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

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.

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Serum Transferrin Receptor Value as a Universal Indicator of Erythropoietic Activity.

Blood ◽

10.1182/blood.v104.11.3682.3682 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3682-3682

Author(s):

Young Soo Lee ◽

Chul Soo Kim ◽

Jong Weon Choi

Keyword(s):

Bone Marrow ◽

Transferrin Receptor ◽

Bone Marrow Cells ◽

Reticulocyte Count ◽

Serum Transferrin Receptor ◽

Group 4 ◽

Universal Indicator ◽

Group 3 ◽

Group 2 ◽

Group 1

Abstract The serum transferrin receptor (sTfR) is thought a sensitive and quantitative parameter of tissue iron deficiency as well as an indicator of erythropoietic activity. This study was aimed at the verification of a hypothesis that sTfR is a general indicator of erythropoiesis regardless whatever the cause is. A total of 173 patients in heterogeneous diseases who underwent bone marrow study as a workup for anemia were measured for sTfR, reticulocyte maturity index (RMI), erythroid element proportion of bone marrow cells, and other hematologic parameters (hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, red cell distribution width, absolute reticulocyte count). By immunoenzymometric method sTfR was measured using IDeATMcTfR kids (Orion Diagnostica, Orion, Finland). Reticulocyte count and proportion was measured manually by one expert examiner after standard blood smear and stain. Reticulocyte subpopulation was automatically analyzed by flow cytometry using R-3000 TM (Sysmex, TOA, Japan). RMI was calculated from the equation of (medium fluorescent reticulocyte fraction + high fluorescent reticulocyte fraction) X 100 / low fluorescent reticulocyte fraction. Correlation analysis was done among the variables including sTfR, RMI, erythroid element proportion of bone marrow cells, and other hematologic parameters using SAS 6.12 soft ware. The analysis was carried out for the whole 173 patients to see the general trends and repeated for 4 groups of disease category, arbitrarily divided to group 1 (n=33, iron deficiency or or disease with no predisposition to anemia of chronic disease), group 2 (n=53, hematologic malignancies), group 3 (n=44, solid tumors), and group 4 (n=43, chronic or infectious disease) to see if the trends may be affected by specific diseases. The results showed a solid correlation of sTfR with RMI as well as erythroid precursors in bone marrow, not only in the whole patient population (e.g. sTfR vs RMI, R=0.587, p=0.0001) but also in individual groups (e.g. sTfR vs RMI, R=0.48, p=0.005 in group 1, R=0.69, p=0.0001 in group 2, R=0.58, p=0.0001 in group 3, R=0.81, p=0.0001 in group 4). These findings indicated the significance of sTfR is valid under any clinical setting as a universal indicator of hematopoietic activity. The sTfR can be used as a useful parameter for monitoring of erythropoiesis in a variety disease.

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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.

Journal of Experimental Medicine ◽

10.1084/jem.169.1.213 ◽

1989 ◽

Vol 169 (1) ◽

pp. 213-238 ◽

Cited By ~ 112

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)

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

Human Immunology ◽

10.1016/s0198-8859(02)00774-7 ◽

2003 ◽

Vol 64 (1) ◽

pp. 21-30 ◽

Cited By ~ 5

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

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Coadministration of bone marrow cells and an inhibitor of apoptosis (Z-VAD) promotes bone marrow cell survival and neurological functional recovery after focal cerebral ischemia in adult rat

Stroke ◽

10.1161/str.32.suppl_1.379-e ◽

2001 ◽

Vol 32 (suppl_1) ◽

pp. 379-380

Author(s):

Yi Li ◽

Jieli Chen ◽

Michael Chopp

Keyword(s):

Bone Marrow ◽

Cerebral Ischemia ◽

Bone Marrow Cells ◽

Inhibitor Of Apoptosis ◽

Apoptotic Cells ◽

Group 3 ◽

Group 2 ◽

Adhesive Removal ◽

Group 1 ◽

Marrow Cells

P222 Most bone marrow cells (BMCs) transplanted into the ischemic brain of adult rodents die shortly after they are grafted, similar to the 90%-95% of the fetal cells that die after transplantation into Parkinson s patients. We tested whether coadministration of BMCs and Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD), an inhibitor of apoptosis, into the ischemic boundary zone of the striatum and the cortex in rat brain promotes BMC survival. Adult Wistar rats were subjected to transient (2 h) middle cerebral artery occlusion (MCAo). At 1 d after ischemia, saline (n=9, Group 1); BMCs (1x10 6 in 10 :l, n=8, Group 2); or BMCs with Z-VAD (50 nM/ml, n=4, Group 3) were injected into brain. BMCs were harvested from donor adult rats injected with bromodeoxyuridine (BrdU, as a tracer). Rats were subjected to rotarod-motor and adhesive-removal somatosensory functional tests before MCAo and at 1 and 7 d after MCAo. Rats in Group 3 exhibited significant improvement (10.3±2.6 seconds, p<0.05) on the adhesive-removal test at 7 d, compared with those in Group 1 (29.3±9.4 seconds) and Group 2 (21.3±5.5 seconds), respectively. Immunohistochemistry staining was employed to identify BrdU-BMCs, and TUNEL staining was used to identify in situ DNA fragmentation of apoptotic cells. Even though the infarct volume in Group 3 (29.9±9.2%) did not change significantly, compared with Group 1 (34.3±4.0%) and Group 2 (26.6±3.8%); the number of BrdU-BMCs (88,200±7,400, ∼8.8% of 10 6 transplanted cells) increased significantly (p<0.05) in rats in Group 3, compared with that in Group 2 (31,700±9,100, ∼3.2% of 10 6 cells) at 7 d. In the grafting areas, apoptotic cells were less clustered (<90 vs.>240 per region) and apoptotic cells were significantly decreased (12.3±1.7/mm 2 vs. 25.9±2.1/mm 2 , ∼47%, p<0.05). Our data suggest that intracerebral coadministration of bone marrow cells and inhibitors of apoptosis enhance cellular survival of bone marrow cells and improves neurological functional recovery after cerebral ischemia.

