Distinct Role of Antigen-Specific Tc1 and Tc17 Cells in Tumor Eradication In Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1910-1910
Author(s):  
Yu Yu ◽  
Hyun Il Cho ◽  
Dapeng Wang ◽  
Kane Kaosaard ◽  
Claudio Anasetti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Tumor Immunity ◽  
Conflicts Of Interest ◽  
B16 Melanoma ◽  
Host Cells ◽  
Tumor Bearing ◽  
Tc17 Cells

Abstract Abstract 1910 Background: Adoptive cell transfer (ACT) of tumor-reactive T cells is one of the most promising approaches for the treatment of established melanoma. Recently, limited studies provide some evidence that Th/Tc17 cells may also have potent anti-tumor activities, but the conclusion is far from reach. Methods: Human gp10025-specific Tc1 or Tc17 cells were generated from pmel-1 transgeneic mice and used as cell source for ACT. Luciferase-transduced B16 melanoma was intravenously injected into C57BL/6 mice to establish lung-metastasis. After 7 days, tumor-bearing mice were lethally irradiated and transferred gp-10025 specific Tc1 or Tc17 cells in the combination of syngeneic bone marrow. Survival of those tumor-bearing mice was monitored daily, and tumor growth was monitored weekly using in vivo bioluminescent imaging (BLI). Donor T-cell expansion and cytokine secretion from the spleen and lung of tumor bearing mice were analyzed using flow cytometry and ELISPOT assays. To evaluate the role of IFNγ in anti-tumor immunity, we used a B16 melanoma cell line that was transduced with a plasmid encoding a dominant-negative IFNγ receptor (B16-IFNγRDN), and IFNγR knockout mice as tumor-bearers. Results: As expected, irradiation and transfer of syngeneic bone marrow had little or no effect on established melanoma. Adoptive transfer of tumor-specific Tc17 cells significantly suppressed the tumor growth, whereas Tc1 cells induced long-term regression of established melanoma. After ACT, Tc1 cells maintained their phenotype to produce IFNγ. However, Tc17 cells largely preserved their ability to produce IL-17, but a subset of them secreted IFNγ, indicating the plasticity of Tc17 cells in vivo. Mechanistically, Tc1 cells executed their anti-tumor immunity primarily through the direct effect of IFNγ on melanoma cells because Tc1 cells had essentially no effect on B16-IFNγRDN tumor. However, Tc1 cells had a similar therapeutic effect on IFNγR knockout as wild type mice, indicating that IFNγ signaling in host cells was not critical. In contrast, despite the fact that Tc17 cells also secreted IFNγ, Tc17-mediated anti-tumor immunity was independent of the effect through IFNγ. Ironically, IFNγ was inhibitory to Tc17-mediated anti-tumor activity. Conclusions: Taken together, these studies demonstrate that both Tc1 and Tc17 cells can mediate effective anti-tumor immunity, but Tc1 is superior to Tc17. These findings also demonstrated for the distinct effect mechanisms of antigen-specific Tc1 and Tc17 cells in anti-tumor response, and direct IFNγ signaling on tumor cells is a key effect to eradicate established tumors mediated by Tc1 cells. Disclosures: No relevant conflicts of interest to declare.

IL-12 Enhances Tumor Immunity Via Differential Effects On Regulatory T Cells and Cytotoxic Lymphocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1656-1656
Author(s):  
Xuefang Cao ◽  
Karen Leonard
Keyword(s):  
Tumor Growth ◽  
Tumor Immunity ◽  
Conflicts Of Interest ◽  
Treg Cells ◽  
Cell Contact ◽  
Cytotoxic Lymphocytes ◽  
Differential Effects ◽  
Killing Activity

