Rapid, B lymphoid-restricted engraftment mediated by a primitive bone marrow subpopulation

Utilizing multiparameter flow cytometry, we have defined a subset of bone marrow cells containing lymphoid-restricted differentiation potential after i.v. transplantation. Bone marrow cells characterized by expression of the Sca-1 and c-kit Ags and lacking Ags of differentiating lineages were segregated into subsets based on allele-specific Thy-1.1 Ag expression. Although hematopoietic stem cells were recovered in the Thy-1.1low subset as previously described, the Thy-1.1neg subset consisted of progenitor cells that preferentially reconstituted the B lymphocyte lineage after i.v. transplantation. Recipients of Thy-1.1neg cells did not survive beyond 30 days, presumably due to the failure of erythroid and platelet lineages to recover after transplants. Thy-1.1neg cells predominantly reconstituted the bone marrow and peripheral blood of lethally irradiated recipients with B lineage cells within 2 weeks, although a low frequency of myeloid lineage cells was also detected. In contrast, myeloid progenitors outnumbered lymphoid progenitors when the Thy-1.1neg population was assayed in culture. When Thy-1. 1low stem cells were rigorously excluded from the Thy-1.1neg subset, reconstitution of T lymphocytes was rarely observed in peripheral blood after i.v. transplantation. Competitive repopulation studies showed that the B lymphoid reconstitution derived from Thy-1.1neg cells was not sustained over a 20-wk period. Therefore, the Thy-1. 1neg population defined in these studies includes transplantable, non-self-renewing B lymphocyte progenitor cells.     

Role of bone marrow stroma in the phenomenon of hybrid resistance]

In the site of hemopoiesis produced by implantation of an irradiated bone marrow plug beneath the kidney capsule all dividing hemopoietic cells were of recipient origin, as shown by karyologic analysis. In such implants hybrid resistance was of the donor's type. It is suggested that the migrating cells of hemopoietic origin, including lymphocytes and macrophages, do not participate in producing the hybrid resistance.     

Strategic nonmyeloablative conditioning: CD154:CD40 costimulatory blockade at primary bone marrow transplantation promotes engraftment for secondary bone marrow transplantation after engraftment failure

There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 costimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T cell depletion inhibits generation of the humoral immune response after primary BMT, as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, because almost all CFSE-labeled donor cells were killed at 0.5 and 3 h in sensitized recipients in in vivo cytotoxicity assay, reflecting Ab-mediated cytotoxicity. CD154:CD40 costimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.     

Abrogation of resistance to the reinduction of experimental allergic encephalomyelitis by pertussigen

Experimental allergic encephalomyelitis is an autoimmune disease initiated by an injection of myelin basic protein in complete Freund's adjuvant. Lewis rats which have recovered from the initial episode of hindquarter paralysis are resistant for at least 6 months to disease reinduction by basic protein-complete Freund's adjuvant, although specific antigen-reactive cells are detectable in convalescent rats. Resistance cannot be attributed to the activity of the adjuvant alone. In contrast, clinical disease could be reinduced by a secondary challenge with spinal cord homogenate and pertussigen (“lymphocytosis promoting factor” of Bordetella pertussis). Disease could also be reinduced by a simultaneous secondary challenge with basic protein-complete Freund's adjuvant along with pertussigen. Vascular permeability increases in the spinal cord paralleled disease induction or reinduction. No definite conclusions can be drawn concerning the mechanism by which pertussigen promotes disease reinduction in convalescent rats.     

CD8(+), alphabeta-TCR(+), and gammadelta-TCR(+) cells in the recipient hematopoietic environment mediate resistance to engraftment of allogeneic donor bone marrow

