Visualization of immune response kinetics in full allogeneic chimeras.

BACKGROUND: Donor Lymphocyte Infusion (DLI) is a well-recognized tool for augmentation of the anti-leukemia effect after mismatched bone marrow transplantation. Experimental results show, however, that DLI efficacy is strongly dependent on the number of donor hematopoietic cells persisting in recipient after transplantation. It is strong in mixed chimeras and relatively weak in full chimeras (FC) that replace host antigen-presenting cells by donor antigen-presenting cells. In this study we applied a new in vivo cytotoxicity monitoring method for evaluation of the changes in FC anti-host immunity after co-transplantation of donor and host hematopoietic cells together. METHOD: Full hematopoietic chimeras and naïve control mice were transplanted with a mixture of equivalent numbers of donor and recipient or donor and third party splenocytes labeled by a cell-permeable fluorescent dye CFDA-SE. The animals were sacrificed at various time points, and their splenocyte suspensions were prepared, depleted of red blood cells, stained with allophycocyanin-labeled anti-H2(b) antibodies, and analyzed using fluorescence-activated cell sorting. The immune response was assessed according to the percentage of single positive CFDA-SE(+)/ H2(b-) cells of all CFDA-SE(+) cells. RESULTS: FC grafted with splenocytes from similar FC mixed with splenocytes from naïve host-type or third-party-type mice rejected host cells within 14 days, and third-party cells within 7 days. NK cell depletion in vivo had no influence on host cell rejection kinetics. Co-infusion of host-type splenocytes with splenocytes obtained from naïve donor-type mice resulted in significant acceleration of host cell rejection (10 days). Naïve mice rejected the same amount of allogeneic lymphocytes within 3 days. CONCLUSIONS: Proposed method provides a simple and sensitive tool to evaluate in vivo post-transplant cytotoxicity in different experimental settings. The method demonstrates that FC is specifically deficient in their ability to reject host lymphocytes even when antigen-presenting host cells are provided. DLI improve anti-host immune response in FC but can not restore it to the level observed in naïve donor-type mice.

Prevention of acute graft-vs-host disease by a single low-dose cyclophosphamide injection following allogeneic bone marrow transplantation.

OBJECTIVE: Previously, we documented that conditioning based on donor-specific cell transfusion (DST) and subsequent selective depletion of activated donor-reactive cells by cyclophosphamide (CY) facilitates alloengraftment in a murine transplantation model. Transplantation event represents a strong immunogenic stimulus for host-reactive donor T cells that induce graft-vs-host disease (GVHD). Therefore, in this study, we addressed the question of whether a single posttransplantation CY administration (CY2) can prevent acute GVHD-related mortality without compromising engraftment of allogeneic transplant. MATERIALS AND METHODS: Splenocyte-enriched C57BL/6 bone marrow was transplanted to BALB/c recipients after mild irradiation, and conditioning with DST and 100 mg/kg CY. Following transplantation, recipients were left untreated or given on a specified day a single CY2 injection (50 mg/kg). All animals were monitored for survival, chimerism, and clinical signs of GVHD. Experimental mice that received BCL1 leukemia cells before transplantation were monitored for leukemia-related mortality as well. RESULTS: Animals that received no CY2 after transplantation died of acute GVHD. A single low-dose CY2 treatment within the first 5 days after transplantation prevented mortality in most recipients. However, only CY2 administration on days +1 or +5 preserved chimerism. Most chimeras survived GVHD-free for >200 days. Prolonged persistence of host-reactive T cells in mice (CY2 on day +5) permitted a reduction to be made in engraftment-essential irradiation dose and preserved a strong graft-vs-leukemia effect of transplantation. CONCLUSION: Acute GVHD can be prevented in mice by a single properly timed posttransplantation low-dose CY administration.

Mesenchymal stromal cells lose their immunosuppressive potential after allotransplantation.

OBJECTIVE: The stem cell fraction of mesenchymal stromal cells (MSCs) is capable of self-renewal and under inductive conditions differentiates into bone, cartilage, hematopoietic stroma, and other mesenchymal tissues. Therefore, MSCs represent a promising source for hard tissue repair therapies. MSCs are also immunosuppressive and prevent activation of allogeneic lymphocytes in vitro. Thus it has been suggested that they might be able to engraft in allogeneic recipients and downregulate recipients' immunity. In this study we examined whether MSCs retain their immunomodulating properties in vivo after allotransplantation. MATERIALS AND METHODS: MSCs were propagated from bone marrow (BM), placenta, or umbilical cord tissues. Using a murine parental-into-F1 model of graft-vs-host disease (GVHD) we tried to control GVHD by intravenous transplanting parental or recipient MSCs together with parental lymphocytes (day 0) and on days +7 and +14. MSCs' immunosuppressive potential in vivo was also examined by comparing their ability to construct ectopic bone after local transplantation with osteogenic inductor (demineralized bone matrix) under the kidney capsule of syngeneic and allogeneic recipients. RESULTS: Repeated IV MSC injections failed to reduce GVHD-related recipient mortality. Local implantation of MSCs propagated from BM, placenta or umbilical cord resulted in ectopic bone formation in syngeneic recipients and in transplant rejection by allogeneic mice. CONCLUSION: MSCs lose their immunosuppressive potential in mismatched setting.