Abstract Abstract 1656 Poster Board I-682 To study the roles of IL-12 and Interferon-gamma (IFNg) in tumor immunity, we used RMAS lymphoma cells to challenge IL-12 receptor beta 2-deficient (IL-12Rb2-/-) and IFNg receptor 1-deficient (IFNgR1-/-) mice that are in the syngeneic C57BL/6J background. We intravenously injected mice with a dose of 1 × 104 RMAS cells that caused death in about 50% of wild-type (WT) mice. As shown in the Figure below, all of the WT mice treated with exogenous IL-12 were rescued from death caused by tumor growth; endogenous IL-12 was not sufficient to impact tumor growth since IL-12Rb2-/- mice showed a survival rate similar to that of WT mice. However, all of the IFNgR1-/- mice succumbed to tumor growth, indicating that endogenous IFNg is required for tumor immunity in this system. Furthermore, IL-12 treatment did not improve the survival of the IFNgR1-/- mice, suggesting that IFNg signaling is required for IL-12's anti-tumor effect. We previously showed that an IL-12/IFNg axis can inhibit tumor-induced regulatory T cell (Treg) proliferation in vitro (Cao et al, 2008 ASH Annual Meeting). We have subsequently examined their effects on Treg cells in vivo. Compared to naive mice, significant Treg expansion (4.9 ± 2.1 fold, n=5, p=0.025) was observed in the peritoneal cavity of WT mice within 2 weeks after an intraperitoneal injection of 1 × 104 RMAS cells. This expansion was completely blocked by treatment with exogenous IL-12. Treg cells in the IL-12Rb2-/- mice expanded to levels comparable to that in WT animals, suggesting that endogenous IL-12 was not sufficient to control Treg expansion. In contrast, significantly higher Treg expansion was observed in IFNgR1-/- mice (36.8 ± 11.8 fold, n=5, p=0.002), which was partially inhibited by IL-12 treatment (13.2 ± 3.5 fold, n=5, p=0.002), suggesting that an IFNg-independent mechanism may also account for IL-12's anti-Treg effect. To further study the effects of IL-12 and IFNg on cytotoxic T lymphocyte (CTL) function, we performed mixed lymphocyte reactions (MLR) and used flow-based killing assays (FloKA) to measure cell contact-dependent killing of allogeneic P815 tumor cells. MLR-activated CTLs were found to kill tumor targets via perforin/granzyme-mediated cytotoxicity. At a 10:1 (effector:target) ratio, granzyme AxB-deficient CTLs and perforin-deficient CTLs displayed significantly reduced killing (8.6 ± 1.2% and 4.5 ± 0.9%, respectively) compared to WT CTLs (36.1 ± 3.5%). IL-12 supplement (2ng/ml) to the MLR significantly increased the killing activity of WT CTLs (65.3 ± 4.2%), but had no significant effect on granzyme AxB-deficient CTLs or perforin-deficient CTLs. In contrast, IFNg supplement (10ng/ml) to the MLR had no significant effect on the killing activity of CTLs. Conversely, MLR-activated IFNgR1-/- CTLs killed P815 cells as efficiently as WT CTLs and responded to IL-12 treatment as efficiently as WT CTLs. Taken together, these data suggest that IL-12 treatment inhibits tumor-induced Treg expansion and stimulates IFNg-dependent anti-tumor immune responses. In addition, IL-12 also activates perforin/granzyme-dependent function of cytotoxic T lymphocytes. These differential effects on diverse immune components may collectively result in enhanced tumor immunity. Disclosures No relevant conflicts of interest to declare.

CD44v6 Is Required For In Vivo Tumorigenesis Of Human AML and MM Cells: Role Of Microenvironmental Signals and Therapeutic Implications

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 605-605 ◽  
Author(s):  
Benedetta Nicolis di Robilant ◽  
Monica Casucci ◽  
Laura Falcone ◽  
Barbara Camisa ◽  
Bernhard Gentner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Cells ◽  
Conflicts Of Interest ◽  
Human Leukemia ◽  
Vegf Receptor ◽  
Durable Response ◽  
Wide Range ◽  
Cd44v6 Expression