Historically, conditioning for engraftment of hematopoietic stem cells has been nonspecific. In the present study, we characterized which cells in the recipient hematopoietic microenvironment prevent allogeneic marrow engraftment. Mice defective in production of alphabeta-TCR(+), gammadelta-TCR(+), alphabeta- plus gammadelta-TCR(+), CD8(+), or CD4(+) cells were transplanted with MHC-disparate allogeneic bone marrow. Conditioning with 500 cGy total body irradiation (TBI) plus a single dose of cyclophosphamide (CyP) on day +2 establishes chimerism in normal recipients. When mice were conditioned with 300 cGy TBI plus a single dose of CyP on day +2, all engrafted, except wild-type controls and those defective in production of CD4(+) T cells. Mice lacking both alphabeta- and gammadelta-TCR(+) cells engrafted without conditioning, suggesting that both alphabeta- and gammadelta-TCR T cells in the host play critical and nonredundant roles in preventing engraftment of allogeneic bone marrow. CD8 knockout (KO) mice engrafted without TBI, but only if they received CyP on day +2 relative to the marrow infusion, showing that a CD8(-) cell was targeted by the CyP conditioning. The CD8(+) cell effector function is mechanistically different from that for conventional T cells, and independent of CD4(+) T helper cells because CD4 KO mice require substantially higher levels of conditioning than the other KO phenotypes. These results suggest that a number of cell populations with different mechanisms of action mediate resistance to engraftment of allogeneic marrow. Targeting of specific recipient cellular populations may permit conditioning approaches to allow mixed chimerism with minimal morbidity and could potentially avoid the requirement for myelotoxic agents altogether.     

Hybrid resistance to precursor cells of bone marrow stroma]

A focusl of hemopoiesis appearing after the transplantation of a bone marrow fragment of C57BL mice to syngeneic mice (under the kidney capsule) contained more hemopoietic cells than in transplantation to the semisyngeneic (CBA X C57BL) FI recipient. Experiments were conducted with a secondary seeding by intravenous injection of hemopoietic cells of the C57BL transplant genotype into the transplant depopulated by irradiation; it was shown that these differences were caused by lesser dimensions of the hemopoietic microenvironment in the focus in the hybrid organism in comparison with such in the syngeneic system. Thus, the hybrid resistance was expressed not only to the hemopoietic cells, but also to the stromal precursors transferring the hemopoietic microenvironment.     

Natural antibodies can inhibit bone marrow engraftment in the rat----mouse species combination.

Specific tolerance can be induced in animals by transplanting hemopoietic cells across concordant species barriers. Despite the fact that the rat-mouse species combination is considered concordant, we have recently demonstrated that normal murine serum contains natural antibodies (nAb), predominantly of the IgM and IgG3 subclasses, with markedly greater binding to rat bone marrow cells (BMC) than to rat splenocytes or thymocytes. Since much greater numbers of rat BMC than of allogeneic murine BMC are required to achieve engraftment in mice, we considered the possibility that these nAbs might be responsible, and that the increased numbers of BMC might be required to absorb these nAb. To evaluate the effect of these nAb on engraftment of rat BMC in mice, we have now performed adoptive transfer studies using T and B cell-deficient severe combined immunodeficiency disease (SCID) mice as recipients. Administration of as few as 5 x 10(5) T cell-depleted rat BMC led to induction of stable xenochimerism in SCID mice conditioned with 4-Gy whole body irradiation. Rat T cells developed after a delay of several weeks, and conferred the ability to reject non-donor-type rat skin grafts, whereas donor-type grafts were accepted. Adoptive transfer of 4 ml of normal BALB/c serum led to a marked reduction in the level of rat chimerism in SCID recipients of 2 x 10(6) F344 BMC. The ability of sera to inhibit engraftment of rat BMC correlated with their cytotoxic nAb content, and the inhibitory effect of highly cytotoxic sera could be overcome by administration of large numbers of rat BMC. Thus, normal mouse serum has a limited ability to hinder engraftment of rat BMC, and this degree of resistance can be overcome by adsorption when large numbers of BMC are administered. Eliminating nAb from serum may be more difficult in discordant species combinations in recipients with functional B cells, but may likewise permit the use of BMT as a means of inducing transplantation tolerance.     

In vitro immune response by murine bone marrow cells stimulated against soluble immune complexes.

Synchronized nonadherent bone marrow lymphocytes were stimulated with soluble immune complexes, in antigen excess formed by C3H/HeJ antibodies and various noncross-reacting protein antigens, in a suspension culture which allowed longterm cultivation. On binding of these complexes, lymphocytes underwent blast transformation with mitosis and formation of plasma cells which secreted specific antibodies to the antigen; a cyclic sequence of lymphocytes, blasts, and plasma cells was observed until the majority of the cell population appeared to be plasma cells. The relative percentage of mature plasma cells then decreased leaving mostly small lymphoid cells among which evidence suggests the presence of memory cells. Complexes at equivalence stimulated for the first few days, whereas antibody excess caused stimulation only initially followed by inhibition of the response. Antibodies passively added to the cultures inhibited the proliferative reaction; free antigen induced a typical secondary-type response.     

