Haploidentical Bone Marrow Cells Research Articles

211At-Anti-CD45 Radioimmunotherapy Can Replace TBI Prior to Haploidentical Bone Marrow Transplantation and Yield Long-Term Hematopoietic Engraftment

Haploidentical bone marrow transplantation (haplo-BMT) is both clinically effective and widely available because related-donors can be identified for nearly all recipients. Despite the curative promise of this approach, many patients with hematologic malignancies will relapse after haplo-BMT and more effective preparative regimens are necessary. We have shown that anti-CD45 radioimmunotherapy (RIT) delivers high-doses of radiation to hematolymphoid organs while minimizing the radiation exposure to non-targeted tissues. The efficacy of beta-emitting radionuclides may be limited by their relatively low decay energies (0.66 – 2.3 MeV). We have thus investigated the higher energy alpha-emitter astatine-211 (211At) (average decay energy of 6.8 MeV), for targeted anti-CD45 radioimmunotherapy (RIT) in lieu of total body irradiation (TBI) prior to haploidentical BMT in a murine leukemia model to decrease relapse rates.Groups of five B6SJLF1/J mice (allotype H2-Db) received escalated activities (20, 30 or 40 μCi) of 211At-anti-CD45 antibody [100 μg (0.67 nmol) of B10-30F11] given by tail vein injections on day -2 in place of TBI prior to BMT. Animals received cyclophosphamide (CY; 200 mg/kg/day) on days –3, –2, or –1, and +2 for graft-versus-host disease prophylaxis, either alone, or with fludarabine (FLU; 100 mg/kg/day) for 4 days starting day -6. Transplanted mice received 1.5 × 107 haploidentical bone marrow cells from CB6F1/J mice (allotype H2-Dd) on day 0. Peripheral blood from recipient mice was then assayed monthly by flow cytometry to measure chimerism as the percentage of donor (H2-Dd) circulating CD8+ cells.The highest activity delivered of 40 µCi 211At-anti-CD45 RIT was uniformly lethal without BMT rescue, whereas 60% of transplanted mice at this dose survived to assessment at 1 month. Mice treated with 30 µCi of 211At-anti-CD45 RIT with pre- and post-transplant CY and without TBI or FLU, had high levels of engraftment with an average of 83.7 ± 5.8% donor CD8+ cells 1 month after haploidentical BMT (Table 1). The addition of FLU to 211At-anti-CD45 RIT with CY did not significantly improve chimerism levels, with mean donor CD8+ cells in mice treated with 40 µCi 211At-anti-CD45 RIT of 64.5 ± 41.6% compared to 60.0 ± 13.9% in the absence of FLU (p=0.8668). In addition, mice that received 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on either day –3, –2, or –1 showed mean donor CD8+ cells of 83.7 ± 5.8%, 49.9 ± 29.6% and 55.0 ± 46.2% 1 month after haploidentical-BMT, respectively. Importantly, chimerism levels remained stable 2 months after haploidentical BMT with mean donor CD8+ cells of 80.4 ± 16.6%, 47.0 ± 37.7% and 63.2 ± 10.7% in mice treated with 30 µCi 211At-anti-CD45 RIT and pre-transplant CY on day –3, –2, and –1, respectively. Engraftment using 40 or 30 µCi 211At-anti-CD45 RIT was comparable to using 850 or 1000 cGy TBI (mean donor CD8+ cells of 70.2 ± 18.8% and 60.0 ± 4.6%, respectively) prior to haploidentical BMT. RIT alone without any chemotherapy was insufficient to facilitate clinically relevant rates of donor engraftment, as mice treated with 30 µCi 211At-anti-CD45 RIT and no FLU, CY or TBI had 15.9 ± 7.1% mean donor CD8+ cells 1 month after haploidentical BMT.These results suggest that 211At-anti-CD45 RIT prior to haploidentical BMT with pre– and post–transplant CY can result in high levels of donor hematopoietic cell engraftment in the absence of TBI and FLU. This conditioning regimen may be less toxic and more effective at preventing relapse than TBI-based approaches due to the high linear energy transfer of the alpha emissions, or the high decay energy of targeted 211At deposited over its short effective path-length. On-going studies are assessing the efficacy and toxicity associated with 211At-anti-CD45 RIT compared to a TBI-based haploidentical BMT using a syngeneic murine leukemia model.Abstract 2417. Table 1Preparative Therapy and CD8+ Donor Chimerism at 1 monthGroupFLU(100mg/kg/d)pre-BMTCY(200mg/kg)TBI211 At-anti-CD45RITpost-BMTCY(200mg/kg)Donor CD8+ %1–day –3–30 µCiday +283.7 ± 5.82–day –2–30 µCiday +249.9 ± 29.63–day –1–30 µCiday +255.0 ± 46.24–day –3–40 µCiday +264.5 ± 41.65d –6 to –3day –3–40 µCiday +260.0 ± 13.96–––30 µCiday +215.9 ± 7.17–day –3––day +24.4 ± 0.68––1000 cGy–day +260.0 ± 4.6 DisclosuresNo relevant conflicts of interest to declare.

