CD8+/TCR− Facilitating Cells Induce Generation of Regulatory T cells in Vivo: Chimeric Regulatory T Cells are Antigen-Specific and Enhance Engraftment of Allogeneic HSC in Non-Obese Diabetic (NOD) Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2318-2318
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Lala-Rukh Hussain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
Regulatory T Cells ◽  
Nod Mice ◽  
Strong Suppression ◽  
Hematopoietic Stem ◽  
Cell Based Therapy

Abstract CD8+/TCR− graft facilitating cells (FC) are a novel tolerogenic cell population in bone marrow that potently enhance engraftment of hematopoietic stem cells (HSC) in allogeneic and syngeneic recipients. The CD11c+/CD11b−/B220+ plasmacytoid precursor dendritic cell (p-preDC) subpopulation of FC (p-preDC FC) comprises over 60% of FC total and plays a critical and nonredundant role in facilitation. FC prevent graft-versus-host disease and remain tolerogenic after in vivo infusion. Regulatory T cells (Treg) are immunomodulatory cells that maintain tolerance in vivo. They can be generated in vitro via co-culture with p-preDC. There is great interest regarding the use of Treg as a cell-based therapy to induce graft/host tolerance in vivo. However, a major challenge to the clinical use of Treg has been to obtain sufficient numbers of cells for in vivo use and maintain their tolerogenic properties in vivo after in vitro expansion. Here, we evaluated whether FC function by inducing the production of Tregin vivo and examined the function of these chimeric Tregin vivo and in vitro. HSC (c-Kit+Sca-1+Lin−; KSL) were sorted from donor B6 and NOD mice. 10,000 B6 HSC and 1,000 NOD HSC were transplanted by tail-vein injection into recipient NOD mice conditioned with 950 cGy of total body irradiation (TBI). Spleen, thymus, and bone marrow were harvested from recipient NOD mice 5 weeks after transplantation. CD4+CD25+Foxp3+ Treg were analyzed by flow cytometry. FC induced the generation of both donor and recipient CD4+CD25+Foxp3+ Tregin vivo; the majority of Treg were recipient-derived (89% to 97%). To test the function of Treg from HSC + FC chimeras (chimeric Treg), CD8− CD4+CD25+ Treg were sorted from the spleen of chimeras 5 weeks after transplantation. 50,000 chimeric Treg plus 10,000 B6 HSC were transplanted into NOD recipients conditioned with 950 cGy TBI. Recipients of 50,000 Treg from naïve B6 spleens (B6 Treg) + HSC or HSC alone served as controls. Five of 26 recipients of HSC alone engrafted and survived up to 100 days. Only 2 of 5 recipients of HSC plus 50,000 B6 Treg engrafted and none of the recipients exhibited durable engraftment beyond 100 days. In striking contrast, 100% (4 of 4) recipients of HSC + 50,000 chimeric Treg engrafted durably, with survival ≥ 100 days. Chimeric Treg function was confirmed in vitro by MLR suppressor assays, as evidenced by strong suppression of T cell proliferation. Sorted chimeric Treg demonstrated an 87.2% suppression of cell proliferation when plated in a 1:1 ratio with naïve NOD responder cells and B6 stimulator cells. Moreover, when plated at a 1:4 and 1:8 ratio with naïve NOD responders, Treg suppressive function titrated to 62.7% and 43.3%, respectively. In contrast, sorted Treg from naïve B6 animals showed 75.8%, 35.4, and 29.4% suppression when plated in ratios of 1:1, 1:4, and 1:8, respectively. Taken together, these data suggest that FC induce the production of antigen-specific Tregin vivo and chimeric Treg are superior to naïve Treg in suppressing the proliferation of effector T cells and potently enhance engraftment of allogeneic HSC.

