Factors that control the differentiation of stem cells. I. The change in direction of hematopoietic stem cell differentiation under the effect of differentiating T-lymphocytes]

A mixed transplantation of bone marrow cells, and lymph nodes or thymic cells of mice CBA strain into lethally irradiated hybrid recipients (CBAXC57B1)F1 is accompanied with changes in the differentiation pattern from a mainly erythroid to a mainly granuloid way. Thymectomy of either donor of bone marrow cells or recipients, or both, destroys the stem cell differentiation in the direction of granulopoieseis. Intact syngeneic lymphocytes normalize differentiation of the stem cells, but in the presence of tissue antigens these provide for the stem cell differentiation mainly in the direction of granulopoiesis. The differentiation of stem haemopoietic cells is accomplished under the thymic and lymphocyte control. T-differentiating lymphocytes (Td) are the lymphocytes controlling the stem cell differentiation.     

Effect of syngeneic lymphocytes, T immunodeficiency and the genotype of bone marrow donors on the differentiation of early and late splenic colonies

The formation of "early" (5-8 days) and "late" (12-14 days) colonies in spleen of lethally irradiated syngeneic or hybrid recipients after transplantation of bone marrow cells has been studied. The differentiation pattern did not depend on bone marrow cell donor's genotype and the donor-recipient combination. Erythroid to granulocyte colonies ratio (E/G) equals 2. Change of direction of bone marrow colony-forming units (CFU) differentiation has the same pattern at different stages of colony-formation. Under the influence of antigen-stimulated lymphocytes the granulopoiesis (E/G 0.3-0.5) dominanted. The thymectomy of adult animals leads to a predominant formation of erythroid colonies (E/G 3.5-5.1). When T-immunodeficiency is reversed with syngeneic lymphocytes, the differentiation of CFU is normalized at all stages of colony-formation. The process of differentiation of haemopoietic precursors, that form "early" and "late" colonies, is under T-lymphocyte control.     

Heterologous antiserum to rat lymphohemopoietic precursor cells.

Lymphohemopoietic precursor cells in rat bone marrow are members of a subset of lymphocyte-like cells that bears the bone marrow lymphocyte antigen (BMLA) and that lacks antigens present on peripheral B and T cells. This was demonstrated by two experimental approaches. In the first, bone marrow cells with the potential to form hemopoietic colonies in spleen (CFU-S), to repopulate lumphoid tissues and blood, and to rescue lethally irradiated recipients were enriched approximately 10-fold by a fractionation procedure designed to isolate a "null" population of bone marro lymphocytes. In the second approach, the lymphohemopoietic precursor cell activity in bone marrow was completely abrogated by opsonization with rabbit antiserum (ALSBM) raised against this "null" population of bone marrow cells. Precursor cell activity was not affected by treatment with antiserum to T and B cells. Quantitative cross-absorption studies showed that the antigen detected by ALSBM on lymphohemopoietic precursor cells had the same cellular distribution as did the previously described bone marrow lymphocyte antigen. It is likely that this antigen is present both on pluripotent stem cells and on committed progenitors of the myelocytic, erythrocytic and lymphocytic series.     

Full reconstitution of the immune deficiency in scid mice with normal stem cells requires low-dose irradiation of the recipients

Mice homozygous for an autosomal recessive mutation for the scid gene exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Such mice can be cured of their lymphoid deficiency by grafts with normal bone marrow, although full reconstitution of lymphoid function is seldom obtained. Long-term bone marrow cultures (LTBMC) are devoid of all mature B and pre-B cells but contain lymphoid stem cells. We therefore reconstituted scid mice with LTBMC cells to study the kinetics of B lymphocyte reconstitution in normal and irradiated (4 Gy) scid recipients and in irradiated (9.5 Gy) co-isogenic C.B-17 mice. Detectable colony-forming B cells rapidly increased in the spleen and bone marrow of irradiated C.B-17 and irradiated scid recipients, reaching normal levels between 4 and 6 wk post-grafting. Unirradiated scid recipients showed limited reconstitution in spleen and very poor reconstitution in bone marrow. Unirradiated scid recipients also had relatively few surface Ig+ cells in spleen or bone marrow, whereas both groups of irradiated recipients had normal numbers between 4 and 6 wk post-reconstitution. Normal levels of cytotoxic T cell activity by 8 wk after reconstitution were observed only in the irradiated C.B-17 and irradiated scid recipients. Analysis of mice reconstituted with cells from LTBMC indicates that these cultures contain lymphoid stem cells with significant proliferative and self-renewal potential, and that full reconstitution of lymphoid function requires prior irradiation of the scid recipient.     

