Regulation of the growth and functions of cloned murine large granular lymphocyte lines by resident macrophages.

Using cloned lines with the morphology of large granular lymphocytes (LGL) from BALB/c mice, we studied the exact requirements for proliferation and their functional characteristics, as well as their regulation. Although these cloned LGL lines were interleukin 2 (IL-2) dependent for growth, experiments using human recombinant IL-2 (rIL-2), known to be active on murine cells, indicated that IL-2 was a necessary but not sufficient factor. Coexistance of normal macrophages in addition to rIL-2 was found to support continuous proliferation of cloned LGL in vitro. This role of macrophages could be replaced by partially purified IL-1 derived from macrophage-conditioned medium. An IL-2 binding assay using 125I-rIL-2 suggested that the role of normal macrophages was to selectively induce and/or maintain high affinity IL-2 receptors (IL-2R) (Kd, 0.2-0.5 nM) without affecting low affinity ones (Kd, 10-30 nM). Functional studies indicated that most of the LGL clones killed various combinations of representative groups of natural killer (NK)-susceptible target cells, including leukemic cells (YAC-1, RL male 1), virus-infected cells (HeLa-measles, HeLa-herpes simplex virus), and normal bone marrow cells (BMC), whereas none of them affected any of NK-resistant target cells, including uninfected HeLa cells. Some of these clones also suppressed in vitro hematopoiesis. Such characteristic cytotoxic spectra, as well as serological phenotypes (Thy-1+, Lyt-1-2-, asialo GM1-positive, T200+, TdT-, Fc receptor-positive) indicated that these LGL clones exactly represent endogenous NK cells, rather than a variety of anomalous killer cells generated in various culture conditions. Although there was significant heterogeneity of cytotoxic spectrum among LGL clones, no clonotypic distribution of specificities was observed. Normal macrophages were found to modulate the functional expression of LGL clones. They augmented the cytotoxic potential of the clones against leukemic and virus-infected targets, but suppressed intrinsic reactivity against normal BMC. Similarly, LGL clones maintained with macrophages showed much less suppressive effect on in vitro hematopoiesis. The present observations on the interaction of cloned LGL and normal macrophages provide a basic explanation for the mechanisms by which the immediate responsiveness to IL-2 of the NK effector system, without exogenous stimulation, and the functional selectivity toward abnormal rather than normal cells, are actively maintained in vivo.

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In Vivo Role of Dendritic Cells in a Murine Model of Pulmonary Cryptococcosis

Infection and Immunity ◽

10.1128/iai.00317-06 ◽

2006 ◽

Vol 74 (7) ◽

pp. 3817-3824 ◽

Cited By ~ 58

Author(s):

Karen L. Wozniak ◽

Jatin M. Vyas ◽

Stuart M. Levitz

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell Activation ◽

Cell Activation ◽

Ex Vivo ◽

Interleukin 2 ◽

Mouse Lung

ABSTRACT Dendritic cells (DC) have been shown to phagocytose and kill Cryptococcus neoformans in vitro and are believed to be important for inducing protective immunity against this organism. Exposure to C. neoformans occurs mainly by inhalation, and in this study we examined the in vivo interactions of C. neoformans with DC in the lung. Fluorescently labeled live C. neoformans and heat-killed C. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeled C. neoformans had been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice with Cryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosing Cryptococcus in vivo and presenting antigen to C. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

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Anti-MY9-blocked-ricin: an immunotoxin for selective targeting of acute myeloid leukemia cells

Blood ◽

10.1182/blood.v77.11.2404.2404 ◽

1991 ◽

Vol 77 (11) ◽

pp. 2404-2412 ◽

Cited By ~ 41

Author(s):

