Intra–bone marrow injection of allogeneic bone marrow cells: a powerful new strategy for treatment of intractable autoimmune diseases in MRL/lpr mice

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3292-3299 ◽  
Author(s):  
Taketoshi Kushida ◽  
Muneo Inaba ◽  
Hiroko Hisha ◽  
Naoya Ichioka ◽  
Takashi Esumi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Autoimmune Diseases ◽  
Bone Marrow Cells ◽  
Donor Cell ◽  
Allogeneic Bone ◽  
Early Recovery ◽  
New Strategy ◽  
Marrow Cells

Abstract Intractable autoimmune diseases in chimeric resistant MRL/lpr mice were treated by a new bone marrow transplantation (BMT) method consisting of fractionated irradiation, 5.5 Gy × 2, followed by intra–bone marrow (IBM) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice (5.5 Gy × 2 + IBM). In MRL/lpr mice treated with this method, the number of donor-derived cells in the bone marrow, spleen, and liver rapidly increased (almost 100% donor-derived cells by 14 days after the treatment), and the number of donor-derived hemopoietic progenitor cells concomitantly increased. Furthermore, donor-derived stromal cells were clearly detected in the cultured bone pieces from MRL/lpr mice treated with 5.5 Gy × 2 + IBM. All the recipients thus treated survived more than 1 year (> 60 weeks after birth) and remained free from autoimmune diseases. Autoantibodies decreased to almost normal levels, and abnormal T cells (Thy1.2+/B220+/CD4−/CD8−) disappeared. Hematolymphoid cells were reconstituted with donor-derived cells, and newly developed T cells were tolerant to both donor (B6)-type and host (MRL/lpr)-type major histocompatibility complex determinants. Successful cooperation was achieved among T cells, B cells, and antigen-presenting cells when evaluated by in vitro antisheep red blood cell responses. These findings clearly indicate that this new strategy (IBM-BMT) creates the appropriate hemopoietic environment for the early recovery of hemopoiesis and donor cell engraftment, resulting in the complete amelioration of intractable autoimmune diseases in chimeric resistant MRL/lpr mice without recourse to immunosuppressants. This strategy would therefore be suitable for human therapy.

scholarly journals Generation of CFU-C/suppressor T cells in vitro: an experimental model for immune-mediated marrow failure

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 491-496
Author(s):  
A Bacigalupo ◽  
M Podesta ◽  
MC Mingari ◽  
L Moretta ◽  
G Piaggio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Suppressor Cells ◽  
Allogeneic Bone ◽  
Suppressor Activity ◽  
Healthy Donors ◽  
Marrow Cells

T cells were derived from the bone marrow of 8 healthy donors and fractionated, according to their receptors for the Fc fragment of IgG, into TG+ and TG- lymphocytes. These were then cocultured with autologous or allogeneic bone marrow cells in agar in the CFU-C assay. No significant suppresion of colony formation could be detected. Total T, TG+, and TG- cells were then incubated for 18 hr with PWM, washed, and cocultured with bone marrow cells. PWM-treated TG- cells showed no significant CFU-C suppressor activity, whereas PWM-treated total T and TG+ cells inhibited colony formation of both autologous and allogeneic marrow cells. The supernatant of PWM-treated total T and TG+ cells also inhibited colony formation. PWM alone enhanced colony formation. The results of this study indicate that normal T cells can be activated in vitro to become CFU-C/suppressor cells after PWM stimulation, and that this effect is mediated by T cells with the Fc receptor for IgG.

scholarly journals Generation of CFU-C/suppressor T cells in vitro: an experimental model for immune-mediated marrow failure

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 491-496 ◽  
Author(s):  
A Bacigalupo ◽  
M Podesta ◽  
MC Mingari ◽  
L Moretta ◽  
G Piaggio ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Colony Formation ◽  
Bone Marrow Cells ◽  
Suppressor Cells ◽  
Allogeneic Bone ◽  
Suppressor Activity ◽  
Healthy Donors ◽  
Marrow Cells

Abstract T cells were derived from the bone marrow of 8 healthy donors and fractionated, according to their receptors for the Fc fragment of IgG, into TG+ and TG- lymphocytes. These were then cocultured with autologous or allogeneic bone marrow cells in agar in the CFU-C assay. No significant suppresion of colony formation could be detected. Total T, TG+, and TG- cells were then incubated for 18 hr with PWM, washed, and cocultured with bone marrow cells. PWM-treated TG- cells showed no significant CFU-C suppressor activity, whereas PWM-treated total T and TG+ cells inhibited colony formation of both autologous and allogeneic marrow cells. The supernatant of PWM-treated total T and TG+ cells also inhibited colony formation. PWM alone enhanced colony formation. The results of this study indicate that normal T cells can be activated in vitro to become CFU-C/suppressor cells after PWM stimulation, and that this effect is mediated by T cells with the Fc receptor for IgG.

