in vivo and in vitro parameters of T cell immunity have been examined. Two anti-Qa-2 mAbs of the same isotype and with similar avidities but directed against distinct epitopes of the same Qa-2 molecules were studied. mAb 1-1-2 was found to induce rapid T cell depletion, with maximal effect observed within 2-3 d, while administration of mAb 1-9-9 caused little or no depletion in the first few days, and reached maximal effect only by day 8. Surprisingly, administration of both antibodies resulted in a depletion pattern similar to that of the nondepleting antibody 1-9-9. Consistent with these effects on T cell depletion, treatment with 1-1-2 caused significant prolongation of survival of allogeneic skin grafts placed 1 d after antibody administration, while treatment with 1-9-9 or with the combination of both antibodies caused no prolongation. In an attempt to determine the mechanism of this phenomenon, we examined Qa-2 expression on the cell surface by flow microfluorometry after treatment with each of the two mAbs. Our data indicate that mAb 1-9-9 mediates significantly greater modulation of Qa-2 expression from the surface of peripheral T cells within 1 d than does mAb 1-1-2. Apparently, therefore, modulation occurs more rapidly than cell clearance, and the efficiency of T cell depletion and consequent immune suppression is correlated inversely with the ability of each mAb to cause modulation. The ability of 1-9-9 to cause Qa-2 modulation suggests that it may react with a determinant on this molecule of physiological relevance to the natural ligand interactions of Qa-2 antigens.


Author(s):  
J. Roemer ◽  
S.R. Simon

We are developing an in vitro interstitial extracellular matrix (ECM) system for study of inflammatory cell migration. Falcon brand Cyclopore membrane inserts of various pore sizes are used as a support substrate for production of ECM by R22 rat aortic smooth muscle cells. Under specific culture conditions these cells produce a highly insoluble matrix consisting of typical interstitial ECM components, i.e.: types I and III collagen, elastin, proteoglycans and fibronectin.


2020 ◽  
Author(s):  
Satsuki Murakami ◽  
Susumu Suzuki ◽  
Ichiro Hanamura ◽  
Kazuhiro Yoshikawa ◽  
Ryuzo Ueda ◽  
...  

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