The role of cytokines and hematopoietic growth factors in the autocrine/paracrine regulation of inducible hematopoiesis.

To elucidate the role of hematopoietic growth factors (HGFs) and other cytokines in the autocrine or paracrine regulation of inducible hematopoiesis we studied cytokine gene expression in the bone marrow (BM) of patients after myelosuppressive treatment. Furthermore, we studied the cytokine gene expression profile in healthy individuals before and after bone marrow harvesting for the purpose of bone marrow transplantation. We speculated that the bone marrow harvesting procedure might induce changes in cytokine gene expression. No induction of G-CSF, GM-CSF, IL-1 alpha, IL-3, IL-5, IL-8, IL-9, and IL-12 was observed in the BM of patients following intensive chemotherapy. Also, no up-regulation of expression of M-CSF, IL-1 beta, IL-4, IL-6, TNF-alpha, TGF-beta, IGF-1, EDF, and EPA gene was found, illustrating that the investigated cytokines probably are not relevant in the presumed autocrine/paracrine regulation of the recovery of hematopoiesis following depletion of hematopoietic progenitor cells (HPCs). Concomitantly, elevated G-CSF plasma levels were found in these patients, suggesting that G-CSF has an endocrine regulatory role in inducible hematopoiesis. Induction of GM-CSF and IL-8, but not of G-CSF or IL-3 gene expression and upregulation of IL-1 beta and IL-6 gene following BM harvesting was observed. This induction of GM-CSF and IL-8 may be attributed to tissue damage rather than to HPC depletion.

Extramedullary hematopoietic growth factor and cytokine gene expression indicate endocrine regulation of hematopoiesis in patients with acute appendicitis.

To analyze the role of hematopoietic growth factors (HGFs) and other cytokines in the regulation of hematopoiesis in vivo, we investigated HGFs and cytokine gene expression in appendices obtained from patients who underwent surgery for suspected appendicitis. Concomitantly, HGF gene expression was studied in bone marrow (BM) biopsy specimens and plasma HGF levels were measured. G-CSF gene expression was detected in inflamed but not in normal appendices. With one exception, GM-CSF was detectable in all appendices whether inflamed or not, whereas IL-3, except for one case, was not expressed in appendices. None of the investigated HGFs appeared to be expressed in BM biopsy specimens concurrently obtained with the appendices. Plasma G-CSF levels were significantly elevated in patients with appendicitis compared with patients without inflamed appendices. Circulating levels of GM-CSF and IL-3 were not increased. Significant up-regulation of IL-8 and IL-6 gene expression was observed in response to inflammation, in contrast to IL-1 alpha and IL-1 beta expression, which appeared not to be influenced by the inflammatory state. These data indicate that G-CSF, and not GM-CSF or IL-3, is essential for the regulation of inducible granulopoiesis in acute inflammatory conditions, and that G-CSF acts in an endocrine fashion.

Generation of CD4+ cytotoxic T-lymphocyte clones from a patient with severe graft-versus-host disease after allogeneic bone marrow transplantation: implications for graft-versus-leukemia reactivity.

HLA-identical bone marrow transplantation (BMT) is associated with both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) reactivity. Different T-cell subsets from the bone marrow (BM) graft may be responsible for GVHD and GVL reactivity after BMT. In the etiology of GVHD, not only CD8+ but also CD4+ donor T lymphocytes may play an important role. Here we report a patient with chronic myeloid leukemia (CML) who was transplanted with the BM from his HLA-genotypically identical sister. After BMT there was complete engraftment, but the patient died because of acute GVHD grade III-IV in complete remission. Cytotoxic T-lymphocyte (CTL) lines were generated after BMT using the irradiated leukemic cells from the patient as stimulator cells and the donor-originated peripheral blood mononuclear cells, procured from the patient after BMT, as responder cells. The generated CTL lines showed specific lysis of the recipient lymphocytes and leukemic cells in a 51Cr release assay. Two types of CTL clones could be established from these CTL lines, both phenotypically CD4+. Clone type I showed male-specific HLA-DQ5-restricted lysis of the recipient lymphocytes, but not of the circulating relatively mature leukemic cells from the patient. This may be explained by the low HLA-DQ5 expression of the more mature CML cells. Clone type II showed HLA-DR2-restricted minor histocompatibility antigen-specific lysis of the recipient lymphocytes and leukemic cells. Both types of CTL clones showed antigen-specific cell-mediated growth inhibition of the recipient clonogenic leukemic precursor cells. These CD4+ CTL clones produced several activating cytokines including tumor necrosis factor alpha, interferon gamma, granulocyte-macrophage colony-stimulating factor (GM-CSF), and macrophage CSF. Our results illustrate that these CD4+ CTL clones may have induced GVHD directly by cytolysis and indirectly by activating cytokines. Because both types of CTL clones recognized the recipient leukemic progenitor cells, they may also contribute to GVL reactivity after BMT.