Treatment of intractable autoimmune diseases in MRL/lpr mice using a new strategy for allogeneic bone marrow transplantation

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1862-1868 ◽  
Author(s):  
Taketoshi Kushida ◽  
Muneo Inaba ◽  
Kenji Takeuchi ◽  
Kikuya Sugiura ◽  
Ryokei Ogawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Autoimmune Diseases ◽  
Marrow Transplantation ◽  
Bone Marrow Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Immune Functions

A new bone marrow transplantation (BMT) method for treating severe autoimmune diseases in chimeric resistant MRL/lpr mice is presented. The method consists of fractionated irradiation (5.5 Gy × 2), followed by portal venous (PV) injection of whole bone marrow cells (BMCs) from allogeneic normal C57BL/6 (B6) mice and intravenous (IV) injection of whole B6 BMCs 5 days after the PV injection (abbreviated as 5.5 Gy × 2 + PV + IV). All recipients survived more than 1 year after this treatment (more than 64 weeks after birth). Abnormal T cells (Thy1.2+/B220+/CD3+/CD4−/CD8−) present in MRL/lpr mice before the treatment disappear, and hematolymphoid cells are reconstituted with donor-derived cells. The treated mice are free from autoimmune diseases. Levels of autoantibodies (IgG/IgM anti-ssDNA antibodies and IgG/IgM rheumatoid factors) decrease to normal levels. Successful cooperation is achieved among T cells, B cells, and antigen-presenting cells (APCs) of the treated MRL/lpr mice when evaluated by in vitro anti-SRBC responses. Newly developed T cells are tolerant to both donor (B6)-type and host (MRL/lpr)-type major histocompatibility complex (MHC) determinants. These findings clearly indicate that severe autoimmune diseases in MRL/lpr mice are completely ameliorated by the treatment without recourse to immunosuppressants, and that the treated MRL/lpr mice show normal immune functions, strongly suggesting that this strategy would be applicable to humans.

scholarly journals Cell-mediated amegakaryocytic thrombocytopenia associated with systemic lupus erythematosus

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 479-483
Author(s):  
T Nagasawa ◽  
T Sakurai ◽  
H Kashiwagi ◽  
T Abe
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Bone Marrow ◽  
T Cells ◽  
Lupus Erythematosus ◽  
Culture System ◽  
Bone Marrow Cells ◽  
Rare Complication ◽  
Systemic Lupus ◽  
Marrow Cells

We studied a patient with a rare complication of amegakaryocytic thrombocytopenia (AMT) associated with systemic lupus erythematosus (SLE). To investigate the underlying pathogenesis of AMT, the effects of peripheral blood T cells and serum on human megakaryocyte progenitor cells were studied using in vitro coculture techniques. Mononuclear bone marrow cells (2 X 10(5) from normal donors produced 33.6 +/- 8.8 (n = 10) colony-forming unit-megakaryocytes (CFU-M) in our plasma clot system. When 2 X 10(5) of the patient's T cells were added to the culture system, the number of CFU-M decreased to only 3.5 +/- 0.6/2 X 10(5) bone marrow cells. No evidence of inhibitory effects was found by the addition of the patient's serum and complement to the culture system. The T cells stored at -80 degrees C on admission were also capable of suppressing autologous CFU-M after recovery from AMT. These results indicate that in vitro suppression of CFU-M from allogenic and autologous bone marrow cells by this patient's T cells provides an explanation for the pathogenesis of AMT associated with SLE.

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 ◽  
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.

scholarly journals Long-lasting skin allograft tolerance in adult mice induced across fully allogeneic (multimajor H-2 plus multiminor histocompatibility) antigen barriers by a tolerance-inducing method using cyclophosphamide.