DC Roy ◽

JD Griffin ◽

M Belvin ◽

WA Blattler ◽

JM Lambert ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemic Cells ◽

Short Exposure ◽

Continuous Exposure ◽

Dependent Manner ◽

Acute Myeloid

Abstract The use of immunotoxins (IT) to selectively destroy acute myeloid leukemia (AML) cells in vivo or in vitro is complicated by both the antigenic similarity of AML cells to normal progenitor cells and the difficulty of producing a sufficiently toxic conjugate. The monoclonal antibody (MoAb) anti-MY9 is potentially ideal for selective recognition of AML cells because it reacts with an antigen (CD33) found on clonogenic AML cells from greater than 80% of cases and does not react with normal pluripotent stem cells. In this study, we describe an immunotoxin that is selectively active against CD33+ AML cells: Anti- MY9-blocked-Ricin (Anti-MY9-bR), comprised of anti-MY9 conjugated to a modified whole ricin that has its nonspecific binding eliminated by chemical blockage of the galactose binding domains of the B-chain. A limiting dilution assay was used to measure elimination of HL-60 leukemic cells from a 20-fold excess of normal bone marrow cells. Depletion of CD33+ HL-60 cells was found to be dependent on the concentration of Anti-MY9-bR and on the duration of incubation with IT at 37 degrees C. More than 4 logs of these leukemic cells were specifically depleted following short exposure to high concentrations (10(-8) mol/L) of Anti-MY9-bR. Incubation with much lower concentrations of Anti-MY9-bR (10(-10) mol/L), as compatible with in vivo administration, resulted in 2 logs of depletion of HL-60 cells, but 48 to 72 hours of continuous exposure were required. Anti-MY9-bR was also shown to be toxic to primary AML cells, with depletion of greater than 2 logs of clonogenic cells following incubation with Anti- MY9-bR 10(-8) mol/L at 37 degrees C for 5 hours. Activity of Anti-MY9- bR could be blocked by unconjugated Anti-MY9 but not by galactose. As expected, Anti-MY9-bR was toxic to normal colony-forming unit granulocyte-monocyte (CFU-GM), which expresses CD33, in a concentration- and time-dependent manner, and also to burst-forming unit-erythroid and CFU-granulocyte, erythroid, monocyte, megakaryocyte, although to a lesser extent. When compared with anti-MY9 and complement (C′), Anti- MY9-bR could be used in conditions that provided more effective depletion of AML cells with substantially less depletion of normal CFU- GM. Therefore, Anti-MY9-bR may have clinical utility for in vitro purging of AML cells from autologous marrow when used at high IT concentrations for short incubation periods. Much lower concentrations of Anti-MY9-bR that can be maintained for longer periods may be useful for elimination of AML cells in vivo.

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Macrophage Antitumor Activity Induced by the Antigenic Lymphoma L5178Y/DTIC Subline

Tumori Journal ◽

10.1177/030089168206800502 ◽

1982 ◽

Vol 68 (5) ◽

pp. 365-371 ◽

Cited By ~ 3

Author(s):

Ornella Marelli ◽

Alberto Mantovani ◽

Paola Franco ◽

Angelo Nicotin

Keyword(s):

Antitumor Activity ◽

Tumor Cell ◽

Tumor Cells ◽

Peritoneal Macrophages ◽

Leukemic Cells ◽

Target Cells ◽

Tumor Target ◽

Growth Inhibitory

Murine leukemic cells, after in vivo treatment with antineoplastic drugs, have been shown to express new antigenic specificities that were not detectable on parental cells and that were heritable after the withdrawal of drug treatment. A study was conducted of macrophage antitumor activity triggered by LY/DTIC cells, a subline of LY murine lymphoma, antigenically altered by the drug DTIC. In vitro non-specific inhibition of tumor cell growth was exhibited by spleen and peritoneal macrophages from mice previously challenged with viable LY/DTIC. Peritoneal macrophages from LY/DTIC immune animals showed moderate, although significant lytic activity against unrelated tumor target cells. Supernatants from mixed lymphocyte-tumor cell cultures, in which LY/DTIC immune lymphocytes and LY/DTIC tumor cells had been cultured, rendered normal macrophages non-specifically growth inhibitory for tumor cells.