Multiple minor histocompatibility antigen-specific cytotoxic T lymphocyte clones can be generated during graft rejection after HLA-identical bone marrow transplantation.

Graft rejection after T cell-depleted HLA-genotypically identical bone marrow transplantation (BMT) is probably mediated by mH antigen-specific cytotoxic T lymphocytes (CTL). We have analyzed peripheral blood mononuclear cells (PBMC) from a female bone marrow graft recipient, collected during graft rejection after a sex mismatched HLA-identical BMT. A CTL line was generated by stimulating recipient PBMC collected during graft rejection with donor PBMC and donor EBV-transformed lymphoblastoid cell lines. From this CTL line a large number of clones of different specificity and phenotype was established by limiting dilution. These clones exhibited several mH antigen specificities, restricted by HLA-B7, -B27 or -DR2 as shown by differential recognition of family members and unrelated individuals sharing potential restriction elements. The CD3+CD4+ and CD3+CD8+ bulk culture was cloned, resulting in 50 HLA-B7 restricted CD3+CD4-CD8+CTL clones, three HLA-B27 restricted CD3+CD4-CD8+CTL clones, one HLA-DR2 restricted CD3+CD4+CD8-CTL clone and two additional HLA class II restricted CD3+CD4+CD8-CTL clones with a different specificity. One representative clone of each specificity was selected for further analysis. The CTL line and the HLA-B7 restricted CD8+CTL clone, but not the HLA class II restricted CD4+ CTL clone, inhibited the growth of donor hematopoietic progenitor cells (HPC). In conclusion, these results show that graft rejection after HLA-identical BMT may be mediated by multiple CTL clones that specifically recognize one mH antigen peptide presented by different HLA molecules or different mH antigens expressed on donor cells and that CTL, but not CD4+ CTL inhibited donor HPC growth.

Minor histocompatibility antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T-cell clones inhibit human hematopoietic progenitor cell growth by a mechanism that is dependent on direct cell-cell contact.

HLA-identical bone marrow transplantation (BMT) may be complicated by graft-versus-host disease or graft rejection. Both complications are thought to be initiated by recognition of minor histocompatibility (mH) antigens by HLA-restricted mH-antigen-specific T lymphocytes. Using HLA-A2-restricted mH antigens HA-1-, -2-, and -4-, and HY-specific cytotoxic T lymphocyte (CTL) clones, we studied the recognition by these CTL clones of interleukin-2 (IL-2)-stimulated T cells (IL-2 blasts), BM mononuclear cells (BMMNCs), and hematopoietic progenitor cells (HPCs). We showed that, when IL-2 blasts from the BM donors who were investigated were recognized by the HA-1-, -2-, and -4-, and HY-specific CTL clones, their BMMNCs and HPCs were recognized as well by these CTL clones, resulting in antigen-specific growth inhibition of erythrocyte burst-forming units (BFU-E), colony-forming units-granulocyte (CFU-G), and CFU-macrophage (CFU-M). the HA-2-specific CTL clone, however, inhibited BFU-E and CFU-G growth from four donors to a lesser extent than from two other donors. We further investigated whether inhibitory cytokines released into the culture medium by the antigen-specific stimulated CTLs or by stimulated BMMNCs were responsible for suppression of HPC growth or whether this effect was caused by direct cell-cell contact between CTLs and HPCs. HPC growth inhibition was only observed after preincubation of BMMNCs and CTLs together for 4 hours before plating the cells in semisolid HPC culture medium. When no cell-cell contact was permitted before plating, neither antigen-stimulated CTL nor antigen-nonstimulated CTLs provoked HPC growth inhibition. Culturing BMMNCs in the presence of supernatants harvested after incubation of BMMNCs and CTL clones together for 4 or 72 hours did also not result in HPC growth inhibition. Both suppression of HPC growth and lysis of IL-2 blasts and BMMNCs in the 51Cr-release assay appeared to be dependent on direct cell-cell contact between target cells and CTLs and were not caused by the release of inhibitory cytokines into the culture medium by antigen-specific stimulated CTLs or by stimulated BMMNCs. Our results show that mH-antigen-specific CTLs can inhibit HPC growth by a direct cytolytic effect and may therefore be responsible for BM graft rejection after HLA-identical BMT.