Abstract Background Targeting the interactions between tumor cells and their microenvironment is an exciting new frontier in cancer therapy. The biology of acute myeloid leukemia (AML) and multiple myeloma (MM) is characterized by addiction to specific signals uniquely provided within the bone marrow (BM), where tumor cells preferentially home and locally thrive. The hyaluronate receptor CD44 was shown to be required for retroviral-induced leukemogeneis in syngeneic mouse models. Conversely, CD44 mAbs interfere with human leukemia initiation in immunocompromised mice by inhibiting leukemia stem cell homing to the bone marrow (BM). The therapeutic potential of CD44 mAbs is also under clinical investigation in humans. Much less is known on the role of the differently spliced CD44 variant isoforms. The expression of exon 6 (CD44v6) conveys additional properties to standard CD44, like binding to osteopontin and cooperation with different tyrosine kinase receptors (RTKs), like VEGF receptor type II and c-Met. Interestingly, CD44v6 is the most abundantly expressed CD44 isoform in both AML and MM, where it correlates with a bad prognosis. Since CD44v6 expression is much more tumor-restricted than CD44, targeting this isoform may have a better efficacy/toxicity profile than targeting the standard molecule. Aim To preclinically validate CD44v6 as a therapeutic target in AML and MM Results By FACS analysis and RT-qPCR, we established CD44v6 over expression in a relevant fraction of leukemic blasts from AML pts (15/25, 60%) with preference for the M4-5 FAB subtypes, and in the majority of malignant plasmacells from MM pts (13/15, 87%). CD44v6 was also over expressed on THP-1, Kasumi and U937 human AML cells, and on MM.1S, XG-6 and XG-7 MM cells. To address the specific role of CD44v6 in BM homing, we pretreated MM1.S cells with either a CD44 mAb (SFF-2) or a CD44v6 mAb (VFF-18) and infused them i.v. in NSG mice. Unexpectedly, while SFF-2 almost completely inhibited early (18hrs) homing to the BM compared with an irrelevant mAb, VFF-18 had no effect. To rule out confounding variables associated with specific mAb clones, we silenced CD44v6 expression in MM1.S cells by lentiviral-mediated shRNA transduction and confirmed no difference in BM homing compared with control LV-transduced cells. Longer follow-ups (4-6 weeks) however revealed that, despite unaltered rates of in vitro proliferation, CD44v6-silenced MM1.S cells were severely hampered in their tumorigenic capacity in vivo (P<0.001). These results were confirmed by using THP-1 cells (P<0.001) and primary leukemic blasts (P<0.01). Hypothesizing that CD44v6 may be crucial for in vivo tumorigenesis by cooperating with RTKs, we set-up a co-culture system with BM-derived mesenchymal stromal cells (MSCs), which are producers of VEGF and the c-Met ligand HGF. MSCs protected a wide range of tumor cells, including primary leukemic blasts, from spontaneous apoptosis (P<0.05) and from apoptosis induced by Ara-C or daunorubicin (P<0.01), or bortezomib in the case of MM cells (P<0.001). Comparable results were obtained by using MSC supernatants, hinting to a causative soluble factor, which was neither VEGF nor HGF, as demonstrated by inhibition experiments with bevacizumab and crizotinib, respectively. Noteworthy, MSCs or their supernatants prompted a significant up-regulation of CD44v6 expression levels (P<0.01). Most importantly, preventing CD44v6 up-regulation on tumor cells by shRNA silencing restored their sensitivity to spontaneous and drug-induced apoptosis (P<0.01). Conclusions These results clearly indicate that CD44v6 is dispensable for BM homing, but responsible for AML and MM addiction to microenvironmental signals. Combining CD44v6 targeting with cytotoxic chemotherapy might interfere with this vicious circle and result in higher and/or more durable response rates. Disclosures: No relevant conflicts of interest to declare.

