Robert Sackstein

Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia

The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.

Mixed lymphohaemopoietic chimerism and graft-ver suslymphoma effects after non-myeloablative therapy and HLA-mismatched bone-marrow transplantation

BACKGROUND HLA-mismatched donor bone-marrow transplantation after standard myeloablative conditioning therapy for haematological malignant disorders has been limited by severe graft-versus-host disease (GVHD) and graft failure. We tested a new approach to find out whether lymphohaemopoietic graft-versus-host reactions could occur without excessive GVHD in mixed haemopoietic chimeras produced across HLA barriers with non-myeloablative conditioning. METHODS Five patients with refractory non-Hodgkin lymphoma underwent bone-marrow transplantation from haploidentical related donors sharing at least one HLA A, B, or DR allele on the mismatched haplotype. Conditioning included cyclophosphamide and thymic irradiation before transplantation, and antithymocyte globulin before and after transplantation. The only other GVHD prophylaxis was cyclosporin. FINDINGS Four of five patients were evaluable and showed engraftment. Mixed haemopoietic chimerism was established, with a predominance of donor lymphoid tissue and varying degrees of myeloid chimerism. Two patients were in GVHD-free states of complete and partial clinical remission at 460 and 103 days after bone-marrow transplantation. INTERPRETATION Mixed chimerism can be induced in adult recipients of HLA-mismatched bone-marrow transplantation by a non-myeloablative conditioning regimen. The antilymphoma responses seen in two patients suggest that allogeneic bone-marrow transplantation without myeloablative conditioning might have potent immunotherapeutic benefits.

Combined histocompatibility leukocyte antigen-matched donor bone marrow and renal transplantation for multiple myeloma with end stage renal disease : The induction of allograft tolerance through mixed lymphohematopoietic chimerism

BACKGROUND Experimental and clinical evidence has demonstrated that the establishment of allogeneic chimerism after bone marrow transplantation may provide donor-specific tolerance for solid organ allografts. METHODS Based on the preliminary results of a clinical trial using nonmyeloablative preparative therapy for the induction of mixed lymphohematopoietic chimerism, we treated a 55-year-old woman with end stage renal disease secondary to multiple myeloma with a combined histocompatibility leukocyte antigen-matched bone marrow and renal transplant after conditioning with cyclophosphamide, antithymocyte globulin, and thymic irradiation. RESULTS The posttransplant course was notable for early normalization of renal function, the absence of acute graft-versus-host disease, and the establishment of mixed lymphohematopoietic chimerism. Cyclosporine, which was the only posttransplant immunosuppressive therapy, was tapered and discontinued on day +73 posttransplant. No rejection episodes occurred, and renal function remains normal on day + 170 posttransplant (14 weeks after discontinuing cyclosporine). Although there is presently no evidence of donor hematopoiesis, there is evidence of an ongoing antitumor response with a recent staging evaluation showing no measurable urine kappa light chains. The patient remains clinically well and is off all immunosuppressive therapy. CONCLUSION This is the first report of the deliberate induction of mixed lymphohematopoietic chimerism after a nonmyeloablative preparative regimen to treat a hematological malignancy and to provide allotolerance for a solid organ transplant.

Intentional induction of mixed chimerism and achievement of antitumor responses after nonmyeloablative conditioning therapy and HLA-matched donor bone marrow transplantation for refractory hematologic malignancies.

Mixed lymphohematopoietic chimerism can be induced in mice with bone marrow transplantation (BMT) after a nonmyeloablative preparative regimen that includes cyclophosphamide, anti-T-cell antibody therapy, and thymic irradiation. These mixed chimeras are resistant to the induction of graft-versus-host disease (GVHD) after delayed donor leukocyte infusions (DLIs), despite a potent lymphohematopoietic graft-versus-host reaction that converts the mixed chimeric state to a full donor one. Based on this animal model, we initiated a trial of nonmyeloablative therapy with HLA-matched or -mismatched donor BMT and DLI for refractory hematologic malignancies. Twenty-one of 36 patients enrolled in this trial received a genotypically (n = 20) or phenotypically (n = 1) HLA-matched donor transplant; results reported here are for those patients only. Preparative therapy consisted of cyclophosphamide in doses of 150 to 200 mg/kg; peritransplant antithymocyte globulin; thymic irradiation (in patients who had not received previous mediastinal radiation therapy); and cyclosporine. Eighteen of 20 evaluable patients developed persistent mixed lymphohematopoietic chimerism as defined by >1% donor peripheral white blood cells until at least day 35 posttransplantation. Ten patients received prophylactic DLI beginning 5 to 6 weeks after BMT for conversion of mixed chimerism to full donor hematopoiesis and to optimize a graft-versus-leukemia effect. Fourteen of 20 evaluable patients (70%) achieved an antitumor response; 8 of these responses were complete, and 6 were partial. Of the 8 evaluable patients who received prophylactic DLI, 6 showed conversion to full donor chimerism. Five of the 9 evaluable patients (56%) who received prophylactic DLI achieved a complete response, compared with 3 of 11 patients (27%) who did not receive prophylactic DLI. Currently 11 patients are alive, and 7 of these are free of disease progression at a median follow-up time of 445 days (range, 105-548 days) posttransplantation. Transplantation-related complications included cyclophosphamide-induced cardiac toxicity in 3 of 21 patients (14%) and grade II or greater GVHD in 6 patients (29%). One patient (5%) died from a complication of BMT, and 1 patient (5%) died from GVHD after 2 prophylactic DLIs were given for conversion of chimerism. In summary, mixed lymphohematopoietic chimerism was reproducibly induced after a novel nonmyeloablative preparative regimen incorporating chemotherapy, peritransplant antithymocyte globulin, and thymic irradiation, allowing for early administration of DLI in 10 of 21 patients. After treatment, striking antitumor responses were observed in the majority of patients with chemotherapy-refractory hematologic malignancies.