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Roles of Osteoclasts in Leukemic Cells

Blood ◽

10.1182/blood.v112.11.3184.3184 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3184-3184 ◽

Cited By ~ 1

Author(s):

Asumi Yokota ◽

Shinya Kimura ◽

Ruriko Tanaka ◽

Rina Nagao ◽

Kazuki Sakai ◽

...

Keyword(s):

Cell Cycle ◽

Bone Resorption ◽

Culture System ◽

Bone Matrix ◽

Collagen Gel ◽

Leukemic Cells ◽

Response To Treatment

Abstract We have previously reported that zoledoronic acid (ZOL) augmented the in vivo effect of imatinib in a murine chronic myeloid leukemia (CML) model (Blood 2003). ZOL alone induces apoptosis in leukemic cells in vitro by inhibiting prenylation of the Ras-related proteins. In addition to this direct anti-leukemic effect, we hypothesized that ZOL also has some influence in leukemic cells in vivo indirectly by destroying osteoclasts (OCs), which is the primary therapeutic activity of ZOL in osteoporosis patients. Supporting this notion is that by mediating bone resorption, OCs release a variety of cytokines such as IGF- 1, TGF-β, etc. that have accumulated in the bone matrix. It has been reported that OCs play an important role in bone metastasis of solid tumor, especially in cancer stem cells. However, little is known about the role of OCs in leukemia. Therefore, we investigated it in vitro and in vivo. For this purpose, we established an in vitro osteoblasts (OBs) and OCs co-culture system. The stable co-culture system that we developed includes collagen gel and murine primary OBs and OCs. In addition, murine femoral bone sections were sometimes added to this culture system so that the OCs could release the cytokines from the bone matrix. Thus, the collagen gel and OBs were placed in 12-well plates with and without bone sections and/or OCs. The transwell chambers over the wells then received 1×104 Ba/F3 cells that had been transfected with wild type bcr-abl (Ba/F3/bcr-abl cells). OBs markedly enhanced the growth of Ba/F3/bcr-abl cells in this indirect contact coculture system whereas the presence of both OBs and OCs slightly suppressed cell growth. Intriguingly, when bone sections were added (OBs+OCs+bone), Ba/F3/bcr-abl cell proliferation was significantly suppressed compared to the effect of OBs alone or OBs+OCs (Figure). Cell cycle analysis revealed that the G0/G1 population was increased in Ba/F3/bcr-abl cells co-cultured with OBs+OCs+bones. We also observed that the p27 protein levels of Ba/F3/bcr-abl cells increased upon co-culture with OCs or OCs+bones, similar to their response to treatment with purified TGF-β. We performed ELISAs to determine the concentrations of cytokines in the supernatants of co-cultured OBs and OCs. There were higher levels of TGF-β1 in the OBs+OCs+bones supernatant than in the OBs+OCs supernatant. Furthermore, OBs produced high levels of IGF-1. These findings suggest that OBs and OCs affect the proliferation and the cell cycle arrest of leukemic cells by releasing soluble factors, respectively. To more comprehensively elucidate the roles OCs play in leukemia cells in vivo, we used reveromycin A (RM-A) which inhibits bone resorption by specifically inducing apoptosis in OCs (Woo et al, PNAS 2006). RM-A did not have any in vitro effects on the proliferation of Ba/F3/bcr-abl cells. Thus, we could know the unalloyed role of OCs in leukemia with RM-A compared with ZOL which inhibited directly both OCs and leukemic cells. Our preliminary data show that RM-A suppresses the engraftment of inoculated Ba/F3/bcr-abl cells to nude mice. We also present data from ongoing studies showing the effect of RM-A on leukemic cells in murine models. These findings suggested that OCs may be an important constituent of leukemia stem cell niche and destruction of OCs by either ZOL or RM-A is a novel strategy for leukemia treatment. Figure Figure

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Dendritic cell-derived IL-2 production is regulated by IL-15 in humans and in mice

Blood ◽

10.1182/blood-2004-03-1059 ◽

2005 ◽

Vol 105 (2) ◽

pp. 697-702 ◽

Cited By ~ 65

Author(s):

Sonia Feau ◽

Valeria Facchinetti ◽

Francesca Granucci ◽

Stefania Citterio ◽

David Jarrossay ◽

...