1989 ◽  
Vol 169 (1) ◽  
pp. 213-238 ◽  
Author(s):  
H Mayumi ◽  
R A Good
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Effector T Cells ◽  
Mixed Chimerism ◽  
Allogeneic Bone ◽  
Spleen Cells ◽  
Skin Allograft ◽  
Cell Injection ◽  
Marrow Cells

A new method of cyclophosphamide (CP)-induced skin allograft tolerance in mice that can regularly overcome fully allogeneic (major H-2 plus non-H-2) antigen barriers in mice has been established. The components of the method are intravenous or intraperitoneal administration of 50-100 micrograms of anti-Thy-1.2 mAb on day -1, intravenous injection of 90 x 10(6) allogeneic spleen cells mixed with 30 x 10(6) allogeneic bone marrow cells from the same donor on day 0, and intraperitoneal injection of 200 mg/kg CP on day 2. In each of four fully allogeneic donor----recipient combinations, including C3H/HeJ (C3H; H-2k)----C57BL/6J(B6; H-2b), B6----C3H, BALB/cByJ (BALB; H-2d)----B6, and BALB----C3H, long-lasting survival of skin allografts was induced in most of the recipient mice. The specific tolerant state induced was dependent on the doses of the antibody and bone marrow cells used. The optimal timing of CP treatment to induce tolerance was found to be 1-3 d after the stimulating cell injection. Treatment with the anti-Thy-1.2 antibody together with CP on day 2 after the cell injection on day 0 also induced profound tolerance. In the B6 mice made tolerant of C3H with antibody, C3H spleen cells plus C3H bone marrow cells, and then CP, a minimal degree of stable mixed chimerism was established and the antitolerogen (C3H) immune responses examined here, including delayed footpad reaction (DFR), CTL activity, and capacity for antibody production against donor-strain antigens were abrogated in a tolerogen-specific manner. From cell transfer experiments, the mechanism of tolerance could be largely attributed to reduction of effector T cells reactive against the tolerogen, and strong suppressive influences that might prolong skin allograft survival directly were not detected in the tolerant mice. Moreover, pretreatment with anti-Thy-1.2 antibody or anti-L3T4 (CD4) antibody was more effective than pretreatment with anti-Lyt-1 (CD5) antibody or anti-Lyt-2 (CD8) antibody as an initial step in tolerance induction. These results suggest that permanent tolerance to fully allogeneic skin grafts may be induced because antibody given before the stimulating cell injection reduces the number of reactive T cells in the recipient mice. This antibody treatment may facilitate an antigen-stimulated destruction of responding and thus proliferating cells with CP by preventing a possibly less proliferative, more rapid maturation of reactive T cells or by destroying residual effector T cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Human donor bone marrow cells induce in vitro “suppressor T cells” that functionally suppress autologous B cells

2003 ◽  
Vol 64 (1) ◽  
pp. 21-30 ◽  
Author(s):  
Manuel R Carreno ◽  
Laphalle Fuller ◽  
James M Mathew ◽  
Gaetano Ciancio ◽  
George W Burke ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Bone Marrow Cells ◽  
Suppressor T Cells ◽  
Human Donor ◽  
Donor Bone Marrow ◽  
Marrow Cells

scholarly journals Cell-mediated amegakaryocytic thrombocytopenia associated with systemic lupus erythematosus

Blood ◽  
1986 ◽  
Vol 67 (2) ◽  
pp. 479-483 ◽  
Author(s):  
T Nagasawa ◽  
T Sakurai ◽  
H Kashiwagi ◽  
T Abe
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Bone Marrow ◽  
T Cells ◽  
Lupus Erythematosus ◽  
Culture System ◽  
Bone Marrow Cells ◽  
Rare Complication ◽  
Systemic Lupus ◽  
Marrow Cells

Abstract We studied a patient with a rare complication of amegakaryocytic thrombocytopenia (AMT) associated with systemic lupus erythematosus (SLE). To investigate the underlying pathogenesis of AMT, the effects of peripheral blood T cells and serum on human megakaryocyte progenitor cells were studied using in vitro coculture techniques. Mononuclear bone marrow cells (2 X 10(5) from normal donors produced 33.6 +/- 8.8 (n = 10) colony-forming unit-megakaryocytes (CFU-M) in our plasma clot system. When 2 X 10(5) of the patient's T cells were added to the culture system, the number of CFU-M decreased to only 3.5 +/- 0.6/2 X 10(5) bone marrow cells. No evidence of inhibitory effects was found by the addition of the patient's serum and complement to the culture system. The T cells stored at -80 degrees C on admission were also capable of suppressing autologous CFU-M after recovery from AMT. These results indicate that in vitro suppression of CFU-M from allogenic and autologous bone marrow cells by this patient's T cells provides an explanation for the pathogenesis of AMT associated with SLE.

scholarly journals Factor-dependent in vitro growth of human normal bone marrow-derived basophil-like cells.