High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response

BACKGROUND In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen. METHODS Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2). RESULTS In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens. CONCLUSIONS We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Coagulation and thrombotic disorders associated with pig organ and hematopoietic cell transplantation in nonhuman primates.

Background. Efforts to achieve tolerance to transplanted pig organs in nonhuman primates by the induction of a state of mixed hematopoietic chimerism have been associated with disorders of coagulation and thrombosis. Activation of recipient vascular endothelium and platelets by porcine hematopoietic cells and/or activation of donor organ vascular endothelium and/or molecular differences between the species may play roles. Irradiation or drug therapy could possibly potentiate endothelial cell activation and/or injury. Methods. We have investigated parameters of coagulation and platelet activation in nonhuman primates after (1) a regimen aimed at inducing mixed hematopoietic chimerism and tolerance (TIR that included total body irradiation, T cell depletion, and splenectomy; (2) pig bone marrow or pig peripheral blood mobilized progenitor cell transplantation (PCTx); and/or (3) pig organ transplantation (POTx). Five experimental groups were studied. Baboons were the recipient subjects in all groups except Group 1. Gp 1 Cynomolgus monkeys (n=6) underwent TIR + allotransplantation of hematopoietic cells and a kidney or heart or TIR + concordant xenotransplantation (using baboons as donors) of cells and a kidney; Gp 2 Baboons (n=4) underwent TIR with or without (±) autologous hematopoietic cell infusion; Gp 3 (n=12) PCTx±TIR; Gp 4 (n=5) POTx±TIR; Gp 5 (n=4) TIR + PCTx + POTx. Platelet counts, with plasma prothrombin time, partial thromboplastin time, fibrinogen levels, fibrin split products and/or D-dimer were measured. Results. In the absence of a discordant (porcine) cellular or organ transplant (Groups 1 and 2), TIR resulted in transient thrombocytopenia only, in keeping with bone marrow depression from irradiation. PCTx alone (Group 3) was associated with the rapid development of a thrombotic thrombocytopenic (TTP)-like microangiopathic state, that persisted longer when PCTx was combined with TIR. POTx (±TIR) (Group 4) was associated with a gradual fall (over several days) in platelet counts and fibrinogen with disseminated intravascular coagulation (DIC); after graft excision, the DIC generally resolved. When TIR, PCTx and POTx were combined (Group 5), an initial TTP-like state was superseded by a consumptive picture of DIC within the first week, necessitating graft removal. Conclusions. Both PCTx and POTx lead to profound alterations in hemostasis and coagulation parameters that must be overcome if discordant xenotransplantation of hematopoietic cells and organs is to be fully successful. Disordered thromboregulation could exacerbate vascular damage and potentiate activation of coagulation pathways after exposure to xenogeneic cells or a vascularized xenograft.