Haploidentical bone marrow cell reconstruction of tumor-bearing murine host

Objective To investigate the roles of chimerism level after haploidentical bone marrow cell transplantation on graft versus tumor effect (GVT). Methods Thirty BALB/C mice were randomly divided into 3 groups: control group (group C, 10), mixed chimerism group (group A, 10), full donor chimerism group (group B, 10). Five BALB/C mice in group C were only inoculated with Renca cells (2.6 × 106). Ten mice in group A were conditioned with 4 Gy irradiation, followed by infusion by bone marrow cells of CB6F1 mice on the day 1, then inoculated with Renca cells (2. 6 × 106 ) on the day 8. Ten mice in group B were conditioned with 8 Gy irradiation, followed by infusion by bone marrow cells (5 ×106 ) of CB6F1 mice on the day 1, then inoculated with Renca cells ( 2. 6 × 106 ) on the day 8. Tumor growth was monitored every 4 day. In groups A and B, all mice were analyzed by FACScan cytometry on the day 14, 21 and 28 after BMT. Mice were killed at the 32nd day after inoculation with tumor cells and blood of all mice was collected. All tumors were taken out to be weighed. Results The results of chimera showed that engraftments in group A remained mixed donor chimerism and unstable, tended to decrease after 14 days. In group B, full donor chimerism developed, and the chimerism of engraftments remained above 90% and was preserved even after 28 days; The size and tumor growth rate in groups B were reduced as compared with groups A and C (P ＜0. 01 ). The tumor inhibitory rate in groups B was 52%. Conclusion Stable full donor chimerism status after haploidentical allogeneic bone marrow cell transplant is the basis of GVT. Key words: Bone marrow; Cell transplantation; Graft-versus-tumor effect; Chimerism

Suppression of tumor growth in renal cancer treatment with tumor vaccination after haploidentical bone marrow cell reconstitution

To investigate whether whole tumor cell vaccination strategies in combination with bone marrow transplantation (BMT) can stimulate graft-versus-tumor effect (GVT). Twenty-six BALB/c mice were randomly divided into 3 groups: BMT group (group A, n = 10), BMT + vaccination group (group B, n = 10), control group (group C, n = 6). (BALB/c × C57BL/6) F1 mice [CB6F1, H-2K(b/d)] were used as donors. BALB/c mice of group C were only inoculated with Renca cell (2.6 × 10(6)). Mice of group A and B were conditioned with 8 Gy irradiation, followed by infusion by bone marrow cell of CB6F1 mice on day 1, then inoculated with Renca cell (2.6 × 10(6)) on day 8. All mice of group B were immunized subcutaneous on the back with 5 × 10(5) irradiated Renca tumor cells on day 9 and day 16. All mice of group C were inoculated with Renca cell (2.6 × 10(6)) on day 8. In group A and B, all mice were analyzed by fluorescence activated cell sorter (FACS) on day 14, and 28 day after BMT. Mice were killed on day 32 after inoculation with tumor cell and collected blood sample. All tumors were taken out to be weighed and then fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 µm slices. The slices were stained with HE and examined by TdT mediated-dUTP nick end labeling (TUNEL). Liver, skin, intestine, and spleen were biopsied for histopathological examination. The results of chimera showed that engraftments of group A, B were full donor chimerism, and the chimerism of those remained above 90% and preserved even after 28 days. The tumor weight, tumor volume increment in the group B was lower than group A and C (P < 0.05). The tumor suppressing rates of the group A and B were 54%, 60% respectively. The area ratio of tumor necrosis and apoptosis index (AI) of the tumor in the group B were higher than group A and C (P < 0.05). Graft-versus-host disease was not observed in each group. The mechanism of GVT after haploidentical allogeneic bone marrow transplantation with tumor vaccination may be the promotion of tumor necrosis and apoptosis.