Suppression of NK Cell-Mediated Bone Marrow Cell Rejection by CD4+CD25+ Regulatory T Cells: Linkage of Adaptive to Innate Responses.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2195-2195
Author(s):  
William J. Murphy ◽  
Isabel Bareo ◽  
Alan M. Hanash ◽  
Lisbeth A. Welniak ◽  
Kai Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Cell Function ◽  
Nk Cell ◽  
Marrow Cell ◽  
Poly I:C ◽  
Hybrid Resistance

Abstract While a link between the innate to adaptive immune system has been established, studies demonstrating direct effects of T cells in regulating Natural Killer (NK) cell function have been lacking. Naturally occurring CD4+CD25+ regulatory T cells (Tregs) have been shown to potently inhibit adaptive responses by T cells. We therefore investigated whether Tregs could affect NK cell function in vivo. Using a bone marrow transplantation (BMT) model of hybrid resistance, in which parental (H2d) marrow grafts are rejected by the NK cells of the F1 recipients (H2bxd), we demonstrate that the in vivo removal of host Tregs significantly enhances NK-cell mediated BM rejection. This heightened rejection was mediated by the specific NK cell Ly-49+ subset previously demonstrated to reject the BMC in this donor/host pairing. The depletion of Tregs could also further increase rejection already enhanced by treating recipients with the NK cell activator, poly I:C. Although splenic NK cell numbers were not significantly altered, increased splenic NK in vitro cytotoxic activity was observed from the recovered cells. The regulatory role of Tregs was confirmed in adoptive transfer studies in which transferred CD4+CD25+ Tregs resulted in abrogation of NK cell-mediated hybrid resistance. Thus, Tregs can potently inhibit NK cell function in vivo and their depletion may have therapeutic ramifications with NK cell function in BMT and cancer therapy.

Chimeric Regulatory T cells Enhance HSC Engraftment in An Antigen-Specific Fashion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2424-2424
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Yujie Wen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Third Party ◽  
T Cell Proliferation ◽  
Hematopoietic Stem ◽  
Mixed Chimeras

Abstract Abstract 2424 Poster Board II-401 We previously reported that CD8+TCR- facilitating cells (FC) induce the generation of chimeric regulatory T cells (Treg) in vivo. Transplantation of a mixture of CD8+/TCR- FC and hematopoietic stem cells (HSC) into ablated recipients results in chimerism and tolerance. Treg harvested from the spleen of chimeras (chimeric Treg) potently increase long-term donor chimerism in secondary NOD recipient mice. Here, we evaluated whether chimeric Treg enhance engraftment of hematopoietic stem cells (HSC) in an antigen-specific manner. To prepare mixed chimeras (B6 → NOD), NOD recipients were conditioned with 950 cGy TBI and transplanted with 10,000 B6 HSC and 1,000 NOD HSC plus 45,000 CD8+TCR- B6 FC. At 5 weeks, CD8-CD4+CD25bright chimeric Treg were sorted from spleens of the mixed chimeras (B6 → NOD). 100,000 chimeric Treg were then mixed with 10,000 B6 HSC (donor-specific) + 10,000 B10.BR HSC (third-party) and transplanted into conditioned NOD recipients in competitive repopulation assays. NOD mice given HSC plus nonchimeric naïve B6 Treg or HSC alone served as controls. Two of the four animals that received HSC alone engrafted and exhibited an average of 6.7% donor B6 chimerism at 30 days, 11.2% at 60 days, and 10.6% at 90 days. Three of five animals given HSC plus naïve B6 Treg engrafted with 21.3% donor B6 chimerism at 30 days, 28.8% at 60 days, and 28.9% at 90 days. In contrast, eight of nine recipients of HSC + chimeric Treg engrafted. These animals exhibited a significantly higher level of donor B6 chimerism, ranging from 56.3% at 30 days, 75.4% at 60 days to 85% at 90 days (P = 0.034). None of the recipients engrafted with the MHC-disparate third-party B10.BR HSC. We then assessed the suppressive function of chimeric Tregin vitro by using MLR suppressor cell assays. CD8-/CD4+/CD25bright Treg were sorted from chimeric spleens 5 wks to 12 wks after HSC + FC transplantation. As shown in the Figure 1, Treg from naïve B6 mice resulted in 1.9 fold; 1.3 fold and 1.1 fold inhibition of proliferation at 1:1, 1:0.25, 1:0.125 responder/Treg ratios (n = 3). In contrast, chimeric Treg potently suppressed T cell proliferation by 10.5 fold; 3.2 fold; and 1.7 fold at responder/Treg ratios of 1:1, 1:0.25, 1:0.125 (n = 4). Chimeric Treg significantly suppressed T cell proliferation at responder/Treg ratios of 1:1 and 1:0.25 compared with naïve B6 Treg (P < 0.05). NOD responder splenocytes remained hypoproliferative in response to B6 stimulator and chimeric Treg compared with stimulator plus B6 Treg, suggesting that chimeric Treg are significantly more potent than naïve B6 Treg in suppressing effector T cell proliferation in vitro. These data show that chimeric Treg enhance donor B6 HSC engraftment but not third-party B10.BR HSC, demonstrating that chimeric Treg function in vivo in an antigen-specific fashion. These data also show that the mechanism of FC function in vivo is associated with the establishment of an antigen-specific regulatory feedback loop. Figure 1 Figure 1. Disclosures: Bozulic: Regenerex: Employment. Ildstad:Regenerex: Equity Ownership.