Keyword(s):

Dendritic Cells ◽

T Cell ◽

Molecular Mechanisms ◽

Interleukin 2 ◽

Adaptive Immune ◽

Deficient Mice ◽

Mouse System

Abstract Dendritic cells (DCs) are involved in the initiation and regulation of innate and adaptive immune responses. Several molecular mechanisms regulate these diverse DC functions, and we have previously reported that mouse dendritic cells (mDCs) can produce interleukin-2 (IL-2) in vitro and in vivo, in response to microbial activation and T-cell-mediated stimuli. This property is shared by different DC subtypes, including Langerhans cells. Here we show that, on appropriate stimulation, human DCs, both plasmacytoid and myeloid subtypes, also express IL-2. Interestingly, the production of IL-2 by myeloid DCs is induced by T-cell-mediated stimuli and depends on the presence of IL-15. The key role of this cytokine in regulating IL-2 production was also confirmed in the mouse system. In particular, we could show that DCs from IL-15-deficient mice were strongly impaired in the ability to produce IL-2 after interactions with different microbial stimuli. Our results indicate that DC-produced IL-2 is tightly coregulated with the expression of IL-15.

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Basal Polarity Complex Scribble Is Required for Leukemic Initiation and Propagation through Negative Regulation of Apical Polarity Complex Activator Cdc42 and Hypoxia Inducing Factor-1α

Blood ◽

10.1182/blood-2018-99-118919 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 551-551

Author(s):

Shailaja N Hegde ◽

Mark J Althoff ◽

Ramesh C. Nayak ◽

Ashley M Wellendorf ◽

Fatima Mohmoud ◽

...

Keyword(s):

Median Survival ◽

Leukemic Cells ◽

Mtorc1 Signaling ◽

Apical Polarity ◽

B Lymphoid ◽

Deficient Mice ◽

Expression And Activity

Abstract Despite the introduction of tyrosine kinase inhibitor and CAR-T cell therapies, the prognosis for Ph+ and Ph-like acute lymphoblastic leukemia remains poor. In the present study, we show the role of the Scribble protein in both lymphoid and myeloid leukemogenesis. The polarity protein Scribble is a member of the basal polarity complex, which is down-regulated in many cancers, suggesting a possible tumor suppressor role, especially in so-called cancer initiating cells. Its effect and mechanisms of activity in leukemic cell fate along with its potential activity on leukemic initiating cells have only been recently started to elucidated. Using interferon-responsive inducible (Mx1-Cre) Scribble-deficient mice, we have characterized the role of Scribble in both retroviral transduction, transplantation animal models and binary, inducible stem cell initiated (Scl-tTA/TRE-BCR-ABL) serial propagation models of BCR-ABL induced leukemia. We found that Scribble expression is upregulated at both transcriptional and translational levels in p210- or p190-BCR-ABL induced leukemic progenitors. In vitro, leukemic colony formation was impaired in Scribble deficient leukemic progenitors (~48% reduction; p≤ 0.05, compared to Wt leukemic progenitors) demonstrating that Scribble is important for leukemogenesis. In vivo, the deletion of Scribble abrogates the development of myeloproliferative disease induced by p210-BCR-ABL (median survival: 70 vs 47 days in Scribble deficient and Wt chimeric mice, respectively; p≤0.05); and significantly impairs B-cell lymphoid leukemogenesis induced by p190-BCR-ABL (median survival: 80 vs 60 days for Scribble deficient and Wt chimeric animals, respectively). Mechanistically, BCR-ABL activates the apical polarity regulator Cdc42 in leukemic progenitors and this activation is inhibited by the deficiency of Scribble. The deficiency of Cdc42 does not impair leukemogenesis but the combined deficiency of Cdc42 and Scribble restores the in vivo survival (median survival: 47 days, p≤0.01 compared to Scribble deficient mice) in chimeric p190-BCR-ABL+ leukemic mice to levels similar to wild-type leukemic cells. These data indicate that Scribble-deficient leukemogenesis is dependent on oncogene induced Cdc42 activity in lymphoid progenitors. Furthermore, Scribble deficiency in leukemic progenitors increases the activation of the AMPK/mTORC1 signaling pathway and the protein expression and transcriptional activity of its downstream effector hypoxia-inducing factor-1α (HIF-1α). HIF-1α silencing by constitutive shRNA expression or inducible deletion in Scribble deleted B-lymphoid leukemic cells restored leukemic progenitor clonogenic efficiency (CFU average: 52 vs 110 per 1,000 B220+/EGFP+ BM cells, in Scribble and double Scribble/HIF-1α deficient, respectively; p≤0.01) and B-lymphoid leukemogenesis in vivo (median survival of 62 days; p≤0.05 compared with Scribble deficient chimeric animals). In addition, double deficiency of Scribble and HIF-1α restored AMPK/mTORC1 signaling to Wt leukemic levels. This data indicates that Scribble is a negative regulator of HIF-1α expression and activity, and the restoration of HIF-1α expression and activity to normal leukemic levels is necessary to restore leukemogenesis. Altogether, our data indicates that Scribble is a positive regulator of oncogenesis in leukemic progenitors, in vitro and in vivo, through Cdc42 and HIF-1α activities. Disclosures No relevant conflicts of interest to declare.