Multiple minor histocompatibility antigen disparities between a recipient and four HLA-identical potential sibling donors for bone marrow transplantation.

A patient with acute leukemia and her family including four HLA-identical siblings were analyzed to select a donor who was not only HLA- but also minor histocompatibility (mH) antigen compatible for allogeneic bone marrow transplantation (BMT). The HLA-A2 restricted mH antigen-specific HA-1, -2, -4, and -5 cytotoxic T-lymphocyte (CTL) clones were used to type the family members for expression of these mH antigens. The patient and one HLA-identical sibling were compatible for these mH antigens. This sibling was selected as the bone marrow donor. The patient engrafted promptly but developed acute and chronic graft-versus-host disease. To study the presence of other mH antigen disparities between recipient and donor, host-versus-graft CTL lines and clones were generated by stimulation of recipient peripheral blood lymphocytes (PBLs) with donor bone marrow cells, and graft-versus-host CTL lines were generated after BMT by stimulation of PBLs of donor origin with recipient bone marrow cells. These CTL lines were cytotoxic to cells from the bone marrow donor and from the recipient, respectively, and to cells from several other family members. T-cell lines, generated from the patient after BMT by stimulation of recipient-derived PBLs with donor bone marrow cells, exhibited no specific cytotoxicity to donor or recipient cells. Chimerism studies after BMT revealed that the PBLs and T-cell lines generated after BMT were of donor origin.(ABSTRACT TRUNCATED AT 250 WORDS)

Minor histocompatibility antigen-specific cytotoxic T cell lines, capable of lysing human hematopoietic progenitor cells, can be generated in vitro by stimulation with HLA-identical bone marrow cells.

Recipient-antidonor alloreactivity before HLA genotypically identical bone marrow transplantation (BMT) between donor-recipient pairs that are negative in the mixed lymphocyte reaction (MLR), the cell-mediated lympholysis (CML) assay, and the lymphocyte crossmatch was not detectable in the majority of cases, using recipient peripheral blood lymphocytes (PBL) collected before BMT as responder cells and donor PBL as stimulator cells. However, when donor bone marrow mononuclear cells (BMMNC) instead of PBL were used as stimulator cells, we could detect donor-specific alloreactivity in 7 of 10 HLA genotypically identical donor-recipient pairs. To demonstrate that this alloreactivity was minor histocompatibility (mH) antigen specific and not directed against HLA class I splits or variants, two cytotoxic T lymphocyte (CTL) lines were tested in further detail against phytohemagglutinin (PHA) blasts from pairs of HLA genotypically identical siblings positive for the HLA class I restriction molecule. Both CTL lines recognized mH antigens, as illustrated by the differential recognition of PHA blasts of one of the two siblings from several pairs. The potential role of these mH antigen-specific CTLs in bone marrow graft rejection was demonstrated by the mH antigen-specific growth inhibition of hematopoietic progenitor cells from the original bone marrow donor and from HLA class I restriction molecule-positive individuals who expressed the mH antigens on their PBL and BMMNC. Our assay can be used in HLA genotypically identical BMT to detect a recipient-antidonor response, directed against cellularly defined mH antigens expressed on donor HPC, BMMNC, and PBL, before transplantation.

Rejection of bone-marrow graft by recipient-derived cytotoxic T lymphocytes against minor histocompatibility antigens.

A female patient showed rejection of a T-lymphocyte-depleted bone-marrow graft from her phenotypically HLA-identical father. Before bone-marrow transplantation, there was strong recipient anti-donor cellular cytotoxic reactivity directed against several minor histocompatibility (mH) antigens, including the male-specific H-Y antigen. After conditioning treatment, no recipient anti-donor cytotoxic activity could be detected, and good graft function was shown a month after transplantation. Thereafter, however, graft function deteriorated rapidly, while recipient-derived anti-donor cellular cytotoxic reactivity, against similar mH antigens, reappeared. The recipient-derived cytotoxic T lymphocytes could completely inhibit growth of donor haemopoietic progenitor cells both before and after bone-marrow transplantation. Thus, cytotoxic T lymphocytes can survive very intensive conditioning regimens, and residual recipient cytotoxic T lymphocytes directed against mH antigens expressed on donor haemopoietic progenitor cells may cause graft rejection after HLA-identical T-lymphocyte-depleted bone-marrow transplantation.