Osteoporosis of hematologic etiology.

OBJECTIVE: Here we present evidence that overexertion of the hematopoietic system following chronic bleeding plays an important role in the etiology of osteoporosis. MATERIALS AND METHODS: C57BL/6 mice were exposed to chronic bloodletting (0.2 mL twice per month for 10 months), total body irradiation (900 cGy), or aging (20-30 months old). Bone marrow from standard untreated donors was transplanted under the kidney capsules of all three categories of recipients to investigate the influence of each of these conditions on new bone marrow formation. Cellularity and histologic structure of developed osteohematopoietic sites and histomorphometry of lumbar vertebrae were studied, thus assessing the role of bleeding, irradiation, and old age on new bone formation and effects on existing bone. RESULTS: Chronic blood loss led to augmented production of hematopoietic microenvironment, relative reduction in the amount of generated bone, and activation of the bone resorptive process in the newly forming osteohematopoietic complex. Similar results were seen in irradiated and senescent mice. Activity, stimulating expansion of hematopoietic microenvironment, was revealed in the plasma of all three categories of experimental mice. Likewise, quantification of the relative amount of bone and hematopoietic areas in skeletal sites showed a significant reduction in bone tissue of the first lumbar vertebrae of chronically bled mice. CONCLUSIONS: Our experimental data, together with existing clinical observations documenting the role of hematopoietic insufficiency in the development of osteoporosis, confirm our working hypothesis that chronic blood loss may be the primary factor responsible for the rapid and consistent development of postmenopausal osteoporosis.

Depletion of alloantigen-primed lymphocytes overcomes resistance to allogeneic bone marrow in mildly conditioned recipients.

OBJECTIVE: Successful implantation of allogeneic bone marrow (BM) cells after nonmyeloablative conditioning would allow to compensate for the inadequate supply of compatible grafts and to reduce mortality of graft-vs.-host disease (GVHD). Recently, we proposed to facilitate engraftment of mismatched BM by conditioning for alloantigen-primed lymphocyte depletion (APLD) with cyclophosphamide (CY). Here we summarize the experimental results obtained by this approach. MATERIALS AND METHODS: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY (day 1) to deplete alloantigen-primed lymphocytes, and given a second C57BL/6 BM transplant (day 2) for engraftment. Recipients were repeatedly tested for chimerism in the blood and followed for GVHD and survival. The protocol was also tested for inducing tolerance to donor tissue and organ allografts, and for treatment of leukemia, breast cancer, and autoimmune diabetes in NOD mice. RESULTS: APLD by 200 mg/kg CY provided engraftment of allogeneic BM from the same donor in 100% mildly irradiated recipients. Eighty percent chimeras remained GVHD-free more 200 days. All chimeras accepted permanently donor skin grafts and donor hematopoietic stromal progenitors. Allogeneic BM transplantation (BMT) after APLD had a strong therapeutic potential in BALB/c mice harboring malignant cells and in autoimmune NOD recipients. Tolerance-inducing CY dose could be reduced to 100 mg/kg. Conditioning for APLD resulted in engraftment of allogeneic BM after a significantly lower radiation dose than treatment with radiation and CY alone. CONCLUSION: Our results demonstrate that conditioning for APLD has a definite advantage over general immunosuppression with CY and radiation therapy.

Depletion of donor-reactive cells as a new concept for improvement of mismatched bone marrow engraftment using reduced-intensity conditioning.

OBJECTIVE: New nonmyeloablative strategies to improve acceptance of mismatched bone marrow (BM) may compensate for the inadequate supply of compatible grafts. Recently we proposed to facilitate engraftment of mismatched BM by selective depletion of activated donor-reactive host cells with cyclophosphamide (CY). Here we have compared engraftment of allogeneic BM after depletion of antigen-activated host lymphocytes by CY, with BM engraftment following general immunosuppression by the same CY dose. MATERIALS AND METHODS: Naive or mildly irradiated BALB/c mice were primed with C57BL/6 BM cells (day 0), treated with CY in order to deplete activated T cells (day 1), and transplanted with a second C57BL/6 BM inoculum (day 2) in order to achieve BM engraftment. Alternatively, mice received an equal dose of donor BM cells in a single injection one day after the same CY dose. Treated animals were repeatedly tested for persistence of donor cells in the blood. RESULTS: Depletion of alloantigen-primed lymphocytes by 200 mg/kg CY provided stable GVHD-free engraftment of allogeneic BM in nonirradiated mice, while immunosuppressive treatment with the same CY dose alone resulted in BM rejection. Low-dose irradiation before priming with donor BM allowed the tolerance-inducing CY dose to be reduced to 100 mg/kg. Alloantigen-primed lymphocyte depletion (APLD) by a reduced CY dose resulted in engraftment of donor BM after a significantly lower irradiation dose than treatment with irradiation and CY alone. CONCLUSION: Our results demonstrate that conditioning that focuses on APLD has a definite advantage over general immunosuppression with CY and radiation therapy.