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LncRNA LINC00998 inhibits the malignant glioma phenotype via the CBX3-mediated c-Met/Akt/mTOR axis

Cell Death and Disease ◽

10.1038/s41419-020-03247-6 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Haiping Cai ◽

Yanjiao Yu ◽

Xiangrong Ni ◽

Cong Li ◽

Yuanjun Hu ◽

...

Keyword(s):

Suppressive Effect ◽

Akt Inhibitor ◽

Precision Therapy ◽

Glioma Cell Proliferation ◽

Underlying Mechanisms ◽

Proliferation In Vitro ◽

Glioma Progression

AbstractLong noncoding RNAs (lncRNAs), once considered to be nonfunctional relics of evolution, are emerging as essential genes in tumor progression. However, the function and underlying mechanisms of lncRNAs in glioma remain unclear. This study aimed to investigate the role of LINC00998 in glioma progression. Through screening using TCGA database, we found that LINC00998 was downregulated in glioblastoma tissues and that low expression of LINC00998 was associated with poor prognosis. Overexpression of LINC00998 inhibited glioma cell proliferation in vitro and in vivo and blocked the G1/S cell cycle transition, which exerted a tumor-suppressive effect on glioma progression. Mechanistically, RNA pull-down and mass spectrometry results showed an interaction between LINC00998 and CBX3. IP assays demonstrated that LINC00998 could stabilize CBX3 and prevent its ubiquitination degradation. GSEA indicated that LINC00998 could regulate the c-Met/Akt/mTOR signaling pathway, which was further confirmed by a rescue assay using siRNA-mediated knockdown of CBX3 and the Akt inhibitor MK2206. In addition, dual-luciferase assays showed that miR-34c-5p could directly bind to LINC00998 and downregulate its expression. Our results identified LINC00998 as a novel tumor suppressor in glioma, and LINC00998 could be a novel prognostic biomarker, providing a strategy for precision therapy in glioma patients.

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CIAPIN1 is a potential target for apoptosis of multiple myeloma

Materials Express ◽

10.1166/mex.2019.1601 ◽

2019 ◽

Vol 9 (9) ◽

pp. 1106-1111

Author(s):

Xiao-Bo Wang ◽

Le-Ping Yan ◽

Li-Hua Yuan ◽

Bo Lu ◽

Dong-Jun Lin ◽

...

