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)

Post-Transplant Cyclophosphamide and Sirolimus Are Synergistic in Preventing Rejection and Inducing Stable Mixed Chimerism Independently of Regulatory T Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3540-3540 ◽  
Author(s):  
Courtney Fitzhugh ◽  
Matthew M. Hsieh ◽  
Oswald Phang ◽  
Camille Madison ◽  
Leo Luznik ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Regulatory T Cells ◽  
Synergistic Effect ◽  
Bone Marrow Cells ◽  
Mixed Chimerism ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Donor Chimerism ◽  
Marrow Cells

Abstract Abstract 3540 Poster Board III-477 A strategy that can induce stable mixed chimerism across human leukocyte antigen (HLA) barriers would be beneficial in extending the application of hematopoietic stem cell transplantation (HSCT) to patients with severe sickle cell disease (SCD) who are in need of this potentially curative procedure. Indeed, we have recently demonstrated the feasibility of an HLA-matched sibling protocol employing low dose total body irradiation (TBI, 300cGy), the lymphocyte depleting agent alemtuzumab, and sirolimus to reverse the phenotype with minimal side effects. Due to the lack of HLA-matched siblings in the majority of patients, our goal is to develop a safe haploidentical regimen. In this work, we focused on determining optimal postgrafting immunosuppression and examined sirolimus and post-transplant cyclophosphamide (PT-cy), agents known to induce transplantation tolerance. To determine the optimal sequence for combining these drugs and whether this combination is synergistic in promoting stable donor chimerism despite the antiproliferative effects of sirolimus, we used a mismatched murine model with BalbC donors and C57Bl6 recipients. Twenty-five to 40 recipient mice received 200cGy TBI and PT-cy (200mg/kg intraperitoneally (IP) 2 days post transplant) with or without sirolimus (3mg/kg IP) for 14 to 30 days starting 1 day before or 4, 6, or 10 days post transplant. We found that in contrast to sirolimus or PT-cy alone, the combination of PT-cy and a limited course of sirolimus resulted in stable mixed chimerism: all mice that received PT-cy and sirolimus starting between 1 day before and 6 days after transplant attained donor chimerism levels ranging from 15-35%. Further, a 14 day course of sirolimus was sufficient to maintain stable mixed chimerism in our model (See Figures 1 and 2). To examine whether this synergistic effect is mediated by regulatory T cells, we administered anti-CD25 monoclonal antibody (CD25 mAb), an agent known to transiently deplete these cells in vivo. Fifteen mice received 200cGy TBI, sirolimus, PT-Cy, and either no CD25 mab, CD25 mab (1mg IP) on 7 and 3 days before and 1 day after transplant, or CD25 mab starting 14 days after transplant. CD25 mab was given biweekly for 5 weeks to mice in both groups. Donor engraftment levels did not differ in the three groups, with donor chimerism levels ranging from 30-40%. Our data show that the anti-proliferative effects of sirolimus do not inhibit the efficacy of the cytotoxic agent cyclophosphamide. Rather, our data demonstrate that the combination of PT-cy and a limited course of sirolimus synergistically promote mixed bone marrow chimerism in a complete mismatched setting. Further, the synergistic effect of this drug combination appears to be mediated independently from CD25+ regulatory T cell expression. In light of our previous success using sirolimus in an HLA-matched HSCT protocol, these findings lay the groundwork for developing PT-cy and sirolimus as a novel, safe, and effective means of promoting stable mixed chimerism in the haploidentical setting and thus greatly enhancing our ability to successfully apply this approach to patients with severe SCD. Figure 1 PT-cy and sirolimus are synergistic. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 30 days starting from 1 day before to 4 days after transplant. Figure 1. PT-cy and sirolimus are synergistic. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 30 days starting from 1 day before to 4 days after transplant. Figure 2 Fourteen days of sirolimus is sufficient to maintain stable mixed chimerism. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 14 days starting from 1 day before to 10 days after transplant. Figure 2. Fourteen days of sirolimus is sufficient to maintain stable mixed chimerism. 25 to 40 C57BI6 mice received 200cGy TBI, 22-25 × 106 bone marrow cells from BalbC mice, and PT-cy (Cy) 200mg/kg IP 2 days post transplant with or without sirolimus (Sir) 3mg/kg IP for 14 days starting from 1 day before to 10 days after transplant. Disclosures: No relevant conflicts of interest to declare.

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.

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

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.

scholarly journals The Positive Impact of Donor Bone Marrow Cells Transplantation into Immunoprivileged Compartments on the Survival of Vascularized Skin Allografts

2021 ◽  
Vol 69 (1) ◽  
Author(s):  
Arkadiusz Jundziłł ◽  
Aleksandra Klimczak ◽  
Erhan Sonmez ◽  
Grzegorz Brzezicki ◽  
Maria Siemionow
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Allogeneic Bone ◽  
Group 4 ◽  
Donor Bone Marrow ◽  
Group 3 ◽  
Group 2 ◽  
Marrow Cells