1983 ◽  
Vol 158 (3) ◽  
pp. 857-871 ◽  
Author(s):  
K Tadokoro ◽  
B M Stadler ◽  
A L De Weck
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Interleukin 2 ◽  
Nonspecific Esterase ◽  
Ionophore A23187 ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Marrow Cells

A factor(s) present in supernatants from lectin-stimulated peripheral blood mononuclear cells promoted the production of basophil-like cells in liquid cultures of normal human bone marrow cells. The cultured basophil-like cells had lobulated or round nuclei, and the cytoplasmic granules stained metachromatically with toluidine blue and azurophilic with Giemsa. 20% of the metachromatically staining cells were peroxidase positive but not positive for nonspecific esterase. The histamine content was 0.5-2 pg/cell. The basophil-like cells released histamine upon challenge with calcium ionophore A23187 but not with compound 48/80. They also released histamine with anti-IgE when passively sensitized with human myeloma IgE. The development of basophil-like cells was promoted in a dose-dependent fashion by a factor(s) in the conditioned medium. Blocking of cell proliferation with hydroxyurea or X irradiation inhibited the development of basophil-like cells. The production of the factor was dependent on the presence of T cells. The factor was different from interleukin 2 and its molecular weight was estimated to be 25,000-40,000 by gel filtration on a Sephacryl S-200 column. Thus, human basophil-like cells derived from normal bone marrow cells can grow and differentiate in vitro under the regulation of T cells.

In Vitro Generation Of Osteoclasts From Interleukin-3 (IL-3)-Dependent Mouse Hematopoietic Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4842-4842
Author(s):  
Shunqing Wang ◽  
Huixian Hong
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Control Culture ◽  
Significant Loss ◽  
Murine Bone Marrow ◽  
New Strategy ◽  
Differentiation And Proliferation ◽  
Marrow Cells

Abstract Objective This work aims to develop a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells Methods 1. Here, we describe an alternative method for in vitro generation of osteoclasts, which involves the use of interleukin (IL)-3-dependent murine bone marrow cells. Bone marrow cells, isolated from 6- to 8-week old C57BL/6 were cultured in α-MEM containing 10% FBS in tissue culture dishes overnight to remove stromal cells. Then, non-adherent bone marrow cells were harvested and continued in α-MEM containing 10% FBS without (control) or with IL-3 (1 ng/ml) for 6 days. While no cells survived in the control culture after the 6-day culturing, the IL-3-treated culture gave rise to a significant number of surviving cells. These IL-3-depedent cells were capable of differentiating to osteoclasts in response to M-CSF and RANKL stimulation. Moreover, these IL-3-dependent cells can be further expanded by plating them in non-treated plastic dishes followed with M-CSF treatment; they continued to survive and proliferate in non-treated plastic dishes in the presence of M-CSF for up to 4 days. After 4-day M-CSF treatment, these cells can be lifted by EDTA, and they were still able to differentiate into osteoclasts upon subsequent stimulation of M-CSF and RANKL. 2. We performed the in vitro bone resorption assay, Semiquantitative Reverse Transcription (RT)-PCR, Western Analysis, Infection of Murine Bone Marrow Cells (BMCs) to test whether the osteoclasts generated from IL-3-dependent murine bone marrow cells are different from the osteoclasts generated from traditional method. Results 1. IL-3 can maintain the survival of murine bone marrow cells for up to 6 days and these cells still keep their capacity to generate osteoclasts. The capacity of IL-3-dependent cells to form osteoclasts decreases with time of IL-3 treatment and IL-3 dependent cells can be further expanded by M-CSF without significant loss of the osteoclastogenic potential. 2. IL-3-dependent cells can form functional osteoclasts. RANKL induces the expression of osteoclast genes in IL-3-dependent cells. RANKL activates some of RANK signaling pathways in IL-3-dependent cells. Importantly, we found that IL-3 dependent murine bone marrow cells can be infected by retrovirus encoding GFP. Conclusions 1) We have developed a new strategy to generate murine osteoclasts in vitro using IL-3-dependent cells prepared by 6-day IL-3 treatment of murine bone marrow cells. 2) IL-3-dependent cells can be infected by retrovirus, permitting further experimental manipulations to express or knock down genes in IL-3-dependent cells for studying the molecular mechanism controlling differentiation and proliferation of osteoclast precursors or delineating molecular events in early osteoclastogenesis. Disclosures: No relevant conflicts of interest to declare.

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