Keyword(s):

Multiple Myeloma ◽

Colony Formation ◽

Bone Marrow Cells ◽

Xenograft Model ◽

Soft Agar ◽

Normal Human ◽

Related Proteins

This study firstly aimed to reveal the gene expression differences of CIAPIN1 between myelomas cells from bone marrow cells of multiple myeloma patients and normal human, and subsequently investigate the regulation role of this gene on tumorigenicity ability of multiple myeloma (MM) cell line U266 via in vitro colony formation and in vivo xenograft studies. RT-PCR results obtained from 18 MM patients and 10 health people showed that the expression of CIAPIN1 gene was 4 times higher in normal human compared to MM patients. Besides, CIAPIN1 siRNA (si-CIAPIN1) transfected U266 cells presented higher proliferation ratio and superior colony forming ability than U266 cells and U266 cells transfected with non-coding siRNA (controls) evaluated by CCK8 test and soft agar colony formation assay, respectively. In a mice MM xenograft model, the si-CIAPIN1 transfected U266 cells induced the biggest tumor compared to the controls. Furthermore, CIAPIN1 overexpressed U266 cells were developed and compared with the si-CIAPIN1 transfected U266 cells to study the role of CIAPIN1 in the production of apoptosis related proteins in U266 cells. Results indicated that CIAPIN1 facilitated apoptosis promoting proteins expression in U266 cells, such as upregulation of BAX, BAK, Bcl-xs and BIM, and downregulation of p38, PKC, Bcl-2 and Bcl-xl proteins. Therefore, CIAPIN1 can be a potential suppression target gene in multiple myeloma.

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In Vitro expanded STAT1−/− Regulatory T Cells Prevent Acute Graft Versus Host Disease (GVHD) and Promote Donor Cell Engraftment In Allogeneic Fully MHC-Mismatched Bone Marrow Transplant

Blood ◽

10.1182/blood.v116.21.732.732 ◽

2010 ◽

Vol 116 (21) ◽

pp. 732-732

Author(s):

Huihui Ma ◽

Caisheng Lu ◽

Judy Ziegler ◽

Suzanne Lentzsch ◽

Markus Y Mapara

Keyword(s):