UV-B modulation of haplo-identical bone marrow cells in the prevention of GVHD and induction of specific transplantation tolerance to intestinal allografts

Ultraviolet B (UV-B) irradiation of BM cells (BMC) prior to transplantation into lethally gamma irradiated allogeneic rats prevents graft-versus-host disease (GVHD), induces stable allogeneic chimerism and specific tolerance to donor-type allografts. This study evaluated the role of UV-B modulation of bone marrow transplant (BMT) in the prevention of GVHD in the parent (P)-to-F1 hybrid rat combination of Lewis-to-Lewis × Brown Norway F1 (LBNF1) and of subsequent tolerance to small bowel transplantation (SBT). Lethally gamma irradiated (10.5 Gy) LBNF1 recipients of unmodified Lewis BMT (admixture of 10 8 BMC and 5 × 10 6 spleen cells) developed lethal GVHD and died within 55 days. Similarly, groups of lethally irradiated LBNF1 recipients of Lewis BMT treated with 100–300 J/m 2 UV-B developed GVHD and died within 47 days. All F1 hybrid recipients of Lewis BMT treated with 700 or 900 J/m 2 of UV-B permanently accepted their BM grafts, gained weight, showed no clinical evidence of GVHD and survived for > 300 days. These stable chimeras expressed 94–98% donor T-cells in their lymph nodes and became tolerant to BM donor-type (Lewis) SBT when grafted 180 days after BMT. In contrast, similarly prepared F1 recipients rejected BN SBT, thus demonstrating donor specificity. The chimeric rats were specifically unresponsive to donor (Lewis) Ag in MLR while they maintained normal alloreactivity to BN stimulators. These results suggest that UV-B irradiation of BMT offers a promising approach to overcoming the limitations of T-cell depletion of BMT and may offer a useful approach for recipient conditioning for induction of transplantation tolerance.

Immunologic reconstitution of foals with combined immunodeficiency

Thirty eight foals with combined immunodeficiency (CID) received transplanted fetal liver cells, fetal liver and thymus cells, histocompatible bone marrow cells, or equine lymphocyte antigen (ELA) haploidentical bone marrow cells in an attempt to reconstitute their deficient immune systems. Engraftment was infrequent, partial, and unpredictable when fetal cells were employed. Three of five CID foals receiving ELA haploidentical bone marrow cells demonstrated partial reconstitution, but engraftment was only temporary. Administration of histocompatible bone marrow cells resulted in rapid, full and sustained engraftment.

Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation.

Recent advances in the prevention of graft-vs-host disease (GVHD) have allowed the use of haploidentical bone marrow cells for correction of lethal genetic defects of the immune system. Sequential analyses of blood lymphocyte phenotypes and functions were done before and after transplantation of haploidentical marrow stem cells into 17 infants with severe primary T cell deficiencies. The marrow was depleted of post-thymic T cells and most other mature marrow cells by soy lectin agglutination and sheep erythrocyte rosetting. The studies were performed to define the time course and extent of appearance of immune function, and to identify factors leading to resistance to engraftment. No pretransplant immunosuppression was used. T cell function was detected between 34 and 287 days after transplantation, but a sharp rise usually occurred between 84 and 115 days, and normal function was reached between 113 and 210 days. Fifteen of the patients are alive from 6 to 41 mo post-transplantation, 12 have improved or have normal T lymphocyte function, and nine have proven T cell chimerism. Increased immunoglobulins of several isotypes have been noted in 11 patients and specific antibodies in seven patients, although B cell chimerism has been detected in only one patient. B cell function required 2 to 2.5 yr for normalization. No GVHD occurred in 14 patients, and the other three had only transient mild skin rashes. Two patients died of viral infections. Failure to engraft was correlated with some pre-transplant lymphocyte responses to mitogens and allogeneic cells (three cases), but not with the presence of pre-transplant natural killer cell function (five cases) nor with the presence of purine salvage pathway enzyme deficiencies (four cases). The latter, however, was associated with poor lymphoid function in two patients. These studies indicate that the thymic microenvironment of most infants with severe combined immunodeficiency disease is capable of differentiating donor stem cells to mature and functioning T lymphocytes which can cooperate with apparently normal host B cells for antibody production.

339 Appearance of multiple paraproteins during early engraftment of soy lectin T cell depleted haploidentical bone marrow cells in severe combined immunodeficiency (SCID)

Recent advances in the prevention of graft-versus-host disease through postthymic T-cell depletion have allowed the use of haploidentical bone marrow cells for immunologic reconstitution of severe combined immunodeficiency disease. We report a male infant with severe combined immunodeficiency (with normal adenosine deaminase) who developed two IgG kappa and one IgA lambda paraproteins 7 weeks following the administration of 1.4×109 maternal bone marrow cells depleted of postthymic T cells by soy lectin agglutination and sheep erythrocyte rosetting. Serum IgG rose from 128 to 820 mg/dl, and IgA from 0 to 2400 mg/dl, peaking at 10 weeks postgrafting. By 14 weeks posttransplantation T-cell numbers and function had risen to normal (all dividing T cells had the donor karyotype) and paraprotein concentrations began to decline. These observations strongly suggest that the later-appearing T cells regulated the B-cell clones from which the paraproteins were derived. Failure of such function to appear could account for the increased incidence of B-cell lymphomas in severe combined immunodeficiency.