Comparison of response to stem cell differentiation signals between normal and autoimmune mouse strains

Normal DBA/2 and autoimmune NZB mice were studied with regard to signals eliciting differentiation and division of bone marrow stem cells. Irradiated (NZB X DBA/2)F1 mice were repopulated with various combinations of T-depleted bone marrow from NZB and DBA/2 mice. In response to the repopulation signal of irradiation, recipients of autoimmune NZB marrow initially demonstrated expansion of LY-5+ lymphoid and hemopoietic cells, particularly of the B cell lineage. The greater the proportion of NZB marrow, the higher the percentage of lymphoid cells observed 2 wk post-repopulation. B cells (ThB-positive cells) were increased in disproportionate numbers in recipients of NZB marrow, even those that had received as little as 20% NZB bone marrow cells. However, by 2 mo, the initially observed increase in lymphoid cells in recipients of NZB marrow was no longer observed. Up to 6 mo post-repopulation, cytogenetic analysis revealed that irradiated recipients were repopulated in the same proportion of DBA/2: NZB as was in the injected marrow. Endogenous colony formation assays indicated that recipients of 100% NZB, 80% NZB, and 20% NZB marrow all had greater numbers of splenic endogenous colonies than did recipients of DBA/2 marrow alone. These studies indicated that autoimmune NZB marrow repopulated irradiated mice in the proportion in which it was injected, but there was a disproportionate early increase in cells of the B lineage as well as a disproportionate increase in splenic colony formation.     

Effect of a neutrophilia-inducing tumor on hemopoietic stem cells in mice

The influence of neutrophilic stimulation on hemopoietic stem cells was studied in mice with tumor-induced neutrophilia. Transfusions of marrow cells from normal and neutrophilic tumor-bearing mice into lethally irradiated normal and tumor-bearing mice were performed. The number and the erythroid:granuloid (E:G) ratio of day 7 colonies in the recipient spleens and bones as well as the size of spleen colonies of recipient animals were determined. The E:G ratio of spleen and bone marrow colonies between normal and tumor-bearing mouse recipients and the number of spleen colonies did not differ significantly in either experiment. However, spleen colonies which developed in tumor-bearing irradiated mice were significantly larger than those which developed in normal recipients in both experiments. These studies indicated that while the line of differentiation taken by hemopoietic stem cells was not affected by the neutrophilic influence of the tumor, the tumor-bearing host environment appeared to enhance proliferation of transfused stem cells and/or their descendants. The stimulators of granulocytopoiesis in this model of neutrophilia appear to act on a population of progenitor cells more mature than the stem cells capable of forming 7-day colonies in the spleen and bone marrow of irradiated recipient mice.     

Hematopoietic factors in graft-versus-host reaction

Graft-versus-host (GVH) reaction has a curious unsolved area in the immunopathogenesis and pathophysiology of the immunohematopoietic system, and GVH disease remains one of the major obstacles in clinical allogeneic bone marrow transplantation. T lymphocytes and T lymphocyte subpopulations are now recognized to be initiators of this GVH reaction and disease. Also, T lymphocytes are known to be accessory cells in the regulation of hematopoiesis, and produce a variety of lymphokines relevant to hematopoiesis. Admittedly, remarkable hematopoietic changes can be found in GVH reaction, but the cellular mechanisms underlying these changes are so complex they have yet to be fully elucidated. In fact, elevated serum levels of myeloid and erythroid colony-stimulating activities were found in mice suffering from GVH disease in which marked granulopoiesis and suppression of erythropoietic differentiation were seen. In addition, each granulocyte/macrophage colony-stimulating factor (GM-CSF) or burst-promoting activity (BPA) could be detected in sera from patients with GVH disease following allogeneic bone marrow transplantation. There seems to be at least two mechanisms involved in the control of hematopoiesis with either humoral or local environmental factor, probably via the T lymphocytes or T lymphocyte subpopulations activated by alloantigens or autologous non-T cells.     