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

Blood ◽

10.1182/blood.v57.3.491.bloodjournal573491 ◽

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.

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Omega-3 From Fish Oil Augments Gvhd Through the Enhancement of Chemotherapy Conditioning Regimen and Selective Foxp3 Depletion

Blood ◽

10.1182/blood.v120.21.4671.4671 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4671-4671

Author(s):

Sulaiman Al Hashmi ◽

Behnam Sadeghi ◽

Zuzana Hassan ◽

Magnus Lindskog ◽

Moustapha Hassan

Keyword(s):

Bone Marrow ◽

Fish Oil ◽

Bone Marrow Cells ◽

Corn Oil ◽

Conditioning Regimen ◽

Treg Cells ◽

Allogeneic Bone ◽

Omega 3 ◽

Group 3 ◽

Marrow Cells

Abstract Abstract 4671 Background: Omega-3 has been reported to enhance the effects of some cancer chemotherapeutic agents and to play a role in the immune system as immunoregulator and immunosuppressant. The effect of a diet rich in omega-3 during immunosuppressed states of anticancer treatment and on bone marrow transplantation (BMT) outcome is not well known. Aims: To study the effect of omega-3 on busulfan-cyclophosphamide (Bu-Cy) conditioning regimen. Moreover, to evaluate the effect of omega-3 on the outcome of BMT after Bu-Cy conditioning. Methods: We evaluated the effects of menhadenfish oil (omega-3 rich) on the myeloablative treatment with Bu-Cy as well as on the outcome of BMT in mice compared to the effects of a diet containing corn oil (omega-6 rich) or to standard chow. Animals were divided into three groups, Group 1, Group 2 and Group 3. In all the groups, female BALB/c mice, 8–12 weeks old, were randomly selected to receive a diet enriched with fish oil, a diet enriched with corn oil or standard chow. The mice were fed for two weeks prior to conditioning. Thereafter, all the mice were injected IP with 80 mg/kg busulfan (Bu) for four days (day –7 to day –4) followed by 200 mg/kg cyclophosphamide (Cy) for two days (days –3 and −2). The mice in groups 1 and 2 were killed on day 0 (the proposed day for transplantation) and spleen and femur bone marrow cells were harvested. Organs were analysed with flow cytometry to evaluate the lymphoid and myeloid cells phenotype. Also, regulatory T cell number and function were studied. Mice in Group 3 were transplanted on day 0 with allogeneic bone marrow cells from 8–12 weeks old male C57BL/6 mice (2.0 × 107 bone marrow cells and 3.0 × 107spleen cells). Results: Fish oil-enriched chow significantly suppressed the function (Figure 1) of CD4+CD25+FoxP3+ regulatory T (Treg) cells compared to standard chow and corn oil fed mice at day 0. However, the percentage of FoxP3+ cells was significantly elevated in mice fed corn oil compared to standard chow. In concert with myeloablative chemotherapy, fish oil enhanced the suppressive effect of Bu-Cy on CD11b+ myeloid and CD11c+CD86+ dendritic cell populations in the bone marrow and spleen compared to the other dietary groups. Recipient mice fed fish oil had lower survival after BMT (Figure 2). A higher survival was observed for corn oil-fed recipients, but this survival rate was still inferior to that of recipients fed standard chow. Feeding recipients omega-3 enriched diet was associated with a more pronounced acute graft versus host disease (aGVHD). BM and spleen from mice fed corn oil demonstrated lower chimerism. Conclusion: Fish oil enhances the immunosuppressive effect of the conditioning regimen (Bu-Cy) in mice. BMT recipients fed a diet rich in polyunsaturated fatty acids either have lower engraftment (corn oil) or react with a lethal aGVHD (fish oil) associated with a suppression of Treg cells. These findings may have clinical implications. PUFA supplementation should be reconsidered and caution is advised in patients undergoing stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v97.10.3292 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3292-3299 ◽

Cited By ~ 196

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.

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

Blood ◽

10.1182/blood.v57.3.491.491 ◽

1981 ◽

Vol 57 (3) ◽

pp. 491-496 ◽

Cited By ~ 44

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.

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

Blood ◽

10.1182/blood.v65.5.1134.bloodjournal6551134 ◽

1985 ◽

Vol 65 (5) ◽

pp. 1134-1140 ◽

Cited By ~ 1

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.

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