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.

Expression of CD11, CDw15, and transferrin receptor antigens on human hematopoietic progenitor cells.

The expression of transferrin receptor-associated antigens and of CD11 and CDw15 antigens was investigated on myeloid committed progenitor cells (CFU-GM day 10, CFU-GM day 7, and cluster-forming cells [CFC] day 4), on erythroid committed progenitor cells (BFU-E and CFU-E), and on multilineage progenitor cells (CFU-GEMM). Both complement-dependent cytotoxicity and fluorescence-activated cell-sorting assays were performed. Complement-dependent cytotoxicity appeared to be the more sensitive assay. Transferrin receptor-associated antigens appeared to be clearly present on all myeloid and erythroid committed progenitor cells, but were found to be only weakly expressed on CFU-GEMM. CD11 antigens appeared to be strongly expressed only on mature granulocytes, monocytes, and certain lymphocytes, but not significantly on myeloid committed precursor cells. Surprisingly, CD11 antigens were weakly, but significantly, present on CFU-E. CDw15 antigens appeared to be restricted to myeloid differentiation and were increasingly expressed from CFU-GM day 10 to CFC day 4. Thus, antitransferrin receptor, CD11, and CDw15 antibodies can be used to separate hematopoietic progenitor cells and may be useful tools in the study of hematopoietic differentiation.

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.

Alloimmunization after leukocyte-depleted multiple random donor platelet transfusions.

In a prospective study we investigated the development and the course of alloimmunization after leukocyte-depleted red cell and multiple random donor platelet transfusions in 335 patients. Of these 335 patients, who had a negative antibody screening on admission and a negative transfusion history, 69 (21%) developed either transient (n = 18) or permanent (n = 51) lymphocytotoxic antibodies, but only 31 patients (9%; 95% confidence limits 6-12%) developed multispecific alloantibodies necessitating HLA-matched platelet transfusions. There was no difference with regard to the development of antibodies and platelet refractoriness between leukemia patients receiving cytostatic treatment and patients with aplastic anemia receiving prednisone and antithymocyte globulin. Females with previous pregnancies developed platelet refractoriness with an increased incidence (Chi 2 13.38; p less than 0.001) compared to females without previous pregnancies, males, and children.

Factors influencing release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes.

Mononuclear phagocytes play an important role in the regulation of hematopoiesis, not only by producing regulatory monokines such as prostaglandins, tumor necrosis factor and interleukin-1 (IL-1), but also by the production of colony-stimulating activity (CSA). Previously, we have demonstrated that granulocyte-macrophage CSA (GM-CSA) production by mononuclear phagocytes can be induced by IL-1. In the present study, the influence of culture conditions on the production of GM-CSA was studied. It was found that both human sera and fetal bovine sera contain constituents - at present undefined - that induce GM-CSA production. These factors are distinct from IL-1 and lipopolysaccharide. In selected experiments, no GM-CSA-inducing effect of serum was found, suggesting that the effect may be donor-related. GM-CSA release in the presence of serum could be reduced by 40% after incubation of mononuclear phagocytes at low cell concentrations in methylcellulose, indicating that intimate cell-cell contact is an additional factor that enhances GM-CSA release.

DFFRY codes for a new human male-specific minor transplantation antigen involved in bone marrow graft rejection.

Graft rejection after histocompatibility locus antigen (HLA)-identical stem cell transplantation results from the recognition of minor histocompatibility antigens on donor stem cells by immunocompetent T lymphocytes of recipient origin. T-lymphocyte clones that specifically recognize H-Y epitopes on male target cells have been generated during graft rejection after sex-mismatched transplantation. Previously, 2 human H-Y epitopes derived from the same SMCY gene have been identified that were involved in bone marrow graft rejection. We report the identification of a new male-specific transplantation antigen encoded by the Y-chromosome-specific gene DFFRY. The DFFRY-derived peptide was recognized by an HLA-A1 restricted CTL clone, generated during graft rejection from a female patient with acute myeloid leukemia who rejected HLA-phenotypically identical bone marrow from her father. The identification of this gene demonstrates that at least 2 genes present on the human Y-chromosome code for male-specific transplantation antigens.