AbstractUsing the vascularized skin allograft (VSA) model, we compared the tolerogenic effects of different allogeneic bone marrow transplantation (BMT) delivery routes into immunoprivileged compartments under a 7-day protocol immunosuppressive therapy. Twenty-eight fully MHC mismatched VSA transplants were performed between ACI (RT1a) donors and Lewis (RT11) recipients in four groups of seven animals each, under a 7-day protocol of alfa/beta TCRmAb/CsA (alpha/beta-TCR monoclonal antibodies/Cyclosporine A therapy). Donor bone marrow cells (BMC) (100 × 106 cells) were injected into three different immunoprivileged compartments: Group 1: Control, without cellular supportive therapy, Group 2: Intracapsular BMT, Group 3: Intragonadal BMT, Group 4: Intrathecal BMT. In Group 2, BMC were transplanted under the kidney capsule. In Group 3, BMC were transplanted into the right testis between tunica albuginea and seminiferous tubules, and in Group 4, cells were injected intrathecally. The assessment included: skin evaluation for signs and grade of rejection and immunohistochemistry for donor cells engraftment into host lymphoid compartments. Donor-specific chimerism for MHC class I (RT1a) antigens and the presence of CD4+/CD25+ T cells were assessed in the peripheral blood of recipients. The most extended allograft survival, 50–78 days, was observed in Group 4 after intrathecal BMT. The T cells CD4+/CD25+ in the peripheral blood were higher after intrathecal BMC injection than other experimental groups at each post-transplant time point. Transplantation of BMC into immunoprivileged compartments delayed rejection of fully mismatched VSA and induction of robust, donor-specific chimerism.

Quantitative assays for detection of residual T cells of T-depleted human marrow

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1134-1140 ◽  
Author(s):  
PJ Martin ◽  
JA Hansen
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Indirect Immunofluorescence ◽  
Bone Marrow Cells ◽  
Allogeneic Bone ◽  
Dilution Assay ◽  
Limiting Dilution Assay ◽  
Limiting Dilution ◽  
Marrow Cells

Preliminary studies have suggested that depletion of T lymphocytes from donor marrow might be an effective method for preventing acute graft-v- host disease (GVHD) after allogeneic bone marrow transplantation in humans (Lancet 1:369, 472, 1984). However, the minimum degree of T cell depletion required to assure the prevention of GVHD in a population of marrow graft recipients has not been defined, largely because quantitative assays with sufficient sensitivity for detecting small numbers of residual viable T cells have not been developed. We have investigated three methods for the detection and enumeration of T cells after treatment of bone marrow with murine monoclonal anti-T cell antibodies and complement. Cell populations prepared by adding graded numbers of T cells to treated bone marrow were analyzed by immediate indirect immunofluorescence, by indirect immunofluorescence after culture of cells in medium containing phytohemagglutinin (PHA), or by a limiting-dilution assay. Immediate indirect immunofluorescence could reliably detect 300 T cells per 10(5) treated bone marrow cells. Indirect immunofluorescence after culture in PHA was tenfold more sensitive and could reliably detect 30 T cells per 10(5) treated bone marrow cells. The limiting dilution assay could be 300-fold more sensitive than immediate indirect immunofluorescence and 30-fold more sensitive than indirect immunofluorescence after culture in PHA. Sensitive, quantitative assays will be useful in guiding the development of methods for efficient removal of T cells in donor marrow, and will be essential for monitoring and interpreting the results of clinical trials.

Critical Roles of Memory T Cells and Antidonor Immunoglobulin in Rejection of Allogeneic Bone Marrow Cells in Sensitized Recipient Mice

2006 ◽  
Vol 82 (5) ◽  
pp. 689-698 ◽  
Author(s):  
Shigeyuki Nagata ◽  
Shinji Okano ◽  
Yoshikazu Yonemitsu ◽  
Kazunori Nakagawa ◽  
Yukihiro Tomita ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Cells ◽  
Memory T Cells ◽  
Allogeneic Bone Marrow ◽  
Allogeneic Bone ◽  
Marrow Cells

scholarly journals Quantitative assays for detection of residual T cells of T-depleted human marrow

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1134-1140 ◽  
Author(s):  
PJ Martin ◽  
JA Hansen
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Indirect Immunofluorescence ◽  
Bone Marrow Cells ◽  
Allogeneic Bone ◽  
Dilution Assay ◽  
Limiting Dilution Assay ◽  
Limiting Dilution ◽  
Marrow Cells

Abstract Preliminary studies have suggested that depletion of T lymphocytes from donor marrow might be an effective method for preventing acute graft-v- host disease (GVHD) after allogeneic bone marrow transplantation in humans (Lancet 1:369, 472, 1984). However, the minimum degree of T cell depletion required to assure the prevention of GVHD in a population of marrow graft recipients has not been defined, largely because quantitative assays with sufficient sensitivity for detecting small numbers of residual viable T cells have not been developed. We have investigated three methods for the detection and enumeration of T cells after treatment of bone marrow with murine monoclonal anti-T cell antibodies and complement. Cell populations prepared by adding graded numbers of T cells to treated bone marrow were analyzed by immediate indirect immunofluorescence, by indirect immunofluorescence after culture of cells in medium containing phytohemagglutinin (PHA), or by a limiting-dilution assay. Immediate indirect immunofluorescence could reliably detect 300 T cells per 10(5) treated bone marrow cells. Indirect immunofluorescence after culture in PHA was tenfold more sensitive and could reliably detect 30 T cells per 10(5) treated bone marrow cells. The limiting dilution assay could be 300-fold more sensitive than immediate indirect immunofluorescence and 30-fold more sensitive than indirect immunofluorescence after culture in PHA. Sensitive, quantitative assays will be useful in guiding the development of methods for efficient removal of T cells in donor marrow, and will be essential for monitoring and interpreting the results of clinical trials.

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

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