Normal hematopoietic progenitor cells and malignant lymphohematopoietic cells show different susceptibility to direct cell-mediated MHC-non-restricted lysis by T cell receptor-/CD3-, T cell receptor gamma delta+/CD3+ and T cell receptor-alpha beta+/CD3+ lymphocytes.

To evaluate the capability of NK cells and cytotoxic T lymphocytes to interact with normal hematopoietic progenitor cells (HPC), as compared to neoplastic lymphohematopoietic cells, we investigated inhibition of colony growth of these cell populations in semi-solid culture systems, after incubation with cloned cytotoxic effector cells. Three different types of cloned effector cells were investigated: TCR-/CD3- NK cells, TCR-gamma delta+/CD3+ cells, and TCR-alpha beta+/CD3+ cytotoxic T lymphocytes. Effector cells showed differential levels of tumor cell colony inhibition, but no MHC-non-restricted lysis of normal HPC was observed. Pre-stimulation of normal HPC by culturing on established stromal layers had no effect. Cell-mediated lysis of HPC only occurred by Ag-specific MHC-restricted lysis by CTL, or by antibody-dependent cellular cytotoxicity. In cell mixing experiments, irradiated tumor cells, but not normal bone marrow cells inhibited tumor cell lysis. Furthermore, cloned effector lymphocytes were able to specifically eliminate malignant cells from tumor contaminated bone marrow without damaging normal HPC. When fresh leukemic cells were used as targets, growth of acute myeloblastic leukemia colonies was inhibited after incubation with several cytotoxic effector clones, whereas chronic myeloid leukemia precursor cells showed limited sensitivity to MHC-non-restricted cytolysis. These results indicate that MHC-non-restricted cytolysis by NK cells is selectively directed against neoplastic cells and not against normal HPC.

Cellularly defined minor histocompatibility antigens are differentially expressed on human hematopoietic progenitor cells.

Previously, five CTL lines directed against minor histocompatibility (mH) antigens designated HA-1-5 have been established from peripheral blood of patients after allogeneic bone marrow transplantation (BMT), and have been characterized using population and family studies. All cell lines showed specific HLA class I-restricted lysis of PHA-stimulated peripheral blood target cells from donors positive for the particular mH antigens. After 4 h of incubation of the mH antigen HA-3-specific CTL line with bone marrow cells from HA-3+ donors, complete class I-restricted inhibition of colony growth of the hematopoietic progenitor cells was observed even at low E/T ratios, indicating that the HA-3 antigen is strongly expressed on hematopoietic stem cells. Therefore, this antigen may be a target structure in the immune-mediated rejection of the hematopoietic graft in case of incompatibility for this determinant between donor and recipient in allogeneic BMT. In contrast, incubation of bone marrow cells with the antigen-specific anti-HA-1, -2, -4, and -5 CTL lines did not result in growth inhibition of the hematopoietic progenitor cells tested. After a prolonged incubation time and using a very high E/T ratio, progenitor cells from HA-2+ or HA-5+ donors were killed to some extent by the anti-mH-specific CTL lines, although the growth inhibition observed was minor and variable. Our results show that mH antigens are differentially expressed on human hematopoietic progenitor cells. Therefore, only some of these antigens may be targets in immune-mediated rejection of the bone marrow graft.

Minor histocompatibility antigen H-Y is expressed on human hematopoietic progenitor cells.

Polymorphic minor transplantation antigens probably play an important role in immune mediated graft rejections of bone marrow transplants. Mapping of these antigens on hematopoietic progenitor cells (HPC) is important since these antigenic determinants may serve as target structures in the rejection process, and it ultimately opens the possibility to match for these antigens. Using a cell-mediated cytotoxicity assay with H-Y-specific cytotoxic T lymphocytes as effector cells, a dose-dependent growth inhibition up to 100% of myeloid (CFU-GM), erythroid (BFU-E) and multipotential (CFU-GEMM) HPC of male donors was obtained, indicating expression of the H-Y antigen on these progenitor cells. In contrast, inhibition of relatively mature erythroid and myeloid progenitor cells was only 40-50%, indicating that the recognition of the H-Y antigen diminished during maturation of erythroid and myeloid HPC. Our results show that the H-Y antigen can be recognized on HPC as a target for cytotoxic T cell responses. This may be important in graft rejection of male donor bone marrow grafts by female recipients.