Induction of Donor-Type Chimerism and Transplantation Tolerance Across Major Histocompatibility Barriers in Sublethally Irradiated Mice by Sca-1+Lin− Bone Marrow Progenitor Cells: Synergism With Non-Alloreactive (Host × Donor)F1 T Cells

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3212-3221 ◽  
Author(s):  
Esther Bachar-Lustig ◽  
Hong Wei Li ◽  
Hilit Gur ◽  
Rita Krauthgamer ◽  
Hadar Marcus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Transplantation Tolerance ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Donor Type ◽  
Total Body ◽  
Veto Cells

Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1+Lin− cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy–irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1+Lin−cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host × donor)F1 T cells, previously shown to enhance T-cell–depleted BM allografts in lethally irradiated mice, synergize with Sca-1+Lin− cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.

scholarly journals Targeting Antigen Presenting   Cells to Treat Autoimmune  Inflammation

2021 ◽  
Author(s):  
◽  
Aras Toker
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Mhc Class Ii ◽  
T Cell Proliferation ◽  
Target Cells ◽  
Antigen Presenting

<p>Glatiramer acetate (GA) is approved for the treatment of relapsing-remitting multiple sclerosis (MS), and can suppress experimental autoimmune encephalomyelitis (EAE), a murine model of human MS. GA treatment is associated with the induction of anti-inflammatory TH2 responses and with the antigen specific expansion of regulatory T cells that counteract or inhibit pathogenic events in MS and EAE. These T cell mediated mechanisms of protection are considered to be a result of modulation of antigen presenting cells (APCs) by GA, rather than direct effects on T cells. However, it is unknown if GA preferentially targets a specific APC subset or can act through multiple APCs in vivo. In addition, GA-modulated innate cells may also exhibit direct antigen non-specific suppression of autoreactive cells. One objective of this study was to identify the in vivo target cell population of GA and to assess the potential of the target cells to antigen non-specifically suppress immune responses. Fluorophor-labelled GA bound to monocytes after intravenous injections, suggesting that monocytes may be the primary target of GA in vivo. In addition, intravenous GA treatment enhanced the intrinsic ability of monocytes to suppress T cell proliferation, both in vitro and in vivo. The findings of this study therefore suggest that GA-induced monocytes may contribute to GA therapy through direct mechanisms of antigen non-specific T cell immunosuppression. A further objective of this work was to investigate the potential of an in vivo drug targeting approach. This approach was hypothesised to increase the uptake of GA by the target cells and substantially improve GA treatment through antigen specific mechanisms such as induction of TH2 or regulatory T cells. Targeting antigens to professional APCs with an anti-MHC class II antibody resulted in significantly enhanced T cell proliferation in vitro. However, no EAE suppression occurred when GA was targeted to MHC class II in vivo. In addition, targeting GA specifically to monocytes also failed to suppress EAE. These findings suggest that GA treatment may selectively modulate monocytes to enhance their ability to inhibit autoreactive T cells, which could be part of the mechanism by which GA ameliorates MS. Targeting GA to a specific cell type may not be a powerful approach to improve treatment, because increased proliferation of GA specific T cells is not sufficient for disease suppression, and conjugation to antibodies may functionally reduce GA to a mere antigen devoid of immunomodulatory capacity.</p>

scholarly journals IL-27 confers a protumorigenic activity of regulatory T cells via CD39