Cellular events during radiation-induced thymic leukemogenesis in mice: abnormal T cell differentiation in the thymus and defect of thymocyte precursors in the bone marrow after split-dose irradiation

Cellular events during the development of thymic lymphomas in young B10.BR mice given leukemogenic split-dose irradiation were studied by examining the differentiation of functional T lymphocyte precursors in the regenerating thymus. It was found that leukemogenic radiation treatment resulted in a sustained depression of the level of thymic cytotoxic T lymphocyte precursors (CTLp) and of mixed lymphocyte reactivity of thymus cells when assessed between 1 and 4 mo after irradiation, in spite of the fact that the total number of thymocytes was restored to the normal level within 2 mo and continued to increase thereafter. In vitro mixing studies of normal thymocytes with thymus cells from split-dose irradiated mice provided no evidence for active suppression as a mechanism for this depressed activity. The ability of bone marrow cells from split-dose irradiated mice to regenerate the thymus and to differentiate into functional CTLp was examined by use of supralethally irradiated Thy-1 congenic recipients. Reconstitution of supralethally irradiated B10.BR Thy-1.2 mice with normal bone marrow from B10.BR Thy-1.1 mice resulted in the complete repopulation of host-thymus with donor-derived cells when assessed at 4 wk after reconstitution. Lymphocytes from the regenerating thymus of these animals were shown to contain high levels of CTLp which were donor-derived. On the other hand, when the recipient mice were reconstituted with bone marrow cells from donor mice which had been split-dose irradiated 1 mo earlier, regeneration of the recipient thymus was severely depressed when assessed at 4 wk to 3 mo after reconstitution. Although variable but small numbers of donor-derived Thy-1+ cells were detected, CTL activity for alloantigen could not be induced in these donor-derived cells. The results suggest that T cell precursors derived from split-dose irradiated donor mice were unable to undergo active proliferation and differentiation into functional CTLp. The significance of these findings on radiation-induced thymic leukemogenesis is discussed.     

B lymphocyte differentiation in lethally irradiated and reconstituted mice. I. The effect of Strontium-89 induced bone marrow aplasia on the recovery of the B cell compartment in the spleen.

The influence of ⁸⁹Sr-treatment on the recovery of the B cell compartment in lethally irradiated, fetal liver reconstituted mice was studied by means of membrane fluorescence. ⁸⁹Sr is a bone-seeking radio-isotope which causes in a dose of 3 μCi ⁸⁹Sr/g body weight a depletion of all nucleated cells, including immunoglobulin-bearing (B) cells, of the bone marrow.Treatment of irradiated and fetal liver reconstituted mice with 3 μCi ⁸⁹Sr/g body weight immediately and at 17 days after irradiation and reconstitution prevented recovery of the nucleated cell population, including B cells, in the bone marrow. In the spleen of such mice both nucleated cells and B cells reappeared at day 7 and 14 respectively. The B cell population in the spleen did not recover up to normal values during the experimental period of 45 days. It is concluded that B cell differentiation in lethally irradiated, fetal liver reconstituted mice can take place outside the bone marrow. The efficiency of this extra-medullary differentiation is discussed. The conclusion was drawn that mice with a ⁸⁹Sr-induced bone marrow aplasia are able to generate B lymphocytes. Consequently the bone marrow microenvironment seems not to be obligate to the differentiation of B lymphocytes. The peripheral lymphoid organs of such mice were found to be unable to compensate completely for the absence of B lymphocyte production in the bone marrow.     

Colony-stimulating factor and the proliferation of X-irradiated myeloid stem cells

Mouse bone marrow cells irradiated in vitro with X-rays (100R or 200R) were cultured for a week in semi-solid agar containing nutrients, horse serum and various amounts of colony-stimulating factor (CSF), and the number of the colonies was counted microscopically. The result showed that the diminution of proliferative activity of myeloid stem cells (CFU-C) induced by X-rays was partly recoverable by increasing the concentration of CSF, and that the irradiated CFU-C required a higher concentration of CSF than did the control CFU-C to produce colonies in the culture. The increased CSF-requirement was not due to the increased liberation of CSF-inactivator or CSF-antagonist in the culture.     

Evidence for the existence of multipotential lympho-hematopoietic stem cells in adult rat

Rats were given near-lethal doses of x-ray to produce clones of hemic cells marked by radiation-induced chromosome abnormalities. Subsequently, bone marrow from these rats was injected into lethally irradiated mice to form erythropoietic spleen colonies; and peripheral blood lymphocytes from the same rats were stimulated to proliferate in a mixed lymphocyte interaction (MLI), an immunological response to histocompatibility isoantigens. Chromosome markers indicated that in several instances the cells of an erythroid spleen colony and a proportion of the lymphocytes reacting in the MLI were progeny of the same stem cell in the donor rat. In addition, lymphocytes of the same radiation-marked clone were shown to proliferate in response to several different histocompatibility isoantigens. The data indicate the presence in the adult rat of a primitive lymphohematopoietic stem cell capable of yielding both erythroid and lymphoid progeny. The findings also suggest that immunological specificity is determined during lymphoid differentiation, subsequent to the stem cell stage.     