Cell-mediated lysis of human hematopoietic progenitor cells.

Several techniques are available for the serological analysis of antigenic determinants on human hematopoietic progenitor cells (HPC). However, techniques for the recognition of cellularly defined antigens on such progenitor cells have not yet been described. We therefore developed an in vitro cellular cytotoxicity assay, with bone marrow cells as target cells. In this assay specific cytotoxic T lymphocyte (CTL) lines are used as effectors for cell-mediated cytolysis of bone marrow mononuclear cells that express the antigens for which the CTLs were primed in a mixed lymphocyte culture. As a model we used CTL lines against HLA-A2 or -B7 determinants. By using effector-target ratios varying from 1:2 to 4:1, 4 hr of incubation of these CTL lines with bone marrow mononuclear cells from HLA-A2 or -B7 positive donors resulted in a specific dose-dependent growth inhibition up to 100% of myeloid (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) HPC. In contrast no inhibition of HPC was observed using mononuclear bone marrow cells from HLA-A2 or -B7 negative individuals as target cells. Experiments in which cell-cell contact was prevented showed that the antigen-specific lysis of HPC was dependent on intimate cell-cell contact between effector-CTLs and bone marrow target cells. Our results show that this cell-mediated cytotoxicity assay can be used as a sensitive and specific tool for the study of cellularly defined antigens on human hematopoietic progenitor cells.

Selective removal of clonogenic neoplastic B cells from human bone marrow using anti-HLA-DQ antibodies and complement.

Polymorphic HLA-DQ (DC/MB) determinants appeared to be not expressed on human hematopoietic progenitor cells (HPC), using several murine monoclonal and human polyclonal antibodies in a complement-dependent cytotoxicity (CDC) assay. Since mature HLA-DR-positive malignant lymphoma cells prove to be HLA-DQ positive, an attempt was made to remove clonogenic neoplastic DQwl-positive B cells selectively from DQwl-positive marrow samples without affecting hematopoietic progenitor cells. Using a combination of a clonogenic tumor cell assay, an HPC culture assay, and a mixed-tumor-cell-HPC culture assay, selective elimination of more than 98% of clonogenic neoplastic cells from tumor-cell-contaminated bone marrow suspensions was achieved with monoclonal anti-DQ antibodies and complement without depletion of HPC. These results indicate that anti-HLA-DQ antibodies can be used in autologous bone marrow transplantation to deplete the bone marrow cell suspension of DQ-positive malignant cells.

Complement-dependent cytotoxicity in the analysis of antigenic determinants on human hematopoietic progenitor cells with HLA-DR as a model.

Complement-dependent cytotoxicity (CDC) assays using anti-Ia antisera have resulted in controversial conclusions about the expression of HLA-DR determinants on human hematopoietic progenitor cells (HPC). The expression of these antigens on CFU-E in particular could often not be demonstrated. Since this is contradictory to our own results, we decided to study the influence of both the antibody and complement concentrations in CDC assays using murine monoclonal and polyclonal anti-Ia (HLA-DR "backbone") antibodies and human anti-HLA-DR typing sera. We found that with use of the same anti-Ia antibody concentrations, elimination of CFU-E required significantly more complement than CFU-GM and BFU-E. In CDC assays with antipolymorphic HLA-DR antisera, complete kill of both CFU-E and BFU-E required significantly more complement than of CFU-GM. Insufficient complement concentration could be partly overcome by increasing the antibody concentration. Intrinsic insensitivity of CFU-E to low concentrations of complement could be excluded by experiments using monoclonal anti-HLA-A/B/C "backbone" antibodies. Furthermore, FACS experiments demonstrated that the density of HLA-DR determinants on CFU-E is lower than on BFU-E and CFU-GM. These data show that in CDC assays, antigens with low expression on HPC can easily be overlooked.

The in vitro growth pattern of human bone marrow in methylcellulose stimulated by different concentrations of conditioned medium.

The proliferation of human bone marrow in methylcellulose stimulated by various concentrations of conditioned medium (CM) and observed at various intervals was studied. The growth kinetics of granulocytic aggregates was found to differ from monocytic clusters and colonies. Granulocytic aggregates showed a consistent and reproducible dose-response relationship; at day 7, the maximum number of granulocytic aggregates was found at 4% CM. At higher levels, the total number of aggregates decreased, while the number of cells per aggregate increased. The number of macrophage aggregates was far less, depending on the CM concentration. Photographic interval studies showed that at high concentrations of CM, clusters and colonies were formed earlier, but also coalesce to form one colony. Our results suggest that the proliferation kinetics of granulocytic aggregates are complex and preclude simple statements about sensitivity to colony-stimulating activity.