2019 ◽  
Vol 116 (8) ◽  
pp. 3106-3111 ◽  
Author(s):  
Young-Jun Park ◽  
Heeju Ryu ◽  
Garam Choi ◽  
Byung-Seok Kim ◽  
Eun Sook Hwang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Antitumor Immunity ◽  
Suppressive Activity ◽  
Independent Manner ◽  
Bone Marrow Chimera ◽  
Cell Responses

Expression of ectonucleotidase CD39 contributes to the suppressive activity of Foxp3+ regulatory T cells (Tregs) by hydrolyzing immunogenic ATP into AMP. The molecular mechanism that drives CD39 expression on Tregs remains elusive. We found that tumor-infiltrating Tregs (Ti-Tregs) failed to up-regulate CD39 in mice lacking EBI3 subunit of IL-27 or IL-27Ra. Mixed bone marrow chimera and in vitro studies showed that IL-27 signaling in Tregs directly drives CD39 expression on Ti-Tregs in a STAT1-dependent, but STAT3- and T-bet–independent, manner. Tregs stimulated with IL-27 showed enhanced suppressive activities against CD8+ T cell responses in vitro. Moreover, IL-27Ra–deficient Tregs and STAT1-deficient Tregs were less efficient than WT Tregs in suppressing antitumor immunity in vivo. CD39 inhibition significantly abolished IL-27–induced suppressive activities of Tregs. Thus, IL-27 signaling in Tregs critically contributes to protumorigenic properties of Tregs via up-regulation of CD39.

scholarly journals Cathepsin K maintains the number of lymphocytes in vivo

2020 ◽  
Author(s):  
Renate Hausinger ◽  
Marianne Hackl ◽  
Ana Jardon-Alvarez ◽  
Miriam Kehr ◽  
Sandra Romero Marquez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cathepsin K ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cell Compartments ◽  
Lymphocyte Homeostasis

AbstractIn this study, we investigated the influence of the loss of Cathepsin K (Ctsk) gene on the hematopoietic system in vitro and in vivo. We found that cultures with Lineage- SCA1+ KIT+ (LSK) cells on Ctsk deficient stromal cells display reduced colony formation and proliferation, with increased differentiation, giving rise to repopulating cells with reduced ability to repopulate the donor LSK and T cell compartments in the bone marrow. Subsequent in vivo experiments showed impairment of lymphocyte numbers, but, gross effects on early hematopoiesis or myelopoiesis were not found. Most consistently in in vivo experimental settings, we found a significant reduction of (donor) T cell numbers in the bone marrow. Lymphocyte deregulation is also found in transplantation experiments, which revealed that Ctsk is required for optimal regeneration not only of T cells, but also of B cells. Interestingly, cell non-autonomous Ctsk regulates both B- and T cell numbers, but T cell numbers in the bone marrow require an additional autonomous Ctsk-dependent process. Thus, we show that Ctsk is required for the maintenance of hematopoietic stem cells in vitro, but in vivo, Ctsk deficiency most strongly affects lymphocyte homeostasis, particularly of T cells in the bone marrow.

scholarly journals D-mannose ameliorates autoimmune phenotypes in mouse models of lupus

2020 ◽  
Author(s):  
Haiting Wang ◽  
Xiangyu Teng ◽  
Georges Abboud ◽  
Wei Li ◽  
Shuang Ye ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cell Proliferation ◽  
T Cells ◽  
Dendritic Cells ◽  
T Cell Proliferation ◽  
Autoantibody Production ◽  
Models Of Lupus