Studies on the organization and regeneration of bone marrow: origin, growth, and differentiation of endocloned hematopoietic colonies

Hematopoietic colonies were studied by light microscopy in the marrow of alternate fraction x-irradiated mice (C576J/B1) to investigate the microenvironmental organization of marrow and identify early hematopoietic cell-stromal cell interactions. Undifferentiated colonies (UC) were detected at 3 days postirradiation, showed a marked predilection for bone surfaces, and disappeared as differentiated colonies developed. Some UC occurred along marrow arteries. Neutrophilic granulocyte colonies (GC) occurred in all areas at 3 days but grew rapidly only subosteally. Few eosinophilic colonies (GCe) occurred. Erythrocytic colonies (EC) appeared at 4 days as dispersed populations of motile cells within a localized area of marrow; these tended to proliferate initially in intermediate and central marrow zones. Macrophage colonies (M phi C) of two "subtypes" were detected, peaking in relative frequency at 4 days. These appeared active in stromal repair and monocytopoiesis. Megakaryocyte colonies (MC) originated along bone and differentiated away from bone. From 3-5 days, the frequency of GC greater than UC greater than M phi C much greater than MC approximately equal to GCe. All colony types except UC, M phi C, and central GC increased in size and became mixed in differentiation by 12-14 days. For several weeks, however, erythropoiesis concentrated toward central areas, whereas granulopoiesis and thrombopoiesis concentrated along bone. Some mixed colonies showed an abrupt transition from erythrocytic, centrally, to granulocytic, subosteally. These results were interpreted as evidence that in x-irradiated marrow: (1) hematopoietic microenvironments (HMs) for stem-cell proliferation and commitment to differentiation, with the possible exception of HMs determining erythroid differentiation, occur in endosteal and periarterial regions; (2) a proliferative and/or chemotactic stimulus to erythroid progenitors exists in intermediate and central marrow regions; and (3) some subosteal regions may exclude erythropoiesis, or preferentially support nonerythroid differentiation. Elaborate associations occurred between macrophages and early UC, GC, and EC, but not MC hematopoietic cells. UC and GC often associated with osteoclasts. Reticular and other fibroblastic cells associated with the cells of all colony types.     

Modification of the regulatory effects of thymocytes on hematopoiesis in irradiated mice as affected by heterologous immunoglobulin G and low-dose ionizing radiation

The administration of heterologous immunoglobulin G (IgG) and/or exposure of mouse thymocyte donors to 1 and 2 Gy radiation were shown to change the regulatory effects of thymus lymphocytes on the recovery of haemopoiesis in syngeneic recipients irradiated with a median lethal dose of 6 Gy. Thymocytes of exposed (2 Gy) donors produced a stimulatory effect on the restoration of the myelokaryocytes number and increased the number of endogenous splenic colonies and bone marrow CFUs in animals exposed to a median lethal dose, whereas the administration of IgG to thymocyte donors given 2 Gy eliminated the stimulatory effect of thymocytes on the number of myelokaryocytes, and the amount of CFUs in irradiated recipients decreased.     

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.     

Identification of a novel bone marrow-derived B-cell progenitor population that coexpresses B220 and Thy-1 and is highly enriched for Abelson leukemia virus targets.

A novel stage in early B-lymphocyte differentiation has been identified in normal mouse bone marrow cells. Earlier work had demonstrated that bone marrow cells characterized by low levels of Thy-1 and lack of a panel of lineage markers (Thy-1lo Lin- cells) were highly enriched for pluripotent hematopoietic stem cells. In this paper, we present evidence that another bone marrow population, which expressed low levels of Thy-1 and coexpressed B220, a B-lineage-specific form of the leukocyte common antigen, contained early and potent precursors for B lymphocytes upon in vivo transfer to irradiated hosts. These Thy-1lo B220+ cells, comprising 1 to 2% of bone marrow cells, were enriched for large cells in the mitotic cycle; the population lacked significant pluripotent hematopoietic stem cell activity and myeloid-erythroid progenitors. Most strikingly, Thy-1lo B220+ cells represented a highly enriched population of bone marrow cells that could be targets of Abelson murine leukemia virus transformation. We propose that Thy-1lo B220+ bone marrow cells represent the earliest stage of committed lymphocyte progenitors, intermediate in differentiation between Thy-1lo Lin- pluripotent stem cells and, in the B lineage, Thy-1- B220+ pre-B cells.     