Mechanism of hybrid resistance. The role of a natural antibody in parental bone marrow cell rejection

Hybrid resistance (HR) to parental bone marrow growth is specifically directed against hemopoietic histocompatibility (Hh-1) Ag that are present in parental bone marrow cells (bmc). The mechanism of HR seems to be a multistep process. According to a model we proposed earlier, a T cell recognizes the Hh-1 Ag and stimulates a macrophage to secrete IFN-alpha/beta (recognition phase). IFN-alpha/beta activates a NK-like cell that specifically kills the parental bmc (effector phase). We have also described in a previous paper that serum from resistant F1 hybrids contains a humoral factor that seems to be involved in the effector phase of HR. In the present work, we study the role and the nature of this humoral factor. Our results show that this humoral factor: 1) is present in all resistant H-2Db heterozygous F1 hybrids we have tested but not in nonresistant H-2Db homozygous mice; 2) seems to recognize the Hh-1b Ag because it is absorbed on bmc from Hh-1b mice but not on bmc from Hh-1d and Hh-1- mice; and 3) is an IgG1 Ig (natural antibody). These results could help us to explain the specificity of HR at the effector phase by supposing that this natural antibody recognize the Hh-1 Ag and enable NK-like cells to kill parental bmc cells in Hh-1 specific manner.     

Acquired immune haemolysis by anti A 1 antibody following bone marrow transplantation

In ABO mismatched organ or bone marrow transplants recently some cases of acquired immune hemolysis have been reported. It was felt that these life threatening complications were due to immunosuppressive treatment with cyclosporin-A. A case of severe hemolysis following mismatched BMT is reported. Here no cyclosporin-A treatment was given since the bone marrow was T-cell deprived by an E-rosetting technique. Apparently T-cell purging can under these conditions become dangerous.     

Successful bone marrow transplantation in a patient with DNA ligase IV deficiency and bone marrow failure

Cirrhotic cardiomyopathy is the term used to describe a constellation of features indicative of abnormal heart structure and function in patients with cirrhosis. These include systolic and diastolic dysfunction, electrophysiological changes, and macroscopic and microscopic structural changes. The prevalence of cirrhotic cardiomyopathy remains unknown at present, mostly because the disease is generally latent and shows itself when the patient is subjected to stress such as exercise, drugs, hemorrhage and surgery. The main clinical features of cirrhotic cardiomyopathy include baseline increased cardiac output, attenuated systolic contraction or diastolic relaxation in response to physiologic, pharmacologic and surgical stress, and electrical conductance abnormalities (prolonged QT interval). In the majority of cases, diastolic dysfunction precedes systolic dysfunction, which tends to manifest only under conditions of stress. Generally, cirrhotic cardiomyopathy with overt severe heart failure is rare. Major stresses on the cardiovascular system such as liver transplantation, infections and insertion of transjugular intrahepatic portosystemic stent-shunts (TIPS) can unmask the presence of cirrhotic cardiomyopathy and thereby convert latent to overt heart failure. Cirrhotic cardiomyopathy may also contribute to the pathogenesis of hepatorenal syndrome. Pathogenic mechanisms of cirrhotic cardiomyopathy are multiple and include abnormal membrane biophysical characteristics, impaired β-adrenergic receptor signal transduction and increased activity of negative-inotropic pathways mediated by cGMP. Diagnosis and differential diagnosis require a careful assessment of patient history probing for excessive alcohol, physical examination for signs of hypertension such as retinal vascular changes, and appropriate diagnostic tests such as exercise stress electrocardiography, nuclear heart scans and coronary angiography. Current management recommendations include empirical, nonspecific and mainly supportive measures. The exact prognosis remains unclear. The extent of cirrhotic cardiomyopathy generally correlates to the degree of liver insufficiency. Reversibility is possible (either pharmacological or after liver transplantation), but further studies are needed.     

Donor-type activated natural killer cells promote marrow engraftment and B cell development during allogeneic bone marrow transplantation.