Impact of prophylactic donor leukocyte infusions on mixed chimerism, graft-versus-host disease, and antitumor response in patients with advanced hematologic malignancies treated with nonmyeloablative conditioning and allogeneic bone marrow transplantation

In an attempt to capture graft-versus-tumor effects without graft-versus-host disease (GVHD), the authors initiated a trial of nonmyeloablative allogeneic bone marrow transplantation (BMT) in patients with advanced hematologic malignancies, with the majority of patients having chemotherapy-refractory disease. Forty-two patients received an HLA-matched related donor BMT after a cyclophosphamide and antithymocyte globulin-based conditioning that also included thymic irradiation for patients who had not received prior mediastinal radiotherapy. Prophylactic donor leukocyte infusion (pDLI) at a dose of 1 x 10(7) CD3(+) cells per kilogram were given beginning 5 weeks post-BMT to 16 patients with mixed chimerism (MC) but without GVHD, whereas 26 patients did not receive pDLI, either because of GVHD or early relapse. Twelve of 16 patients (75%) receiving pDLI had T cell chimerism at the time of pDLI >/=40%. These patients, by day 100 post-BMT, either converted to full donor chimerism (FDC) (n = 10) or had an increase in or stable donor chimerism (n = 2) after pDLI. Four of 4 patients whose T cell chimerism was </=20% at the time of pDLI, lost the graft. In contrast, only 5 of 18 evaluable patients (28%) not receiving a pDLI converted to FDC by day 100 post-BMT, 7 maintained MC, and 10 of an evaluable 22 lost the graft. Patients who had undergone a previous autologous stem cell transplant had a higher rate of conversion to FDC (69% v 31%) and higher incidence of GVHD (69% v 34%) compared with those who did not have a previous autologous SCT. Eleven of 16 patients (69%) who received a pDLI achieved a remission with 50% 1-year progression-free survival rate and 44% 3-year overall survival rate. Nineteen of 42 patients (45%) had >/=grade II acute GVHD, including 12 after BMT and 7 after pDLI. Approximately one third of patients, after having initial MC, eventually lost their donor graft. The authors conclude that (1) pDLI has the potential to convert MC to FDC; (2) sustained remissions can be achieved in patients with chemorefractory hematologic malignancies who receive a pDLI, albeit with a significant risk of acute GVHD; and (3) the degree of donor T cell chimerism at the time of pDLI is predictive of the fate of MC, ie, donor T cell chimerism >/=40% or </=20% at the time of pDLI correlates with conversion of MC or loss of the graft, respectively.

A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, ∼50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-γ and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d+ lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.

The lymphocyte homing receptors: gatekeepers of the multistep paradigm.

Purpose of reviewLymphocyte trafficking is regulated by adhesion molecules mediating the initial shear-resistant binding of circulating cells to target tissue endothelium (Step 1). This review focuses on the current and emerging perspectives of the biology of these ‘homing receptors.’ Recent findingsThe conventional multistep paradigm holds that leukocyte migration represents a cascade of events, initiated by tethering and rolling interactions of leukocytes on the endothelial surface (Step 1). These interactions are indispensable, required to dampen velocities sufficiently to allow cells to sense the local chemokines and/or other inflammatory signals resulting in activation of integrin adhesiveness (Step 2), with ensuing firm adherence on the vessel wall (Step 3) followed by endothelial transmigration (Step 4). Mechanistic studies now suggest that some effectors of Step 1 interactions themselves activate integrin adhesiveness and trigger transmigration - in some cases by forming complexes with integrins - thus bypassing the need for chemokine signaling. These findings force a reconsideration of the multistep paradigm, and shift focus now to identifying all relevant effectors of Step 1 interactions using adherence assays performed under shear stress to mimic the dynamic conditions of blood flow. SummaryRecent findings suggest that homing receptors are not merely molecular brakes. The cross-talk among the homing receptors and integrins opens a new ‘avenue’ to lymphocyte migration, suggesting that homing receptors may be sufficient alone, in some cases, to perform the function the name implies.

CD44 on LS174T colon carcinoma cells possesses E-selectin ligand activity

Metastasis of circulating tumor cells requires a multistep cascade of events initiated by adhesion of tumor cells to the vascular endothelium of involved tissues. This process occurs under the forces of blood flow and is promoted by adhesion molecules specialized to interact under shear conditions. The endothelial molecule E-selectin is a major mediator of these adhesive events, and there is strong evidence that E-selectin receptor-ligand interactions contribute to the formation of metastasis. However, little is known about the identity of E-selectin ligand(s) expressed on cancer cells. To address this issue, we did SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, and blot rolling assays of LS174T, a colon carcinoma cell line known to interact with E-selectin under physiologic flow conditions. Our studies show that LS174T cells express the hematopoietic cell E/L-selectin (HCELL) glycoform of CD44, which functions as a high-affinity E-selectin glycoprotein ligand on these cells. However, in contrast to the HCELL glycoform on human hematopoietic progenitor cells, which expresses carbohydrate-binding determinant(s) for E-selectin primarily on N-glycans of standard CD44, the relevant determinant(s) on LS174T cells is expressed on O-glycans and is predominantly found on variant isoforms of CD44 (CD44v). Our finding that tumor-associated CD44 splice variant(s) express E-selectin ligand activity provides novel perspectives on the biology of CD44 in cancer metastasis.