B lymphocyte production in the bone marrow of mice with X-linked immunodeficiency (xid)

CBA/N mice carry an X-linked recessive immunodeficiency (xid) gene manifested by the absence of a B lymphocyte subpopulation, but the manner in which the xid gene exerts its effect on B lymphocyte development is unknown. The production of B lymphocytes in the bone marrow of CBA/N mice has now been compared with that of normal CBA/J mice by using two in vivo assays: immunofluorescence stathmokinetic studies measured pre-B cell proliferation, whereas radioautographic [3H]thymidine labeling was used to evaluate small lymphocyte turnover. Although the total cellularity of CBA/N mouse bone marrow was greater than normal, the absolute number of marrow small lymphocytes, pre-B cells, and B lymphocytes were all similar to those in CBA/J controls. Furthermore, in the bone marrow of CBA/N mice, the proliferation rate of pre-B cells, calculated from their rate of entry into mitosis, and the turnover rate of small lymphocytes, derived from their rate of [3H]thymidine labeling, were not significantly different from those seen in nondefective mice. The present findings that pre-B cell proliferation and small lymphocyte production proceed at similar rates in the bone marrow of xid and normal mice suggest that the xid gene does not act at the level of primary B cell genesis in the bone marrow. The findings are in accord with the view that the xid gene produces a maturation block or a functional abnormality among B lymphocytes in the peripheral lymphoid tissues rather than the deletion of a sublineage of B lymphocytes in the bone marrow.     

Proliferative and differentiation properties of bone marrow hematopoietic stem cells in CBA mice of various ages

Hemopoietic bone marrow stem cells (CFCs) of young (3-5 months) and old (23-24 months) were studied according to their ability to form colonies in spleens of the lethally irradiated animals. The number, morphology and volume of CFCs were microscopically examined. The content of CFCs remained unchanged during aging. The number of erythroid colonies decreased in old mice, while that of granulocyte-macrophage and mixed colonies did not change. The megakaryocyte colonies showed even an increase with age. Volumes of all the colony types, except megakaryocyte, reduced. The results obtained thus reflect some age-related features of early stages of the stem-cell differentiation.     

Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions.

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34(+) population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.     

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.     

Opposing roles of CD28:B7 and CTLA-4:B7 pathways in regulating in vivo alloresponses in murine recipients of MHC disparate T cells.

Blockade with B7 antagonists interferes with CD28:B7 and CTLA-4:B7 interactions, which may have opposing effects. We have examined the roles of CD28:B7 and CTLA-4:B7 on in vivo alloresponses. A critical role of B7:CD28 was demonstrated by markedly compromised expansion of CD28-deficient T cells and diminished graft-versus-host disease lethality of limited numbers of purified CD4+ or CD8+ T cells. When high numbers of T cells were infused, the requirement for CD28:B7 interaction was lessened. In lethally irradiated recipients, anti-CTLA-4 mAb enhanced in vivo donor T cell expansion, but did not affect, on a per cell basis, anti-host proliferative or CTL responses of donor T cells. Graft-versus-host lethality was accelerated by anti-CTLA-4 mAb infusion given early post-bone marrow transplantation (BMT), mostly in a CD28-dependent fashion. Donor T cells obtained from anti-CTLA-4 mAb-treated recipients were skewed toward a Th2 phenotype. Enhanced T cell expansion in mAb-treated recipients was strikingly advantageous in the graft-versus-leukemia effects of delayed donor lymphocyte infusion. In two different systems, anti-CTLA-4 mAb enhanced the rejection of allogeneic T cell-depleted marrow infused into sublethally irradiated recipients. We conclude that blockade of the selective CD28-B7 interactions early post-BMT, which preserve CTLA-4:B7 interactions, would be preferable to blocking both pathways. For later post-BMT, the selective blockade of CTLA-4:B7 interactions provides a potent and previously unidentified means for augmenting the GVL effect of delayed donor lymphocyte infusion.