Abstract Background: Systemic lupus erythematosus is a disorder of immune regulation characterized by overproduction of autoantibodies. D-mannose is a C-2 epimer of glucose that exhibits immunoregulatory effects in models of autoimmune diseases, such as type 1 diabetes, induced rheumatoid arthritis, and airway inflammation. This study was conducted to evaluate the efficacy of D-mannose treatment in mouse models of lupus.Methods: The effect of D-Mannose was evaluated by flow cytometry on the in vitro activation of C57BL/6 (B6) murine bone marrow derived dendritic cells and their ability to induce antigen specific CD4+ T cell proliferation and activation. The effect of D-mannose administration in vivo on the frequency of Foxp3+ regulatory T cells in B6 mice was assessed by flow cytometry. D-mannose was administered to two models of lupus: the chronic graft-versus-host disease (cGVHD) induced model and the B6.lpr spontaneous model. Autoantibody production was measured by ELISA and immune activation by flow cytometry. Results were compared by two-tailed statistics: unpaired or paired t tests, or Mann-Whitney U tests depending on whether the data was normally distributed.Results: D-mannose inhibited the maturation of bone marrow dendritic cells and their induction of antigen-specific T cell proliferation and activation in vitro. In vivo, D-mannose increased the frequency of Foxp3+ regulatory T cells in unmanipulated control mice. In the cGVHD model of induced lupus, D-mannose treatment decreased autoantibody production, with a concomitant reduction of the frequency of effector memory and follicular helper T cells as well as germinal center B cells and plasma cells. These results were partially validated in the B6.lpr model of spontaneous lupus. Conclusion: Overall, our results suggest that D-mannose ameliorates autoimmune activation in models of lupus, at least partially due to its expansion of Treg cells, the induction of immature conventional dendritic cells and the downregulation of effector T cells activation. D-Mannose showed however a weaker immunomodulatory effect in lupus than in other autoimmune diseases.

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 63-63 ◽  
Author(s):  
Johannes L. Zakrzewski ◽  
Adam A. Kochman ◽  
Sidney X. Lu ◽  
Theis H. Terwey ◽  
Theo D. Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Hematopoietic Stem ◽  
T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of &gt;95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

scholarly journals Interleukin 10 Gene-Modified Bone Marrow-Derived Dendritic Cells Attenuate Liver Fibrosis in Mice by Inducing Regulatory T Cells and Inhibiting the TGF-β/Smad Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Yejin Xu ◽  
Xinyue Tang ◽  
Min Yang ◽  
Shengguo Zhang ◽  
Shanshan Li ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Liver Fibrosis ◽  
Regulatory T Cells ◽  
Interleukin 10 ◽  
Smad Signaling Pathway ◽  
Culture Supernatants

Aim. To explore the therapeutic effects and mechanisms of interleukin 10 gene-modified bone marrow-derived dendritic cells (DC-IL10) on liver fibrosis. Methods. In vitro, BMDCs were transfected with lentiviral-interleukin 10-GFP (LV-IL10-GFP) at the MOI of 1 : 40. Then, the phenotype (MHCII, CD80, and CD86) and allo-stimulatory ability of DC-IL10 were identified by flow cytometry, and the levels of IL-10 and IL-12 (p70) secreted into the culture supernatants were quantified by ELISA. In vivo, DC-IL10 was injected into mice with CCl4-induced liver fibrosis through the tail vein. Lymphocytes were isolated to investigate the differentiation of T cells, and serum and liver tissue were collected for biochemical, cytokine, histopathologic, immune-histochemical, and Western blot analyzes. Results. In vitro, the expressions of MHCII, CD80, and CD86 in DC-IL10 were significantly suppressed, allogeneic CD4+T cells incubated with DC-IL10 showed a lower proliferative response, and the levels of IL-10 and IL-12 (p70) secreted into the DC-IL10 culture supernatants were significantly increased and decreased, respectively. In vivo, regulatory T cells (Tregs) were significantly increased, while ALT, AST, and inflammatory cytokines were significantly reduced in the DC-IL10 treatment group, and the degree of hepatic fibrosis was obviously reversed. The TGF-β/smad pathway was inhibited following DC-IL10 treatment compared to the liver fibrosis group. Conclusion. IL-10 genetic modification of BMDCs may maintain DC in the state of tolerance and allow DC to induce T cell hyporesponsiveness or tolerance. DC-IL10 suppressed liver fibrosis by inducing Treg production and inhibiting the TGF-β/smad signaling pathway.

scholarly journals Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4063-4063 ◽  
Author(s):  
Renier Myburgh ◽  
Jonathan Kiefer ◽  
Norman F Russkamp ◽  
Alexander Simonis ◽  
Surema Pfister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these. Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv. In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks. To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML. Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. Disclosures Neri: Philochem AG: Equity Ownership.

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