Magnesium protects against cisplatin-induced acute kidney injury without compromising cisplatin-mediated killing of an ovarian tumor xenograft in mice

2015 ◽  
Vol 309 (1) ◽  
pp. F35-F47 ◽  
Author(s):  
Malvika H. Solanki ◽  
Prodyot K. Chatterjee ◽  
Xiangying Xue ◽  
Madhu Gupta ◽  
Ivy Rosales ◽  
...  
Keyword(s):  
Acute Kidney Injury ◽  
Tumor Growth ◽  
Mrna Expression ◽  
Ovarian Tumor ◽  
Magnesium Deficiency ◽  
Kidney Injury ◽  
Tumor Bearing ◽  
Magnesium Status

Cisplatin, a commonly used chemotherapeutic for ovarian and other cancers, leads to hypomagnesemia in most patients and causes acute kidney injury (AKI) in 25–30% of patients. Previously, we showed that magnesium deficiency worsens cisplatin-induced AKI and magnesium replacement during cisplatin treatment protects against cisplatin-mediated AKI in non-tumor-bearing mice (Solanki MH, Chatterjee PK, Gupta M, Xue X, Plagov A, Metz MH, Mintz R, Singhal PC, Metz CN. Am J Physiol Renal Physiol 307: F369–F384, 2014). This study investigates the role of magnesium in cisplatin-induced AKI using a human ovarian tumor (A2780) xenograft model in mice and the effect of magnesium status on tumor growth and the chemotherapeutic efficacy of cisplatin in vivo. Tumor progression was unaffected by magnesium status in saline-treated mice. Cisplatin treatment reduced tumor growth in all mice, irrespective of magnesium status. In fact, cisplatin-treated magnesium-supplemented mice had reduced tumor growth after 3 wk compared with cisplatin-treated controls. While magnesium status did not interfere with tumor killing by cisplatin, it significantly affected renal function following cisplatin. Cisplatin-induced AKI was enhanced by magnesium deficiency, as evidenced by increased blood urea nitrogen, creatinine, and other markers of renal damage. This was accompanied by reduced renal mRNA expression of the cisplatin efflux transporter Abcc6. These effects were significantly reversed by magnesium replacement. On the contrary, magnesium status did not affect the mRNA expression of cisplatin uptake or efflux transporters by the tumors in vivo. Finally, magnesium deficiency enhanced platinum accumulation in the kidneys and renal epithelial cells, but not in the A2780 tumor cells. These findings demonstrate the renoprotective role of magnesium during cisplatin AKI, without compromising the chemotherapeutic efficacy of cisplatin in an ovarian tumor-bearing mouse model.

scholarly journals Src-like Adaptor Protein Promotes the CD103+ dendritic Cell Homeostasis in the Lung

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1339-1339
Author(s):  
Namit Sharma ◽  
Pan Zhongda ◽  
Tracy Lauren Smith ◽  
Savar Kaul ◽  
Emilie Ernoult ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Immune Response ◽  
Immune Cell ◽  
Conflicts Of Interest ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Apoptotic Cells ◽  
Direct Role