Polymorphic and monomorphic HLA-DR determinants on human hematopoietic progenitor cells.

The expression of monomorphic Ia-like antigens and polymorphic (allotypic) HLA-DR determinants on CFU-GM, BFU-E, CFU-E, and CFU-GEMM was studied in bone marrow and peripheral blood cells from normal healthy individuals. Using various polyclonal and monoclonal anti-Ia-like antibodies, the presence of HLA-DR backbone antigens was shown on all hematopoietic progenitor cells (HPC) studied, both in complement-dependent cytotoxicity assays and in fluorescence-activated cell sorting (FACS). The expression of allotypic determinants was demonstrated on all HPCs, using the HLA-DR typing sera anti-HLA-DR1, 2, 3, 4, 5, and 7. The Class II antigen MT-2 was also shown on all HPCs, using both monoclonal and alloantisera, whereas the MB-1 (DC-1) determinant could not be demonstrated on HPCs. This might open the possibility of removing MB-1-positive malignant cells from the graft in autologous bone marrow transplantation.

The impact of the composition of the bone marrow graft on engraftment and graft-versus-host disease.

To study the impact of the composition of the bone-marrow graft on engraftment and graft-versus-host disease (GvHD), we analyzed the data on 29 patients with acute leukemia in remission and 11 patients with aplastic anemia. All of them received bone-marrow grafts from HLA matched, MLC nonreactive, sibling donors, were nursed in laminar down-flow isolators with selective gut decontamination, and received GvHD prophylaxis with methotrexate. The number of nucleated cells in the marrow graft/kg body weight of the recipient had no relation with the rapidity of engraftment or with the occurrence and severity of GvHD. The number of hematopoietic progenitor cells (CFUc)/kg had a weak, but significant, correlation with both the number of neutrophils at day 30 post BMT and with the day at which the reticulocytes passed the 10% level. The number of T cells/kg did not show any correlation with either the occurrence or the severity of GvHD. Our data show that the concentration of hematopoietic progenitor cells in the graft correlates with the rapidity of engraftment. However, within the range of numbers of T cells infused in this study, no correlation is present between T cells in the graft and GvHD. Therefore, nearly complete depletion of marrow grafts of T cells is probably necessary to effectively decrease the incidence of GvHD.

Serum inhibitors in aplastic anaemia.

To study the frequency and clinical importance of serum factors inhibiting CFU-c in patients with aplastic anaemia, sera from 21 patients were analysed. The sera of eight patients showed inhibition of at least one normal bone marrow, but strong and consistent inhibition of two or more normal bone-marrow samples was found in only two cases. In both of these two patients the inhibition disappeared after successful therapy. Fair, but not complete, correlation was found between serum inhibitors and HLA-antibodies, suggesting that at least part of these inhibitors are antibodies directed against HLA-antigens present on myeloid progenitor cells. Serum inhibitors appear to be infrequent in the population of patients with aplastic anaemia seen in our hospital and appear to have no major implications for therapy or outcome of the disease.

Anti-thymocyte globulin effective in the treatment of aplastic anemia does not stimulate granulocyte precursor cells.

To find out whether the preparations of anti-lymphocyte or anti-thymocyte globulin (ATG), successfully used in our center for the treatment of patients with aplastic anemia, were stimulatory for hematopoietic precursor cells, we studied the effect on CFUC in 30 normal bone marrow samples. Although in 2 out of 30 cases stimulation was observed, the overall result for both the horse anti-lymphocyte globulin and the rabbit anti-thymocyte globulin was dose-dependent and complement-dependent inhibition. Neither in the absence, nor in the presence of complement was there any indication of consistent stimulation of CFUC. When bone marrow was depleted of E-rosette forming cells, the incubation of the depleted fraction with ATG did not result in stimulation. The incubation of the E-rosette positive fraction with ATG, followed by the addition of these treated cells to untreated E-rosette depleted cells was equally ineffective in giving any stimulation of CFUC. Our data suggest that the effect of ATG in the treatment of aplastic anemia is not due to direct stimulation of hematopoietic precursor cells.