Bone Marrow ◽

T Cells ◽

Bone Marrow Cells ◽

Donor Cell ◽

Treg Cells ◽

Cell Engraftment ◽

Lethal Irradiation

Abstract Abstract 732 Treg cells have been recognized as critical regulators of the immune response and shown to prevent the development of GVHD. However, little is known about of the role of STAT1 signaling in Treg cells during the development of GVHD. In this study, we tried to investigate how STAT1 signaling controls donor Treg development and function in the setting of GVHD. For this purpose we studied the role of STAT1 in natural and inducible Treg (nTreg and iTreg, respectively). To better understand the influence of STAT1-deficiency on the proliferation of nTreg cells, purified splenic STAT1−/− or STAT1+/+ CD4+CD25+ cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and cultured on anti-CD3 coated plates in the presence of anti-CD28 and IL-2 for 3 days and analyzed for proliferation and viability. After 72h of in vitro culture 50% of the STAT1+/+ starting population were no longer viable compared to only 10% of STAT1−/− cells. Furthermore, we noted a significantly increased expansion of STAT1-deficient CD4+CD25+Foxp3+ Treg cells compared to STAT1+/+ Treg cells (p<0.001). In line with these findings, STAT1-deficiency resulted in a significantly higher proportion of CFSElo cells indicating vigorous proliferation (85% Foxp3+CFSElo in STAT1−/− compared to only 65% Foxp3+CFSElo in STAT1+/+ Treg cells. Furthermore, at the end of the culture 30% of the STAT1+/+ CD4+CD25+ population were Foxp3-negative compared to only 10% of the STAT1−/− cells. We next determined the impact of STAT1 on the generation of iTreg cells in vitro. For this purpose CD4+CD25− cells from STAT1−/− or STAT1+/+ mice were cultured for 3 days on anti-CD3 coated plates in the presence of anti-CD28 antibodies, hTGF-β, mIL-2, anti-IFN-γ and anti-IL-4 for 3 days. Compared to STAT1+/+, we observed significantly enhanced generation of iTregs from STAT1−/− splenocytes (19.9%±3.0% vs. 10.6%±1.3%, p=0.008). We then performed studies to assess the in vivo generation of iTreg. For that purpose BALB/c mice were reconstituted with T Cell Depleted (TCD) 129.STAT1+/+Bone Marrow Cells (BMC) following lethal irradiation and recipients were co-injected with CD4+CD25− cells purified from either 129.STAT1+/+ or 129.STAT1−/− splenocytes. We again noted a significantly higher proportion of CD4+CD25+ Foxp3+ cells in recipients of CD4+CD25−STAT1−/− cells compared to recipients of STAT1+/+ T cells indicating a significantly increased conversion of CD4+CD25- cells into Treg cells. To confirm the in vitro results we tested the functional ability of in vitro expanded (using anti-CD3, anti-CD28, IL-2 and TGF-β) STAT1+/+ or STAT1−/− Treg cells to block induction of GVHD. GVHD was induced in BALB/c mice following lethal irradiation (800rad) and fully MHC-mismatched BMT using 129.STAT1+/+ bone marrow cells plus 129.STAT+/+ conventional T cells (Tcon). Animals were co-injected with expanded Treg cells from either 129.STAT1+/+ or 129.STAT1−/− donors at a ratio of 1:1 or 1:4 (Treg:Tcon). STAT1−/− or STAT1+/+ Treg cells were equipotent in completely preventing GVHD mortality. However, compared to recipients of STAT1+/+ Treg recipients of STAT1−/− Treg showed reduced signs of GVHD morbidity as determined by a significantly improved weight development. Furthermore, recipients of STAT1−/− Treg showed significantly increased donor cell engraftment compared to recipients of STAT1+/+Treg (donor CD4+ [87% vs. 60%, p=0.03], CD8+[99% vs. 96%, p=0.04], Mac1+[96% vs. 77%, p=0.02] and B220+[100% vs. 96%, p=0.007]) cells in the recipient spleen. These observations clearly demonstrate that STAT1 is a critical regulator of Treg cell development and expansion and that targeting STAT1 in CD4+ T cells may facilitate in vitro and in vivo generation/expansion of Treg cells for therapeutic use in GVHD while also promoting donor cell engraftment. Disclosures: Lentzsch: Celgene Corp: Research Funding. Mapara:Resolvyx: Research Funding; Gentium: stocks.

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Hematopoiesis in vitro coexists with natural killer lymphocytes

Blood ◽

10.1182/blood.v74.7.2376.2376 ◽

1989 ◽

Vol 74 (7) ◽

pp. 2376-2382 ◽

Cited By ~ 8

Author(s):

CM Niemeyer ◽

CA Sieff ◽

BR Smith ◽

KA Ault ◽

DG Nathan

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Colony Formation ◽

Progenitor Cell ◽

Marrow Cell ◽

Interleukin 2 ◽

Target Cells ◽

Cell Lineages

Abstract The role of natural killer (NK) lymphocytes in the regulation of human hematopoiesis is controversial. NK-mediated inhibition of colony formation of hematopoietic progenitor cells has been irregularly reported for various cell lineages. In an effort to clarify such disparate findings, we studied the interaction of clearly defined NK and partially purified progenitor cell populations. Cell sorter purified CD16 positive blood NK cells and enriched autologous marrow progenitors were co-incubated at various lymphocyte to marrow cell ratios and then cultured in methylcellulose. There was no inhibition of myeloid, erythroid, or mixed colony formation. Similarly, activation of CD16 positive lymphocytes by interleukin-2 (IL-2) before co-incubation and co-culture did not result in inhibition of colony formation. Furthermore, in a newly designed assay system, we demonstrated that NK cells, which did not modulate colony-formation, remained capable of recognizing and killing rare K562 target cells seeded within the marrow cell population. Our results indicate that unstimulated and IL-2 activated isolated blood NK cells coexist with functioning autologous marrow progenitors in vitro.

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