Abstract Dendritic cells (DCs) along with mast cells function as sentinels for the innate immune system and perform as antigen presenting cells (APCs) to mount an adaptive immune response against invading pathogen. FLT3 receptor tyrosine kinase signaling has been shown to regulate the homeostatic mechanisms of subsets of DCs particularly, CD103+DCs compared to CD11b+DCs. CD103+DCs are regarded as APCs with superior capabilities to mount an effective immune response, thus understanding their homeostasis mechanism(s)/function is of paramount importance to devise effective therapeutics including DC vaccines. The Src-like adapter protein (SLAP) has been shown to dampen the signaling downstream of receptor tyrosine kinases including FLT3, cKit, and immune cell receptors including T cell receptor, B cell receptor, and Granulocyte-monocyte colony stimulating factor receptor via by recruiting c-Cbl, an ubiquitin ligase. Here, we report that SLAP deficient mice (KO) have reduced numbers of CD103+DC in lung while equal numbers in liver and kidney compared to control mice. To further confirm reduced CD103+DC in the lung, efferocytosis assays that are dependent upon CD+103 DC in lung epithelium to cleanse the apoptotic cells were performed. Flow cytometric quantification of CD103+DCs that uptake fluorescently labeled apoptotic cells administered via intranasal route and migrate to mediastinal lymph nodes confirmed reduced number of CD103+DCs in SLAP KO mice. Further analysis of DC progenitor populations showed reduced pre-DC progenitor in the lung in SLAP KO mice while bone marrow compartment showed equal progenitor populations including pre-DC and common dendritic progenitors suggesting the role of SLAP in localized FLT3 signaling in the lung. Consistently, DCs in lymphoid compartment including spleen, thymus, inguinal and popliteal lymph node did not show any defects. Upon further dissecting the cellular mechanism, SLAP KO DCs showed increased apoptosis while having similar proliferation potential in vivo at steady state.Bone marrow progenitors from SLAP KO mice failed to generate mature DCs in the presence of FLT3 ligand in vitrodue to enhanced apoptosis at early time points. Also, submaximal inhibition of FLT3 with an inhibitor, quizartinib partially rescues the apoptotic phenotype of SLAP KO bone marrow progenitors suggesting a cell-intrinsic role of SLAP in the survival of DCs. Biochemical analysis revealed that SLAP is directly recruited to the juxta-membrane residues of the FLT3 receptor in an inducible manner suggesting a direct role of SLAP in the regulation of FLT3 signaling. Phosphoflow analysis of DCs generated in the combined presence of GMCSF and FLT3 ligands showed that SLAP promotes the signaling to SHP2 while perturbs signaling to the mTOR pathway. Together these results suggest that SLAP is a critical regulator of CD103+DCs homeostasis in selective peripheral organs including the lung. Disclosures No relevant conflicts of interest to declare.

Study of migration and distribution of bone marrow cells transplanted animals with B16 melanoma

2017 ◽  
pp. 10-21
Author(s):  
А.Ф. Повещенко ◽  
А.О. Соловьева ◽  
К.Э. Зубарева ◽  
Д.Н. Стрункин ◽  
О.Б. Грицык ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Y Chromosome ◽  
Bone Marrow Cells ◽  
Tumor Formation ◽  
B16 Melanoma ◽  
Light Cycler ◽  
Migration Of Cells ◽  
Marrow Cells