Purified NK cells were obtained from mice with severe combined immune deficiency and were activated with human IL-2 (hrIL-2) in vitro to determine if, once activated, these cells could be transferred with compatible bone marrow cells (BMC) and promote marrow engraftment in irradiated allogeneic recipients. After culture with hrIL-2, these cells maintained a phenotypic and lytic spectrum consistent with a pure population of activated NK cells. These activated NK cells were then adoptively transferred with the donor BMC and rhIL-2 into lethally irradiated allogeneic hosts. The addition of NK cells with the BMC allowed for more rapid hematopoietic engraftment as determined through short term studies, and greater donor-derived chimerism with accelerated reconstitution of the B cell population as determined with long term analysis. No evidence of graft-vs-host disease was detected in the recipients receiving the activated NK cells with allogeneic T cell replete BMC and hrIL-2. The mechanism by which the transferred NK cells improved BMC engraftment was at least partly through the abrogation of the host effector cell's ability to mediate resistance to the marrow graft. Thus, the administration of donor-type activated NK cells with BMC and hrIL-2 may significantly augment hematopoietic engraftment and immune reconstitution in the clinical setting of allogeneic BMT without giving rise to graft-vs-host disease.     

FADD deficiency impairs early hematopoiesis in the bone marrow

Signal transduction mediated by Fas-associated death domain protein (FADD) represents a paradigm of coregulation of apoptosis and cellular proliferation. During apoptotic signaling induced by death receptors including Fas, FADD is required for the recruitment and activation of caspase 8. In addition, a death receptor-independent function of FADD is essential for embryogenesis. In previous studies, FADD deficiency in embryonic stem cells resulted in a complete lack of B cells and dramatically reduced T cell numbers, as shown by Rag1(-/-) blastocyst complementation assays. However, T-specific FADD-deficient mice contained normal numbers of thymocytes and slightly reduced peripheral T cell numbers, whereas B cell-specific deletion of FADD led to increased peripheral B cell numbers. It remains undetermined what impact an FADD deficiency has on hematopoietic stem cells and progenitors. The current study analyzed the effect of simultaneous deletion of FADD in multiple cell types, including bone marrow cells, by using the IFN-inducible Mx1-cre transgene. The resulting FADD mutant mice did not develop lymphoproliferation diseases, unlike Fas-deficient mice. Instead, a time-dependent depletion of peripheral FADD-deficient lymphocytes was observed. In the bone marrow, a lack of FADD led to a dramatic decrease in the hematopoietic stem cells and progenitor-enriched population. Furthermore, FADD-deficient bone marrow cells were defective in their ability to generate lymphoid, myeloid, and erythroid cells. Thus, the results revealed a temporal requirement for FADD. Although dispensable during lymphopoiesis post lineage commitment, FADD plays a critical role in early hematopoietic stages in the bone marrow.     

The facilitation of enduring engraftment of allogeneic bone marrow and avoidance of secondary disease in mice

A method for transplantation of allogeneic bone marrow has been developed and tested in mice. It consists of a treatment preceding supralethal, total-body irradiation (preconditioning) in which a combination of three drugs acting on neuroendocrine regulation are administered, followed by inoculation of a large number of allogeneic bone marrow cells. A second inoculation of allogeneic marrow from the same immunogenetically different donor is given after irradiation. This system provides a high level of protection to mice against radiation damage and facilitates engraftment of the foreign marrow. A large proportion of the engrafted mice become enduring chimeras, manifesting no secondary disease.     

Restoration of the immune balance by autologous bone marrow transplantation in juvenile idiopathic arthritis

Juvenile idiopathic arthritis (JIA) is one of the most frequent autoimmune diseases in childhood and is characterized by chronic inflammation of the synovial fluid in joints. Several drugs are available for the treatment of JIA, including various biological agents that interfere with critical cytokine pathways. Though very effective in suppressing disease activity, none of these drugs can cure the disease and induce a lasting medication free remission. A small proportion of JIA patients will become or are unresponsive to any form of medical treatment. For these severely ill patients autologous bone marrow transplantation (aBMT) is a last resort treatment. aBMT is remarkably effective in suppressing disease activity, with beneficial outcome reported in around 70% of these previously refractory patients. Moreover aBMT is the only treatment that can induce a lasting medication-free-disease remission in these patients. In the very long term (after 7 years of remission) however, some disease relapses are observed, with the disease returning in a less severe form compared to prior aBMT. The exact mechanism of how aBMT is inducing this lasting disease remission is still largely unknown, but data from both animal models and humans suggest a prominent role for regulatory T cells. In this review we reviewed the current views of the cellular mechanisms that lay beneath disease induction of JIA and the disease remission caused by aBMT therapy.