Цель - выявление особенностей миграции и распределения сингенных клеток костного мозга (ККМ) и его субпопуляции (МСК) после их трансплантации в органах реципиента-носителя меланомы В16. Методика. В работе использовались мыши самцы и самки линии С57Вl/6. Индукция опухолевого роста: имплантировали клетки меланомы В16 подкожно в заднюю правую лапу самок мышей С57Bl/6 в дозе 2,5 х 10 клеток/мышь. Изучение миграции и распределения in vivo ККМ и МСК осуществляли при помощи генетического маркера - специфической последовательности Y-хромосомы самцов линии С57Bl/6 при сингенной внутривенной трансплантации самкам с использованием полимеразной цепной реакции (ПЦР) в реальном времени на Authorized Termal Cycler - Light Cycler 480 II/96 (Roche). Введение суспензии неразделенных клеток костного мозга, мезенхимальных стволовых клеток от самцов-доноров мышам-реципиентам (сингенным реципиентам самкам С57Вl/6) с последующим выделением органов реципиентов проводилось через определенные временные интервалы, затем из органов реципиентов выделяли ДНК. Результаты. Показано, что клетки костного мозга, позитивные по Y-хромосоме, мигрируют как в лимфоидные (лимфатические узлы, селезенку, костный мозг), так и в нелимфоидные органы (печень, сердце, головной мозг, кожу) сингенных реципиентов. Помимо миграции клеток из костного мозга в другие органы, существует и обратный путь миграции клеток из кровотока в костный мозг. Развитие у интактных мышей линии С57Вl/6 меланомы В16 стимулирует процессы миграции трансплантированных ККМ и МСК в костный мозг. Установлено, что при опухолевом росте усилена миграция трансплантированных клеток костного мозга, в том числе и популяции МСК, в костный мозг. На ранней стадии формирования опухоли миграционная активность МСК в опухоль выше по сравнению с неразделенной фракцией костного мозга. На поздних стадиях формирования опухоли неразделенная популяция клеток костного мозга интенсивнее мигрирует в опухоль по сравнению с популяцией МСК. Заключение. Обсуждается возможность использования МСК костного мозга для таргетной терапии опухолевых заболеваний, так как миграция МСК в опухолевую ткань может быть использована для эффективной доставки противоопухолевых препаратов. Purpose. Reveal features migration and distribution of syngeneic bone marrow cells (BMC) and subpopulations (MSC) after transplantation into the recipient carrier B16 melanoma bodies. Methods. We used mouse male and female C57BL/6 mice. Induction of Tumor Growth: B16 melanoma cells implanted subcutaneously into right hind paw of female C57BL/6 mice at a dose of 2.5 x 105 cells / mouse. migration study in vivo distribution and BMC and MSC was performed using genetic markers - Y-chromosome specific sequence line male C57Bl/6 syngeneic intravenous transplantation in females using the polymerase chain reaction (PCR) in real time on Authorized Termal Cycler - Light Cycler 480 II / 96 (Roche). Introduction suspension of unseparated bone marrow cells, mesenchymal stem cells from donor to recipient male mice (syngeneic recipient female C57BL/6), followed by isolation of recipients of organs was performed at regular intervals, then of organ recipients isolated DNA. Results. It was shown that bone marrow cells positive for Y-chromosome in migrate lymphoid (lymph nodes, spleen, bone marrow) or in non-lymphoid organs (liver, heart, brain, skin) syngeneic recipients. In addition to the migration of cells from the bone marrow to other organs, there is a way back migration of cells from the circulation to the bone marrow. B16 melanoma stimulates the migration of transplanted MSCs and BMC in bone marrow. It is found that tumor growth enhanced migration of transplanted bone marrow cells, including populations of MSCs in the bone marrow. In the early stages of tumor formation MSC migration activity higher than the BMC. In the later stages of tumor formation undivided population of bone marrow cells migrate to the intense swelling compared with a population of MSCs. Conclusion. The possibility of using bone marrow MSCs for targeted therapy of tumor diseases, because migration of MSCs in tumor tissue can be used to effectively deliver anticancer drugs.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Double Haploidentical Hematopoietic Stem Cell Transplantation (HSCT) Enhances Anti-Tumor Activity After Transplant

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1897-1897
Author(s):  
Tulin Budak-Alpdogan ◽  
Cavan P Bailey ◽  
Michelle Panis ◽  
Christopher Sauter ◽  
Vikas Agrawal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Tumor Growth ◽  
Low Dose ◽  
Tumor Model ◽  
Tumor Escape ◽  
Tumor Bearing ◽  
Mhc Haplotype ◽  
Anti Tumor Activity

Abstract Abstract 1897 We have previously shown that haploidentical (HI) HSCT with low dose donor T-cell infusion provides a survival advantage in tumor bearing mice when compared to parent F1 or MHC-matched transplant models. We suggest that MHC difference in HI-HSCT generates early T-cell clonal activation against the unshared MHC haplotype, which eliminates residual tumor cells that express the unshared MHC haplotype. However, alteration in MHC antigen expression is a significant contributor to tumor escape from graft-versus-tumor (GVT) activity. Recent haploidentical transplant data revealed that uniparental disomy, the loss of the HLA haplotype, is a clinically relevant mechanism of tumor escape that leads to post-transplant leukemia relapse. Murine renal cell carcinoma, RENCA-TGL, cell line normally expresses only H2Kd as a MHC molecule. Therefore, in our haploidentical transplant model, T cell clonal activity is usually restricted against H2Kd molecule only. For circumventing the single haplotype expression of tumor model, we transfected this cell line with a H2Kb expressing vector, pAcGFP-NeoR-H2Kb, and generated stable clones with G418 selection. The clone that has more than 95% H2Kb expression used for in vivo experiments. Both tumor cell lines, i.e. parental and transfected clone, had similar in vivo tumor growth acceptance and growth rate. We then used two different haploidentical donors that were targeting different MHC haplotypes. Lethally irradiated B6D2F1 (H2Kb/d) mice were transplanted with T cell depleted bone marrow (TCD-BM) from either B6C3F1 (H-2Kb/k) (single haplo-1; SH1), or C3D2F1 (H2Kk/d) (single haplo 2; SH2) or both donor mice with low-dose (1×105) T-cells. In some experiments, animals were also injected either H2Kd or H2Kb/d expressing RENCA-TGL cells for the evaluation of GVT activity. Bone marrow (BM), spleens and thymi were harvested from recipients of single and double HI-HSCT at day 35 and showed similar cellularities. Interestingly, spleen and bone marrow had similar chimerism from both donors in DH-HSCT. There were no early transplant mortality, graft failure, weight loss and GVHD scoring difference among the double or single-haploidentical transplant recipients. In two other sets of experiments, we followed the tumor growth and the survival of tumor bearing mice after transplant. The recipients of DH-HSCT showed a better survival and GVT activity than the recipients of SH-HSCT in RENCA-TGL (H2Kb/d) bearing tumor model. These observations confirmed that MHC targeting plays a prominent role in tumor surveillance, and immune targeting the unshared MHC haplotype with haploidentical transplant induce remarkable survival advantage. Double HI-HSCT provides an unique anti-tumor activity that continues to exert GVT effect, even in case of MHC haplotype loss. Disclosures: No relevant conflicts of interest to declare.

Heterogeneity in the Making of Blood

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. SCI-27-SCI-27
Author(s):  
David T. Scadden
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Bone Marrow Niche ◽  
Cell Selection ◽  
Hematopoietic Progenitor ◽  
Marrow Space ◽  
And Function ◽  
Osteolineage Cells

It is increasingly clear that the bone marrow is comprised of a heterogeneous complex of niches for hematopoietic cells, some for stem cells in the perivascular space and some for progenitors. We have used two approaches to define the role of specific cells in the marrow. First, single cell selection and characterization based on in vivo proximity to HSPC. This method has defined a subset of endosteal lining cells that can be immunophenotypically defined and isolated and reveals IL-18 as a regulator of hematopoietic progenitor quiescence. Second, candidate cell depletion that revealed mature osteolineage cells expressing osteocalcin as regulating the production of thymic emigrants through the expression of Dll4. Deletion of these cells reduces the number and function of T-biased lymphoid progenitors in the marrow space as well as thymic populations and mature T cells in the blood. These data suggest that specific niche subsets can be defined and through them, novel molecular regulators of HSPC function. The bone marrow niche is a heterogeneous composite of distinctive niches. Disclosures No relevant conflicts of interest to declare.

scholarly journals Platelet-derived thrombospondin-1 is a critical negative regulator and potential biomarker of angiogenesis

Blood ◽  
2010 ◽  
Vol 115 (22) ◽  
pp. 4605-4613 ◽  
Author(s):  
Alexander Zaslavsky ◽  
Kwan-Hyuck Baek ◽  
Ryan C. Lynch ◽  
Sarah Short ◽  
Jenny Grillo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Angiogenesis Inhibitor ◽  
Negative Regulator ◽  
Mouse Tumor ◽  
Tumor Bearing ◽  
Potential Biomarker ◽  
Thrombospondin 1

Abstract The sequential events leading to tumor progression include a switch to the angiogenic phenotype, dependent on a shift in the balance between positive and negative angiogenic regulators produced by tumor and stromal cells. Although the biologic properties of many angiogenesis regulatory proteins have been studied in detail, the mechanisms of their transport and delivery in vivo during pathologic angiogenesis are not well understood. Here, we demonstrate that expression of one of the most potent angiogenesis inhibitors, thrombospondin-1, is up-regulated in the platelets of tumor-bearing mice. We establish that this up-regulation is a consequence of both increased levels of thrombospondin-1 mRNA in megakaryocytes, as well as increased numbers of megakaryocytes in the bone marrow of tumor-bearing mice. Through the use of mouse tumor models and bone marrow transplantations, we show that platelet-derived thrombospondin-1 is a critical negative regulator during the early stages of tumor angiogenesis. Collectively, our data suggest that the production and delivery of the endogenous angiogenesis inhibitor thrombospondin-1 by platelets may be a critical host response to suppress tumor growth through inhibiting tumor angiogenesis. Further, this work implicates the use of thrombospondin-1 levels in platelets as an indicator of tumor growth and regression.

Novel Pleiotropic Effects of Thrombin in Regulation of Metastatic Potential of Human Rhabdomyosarcoma (RMS) Cells – Identification of Negative PAR1 and PAR3 Receptor Crosstalk.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 336-336
Author(s):  
Marcin Wysoczynski ◽  
Rui Liu ◽  
Mariusz Z Ratajczak
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Metastatic Potential ◽  
Cell Interaction ◽  
Lymphocytic Leukemia ◽  
Coagulation Cascade ◽  
Receptor Crosstalk

Abstract Abstract 336 Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of adolescence and childhood that frequently infiltrates bone marrow (BM) to this degrees that it may mimic acute lymphocytic leukemia. We identified chemokines and growth factors (e.g., SDF-1, HGF) that play an important role in RMS metastasis (Blood 2002;100:2597, Cancer Res. 2003;63:7926, Cancer Res. 2007;67:2131). Novel evidence however accumulates that metastatic process for many tumors may be modulated by the components of coagulation cascade (CC) (e.g., thrombin, activated platelets). Thus, we become interested on a role of CC in modulating metastasis of RMS cells. First, we learned that RMS cells express tissue factor (TF) and thus may activate coagulation by generation of thrombin. Thrombin activated in tumor microenvironment activates platelets that release microvesicles. We observed that platelet derived microvesicles (PMV) transfer to RMS cells several platelet integrin receptors (e.g., α2β3) important for RMS cell interaction with endothelium, and thus increase their adhesive potential to endothelial cells. To support this, we noticed that RMS cells covered with PMV showed higher metastatic potential after intravenous injection into immunodeficient SCID mice. We also found that PMV also directly chemoattracted RMS cells and activated MAPKp42/44 and AKT. Next we learned that all 10 human RMS cell lines investigated in our studies express functional PAR1 and PAR3 receptors. To support this, we observed in thrombin stimulated RMS cells phosphorylation of MAPKp42/44 and MAPKp38. To our surprise however, in in vitro experiments thrombin decreased RMS chemotactic response to conditioned media from bone marrow fibroblast and PMVs. Furthermore, we didn't observe any effect of thrombin on proliferation, survival and expression of pro-angiogenic factors in RMS cells. Thrombin also decreased adhesion of RMS cells to fibronectin and bone marrow stroma cells. In contrast PAR1 specific agonist TRAP-6 stimulated proliferation of RMS cells. Different responsiveness to thrombin and TRAP-6 stimulation could be explained by negative modulatory role of PAR3 receptor in response to stimulation by thrombin. Thus, to learn more on a role of PAR1 and PAR3 in RMS proliferation/metastasis we knock-down both receptors by employing shRNA strategy. We observed that PAR1-/- receptor RMS cells that express intact PAR3 cells formed in vivo smaller tumors as compared to unmodified control cells. On the other hand, PAR3-/- RMS cells that express functional PAR1 began to proliferate robust in response to thrombin. In conclusion, we demonstrate that RMS-expressed TF activates prothrombin and that thrombin is a novel, underappreciated, pro-metastatic factor for these cells. Activated in tumor proximity by thrombin, platelets release PMVs that chemoattract and transfer several platelet-derived receptors/adhesion molecules to RMS cells that are crucial for adhesion/interaction with the endothelium. Conversely, by decreasing the responsiveness of RMS cells to local chemoattractants and decreasing adhesiveness of RMS cells, thrombin promotes their release from the primary tumor into circulation. Consequently, RMS cells that are covered by PMVs release into circulation and respond to chemoattractants in distant organs for metastasis. Finally, our data also supports a negative regulatory role of thrombin-PAR3 axis in proliferation of RMS. Disclosures: No relevant conflicts of interest to declare.

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