Interleukin-1 (22-K factor) induces release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes.

An electrophoretically pure preparation of natural human interleukin-1 (IL-1) was shown to stimulate in vitro colony formation in human bone marrow cultures. Day 4 myeloid cluster-forming cells (CFC), as well as early (day 7) and late (day 10) granulocyte-macrophage colony-forming units (CFU-GM) were stimulated in a dose-dependent fashion. At optimal concentrations of IL-1, the number of day 4 CFC reached 72%, the number of day 7 CFU-GM reached 32%, and the number of day 10 CFU-GM reached 80% of the respective numbers of colonies obtained by addition of crude leukocyte-conditioned medium (LCM). The IL-1-induced stimulatory effect on CFU-GM growth could be completely neutralized by a rabbit anti-IL-1 antiserum. Colony growth was abrogated by depleting the marrow cell suspensions of phagocytic cells prior to IL-1 addition. Conversely, the effect could be reintroduced by addition of marrow-derived adherent cells to bone marrow cell suspensions that had been depleted of both phagocytic and E rosetting T cells. Furthermore, media conditioned by bone marrow-derived adherent cells or by peripheral blood mononuclear phagocytes in the presence but not in the absence of IL-1, stimulated in vitro colony growth of phagocyte-depleted bone marrow cell suspensions. These results indicate that IL-1 induces release of granulocyte-macrophage colony-stimulating activity (GM-CSA) from human mononuclear phagocytes.

Cerebral venous and sinus thrombosis with cerebrospinal fluid circulation block after the first methotrexate administration by lumbar puncture.

We report a patient treated for small lymphocytic lymphoma/leukemia with cerebral venous and sinus thrombosis (CVST) after lumbar puncture with intrathecal administration of methotrexate (MTX). He also developed a cerebrospinal fluid flow block. This is the first report of an association between lumbar puncture and intrathecally administered MTX and the development of CVST. Intrathecal treatment in this patient was discontinued and he was successfully treated with high-dose low-molecular-weight heparin subcutaneously.

Perioperative blood transfusion and cancer prognosis. Different effects of blood transfusion on prognosis of colon and breast cancer patients.

A detailed retrospective analysis was undertaken of the effect of perioperative blood transfusion on long-term survival of 113 patients with Dukes' Stages A, B and C1 cancer of the colon and 383 patients with invasive cancer of the breast who were treated in our institution between 1973 and 1978 and followed for 5 to 10 years. In the patients with colon cancer, a significant adverse effect of transfusion on long-term survival was seen. In this group there was a cumulative 5-year overall survival of 48% for the transfused and 74% for the nontransfused patients (P = 0.007, log-rank test). Perioperative blood transfusion was associated with a relative risk of 3.42 for all deaths (P = 0.005) and 4.25 for death due to cancer (P = 0.03), after adjustment for other important variables such as age, sex, stage, location of tumor, surgical procedure, and preoperative hemoglobin level. In contrast, in our study group of patients with breast cancers, who all underwent a modified radical mastectomy, no effect of blood transfusion on long-term survival was seen. Multivariate analysis adjusting for size of tumor, number of positive regional lymph nodes, menopausal status, estrogen receptor status and the addition or absence of chemotherapy, did not show any increased risk in all deaths or death due to cancer associated with blood transfusion. Although no definite explanation is available, our data show that there seems to be a difference in the relationship between perioperative blood transfusion and survival for colon and breast cancer patients.

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta.

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1-stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.

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.

Interleukin 1 induces human marrow stromal cells in long-term culture to produce granulocyte colony-stimulating factor and macrophage colony-stimulating factor.

Pure interleukin 1 (IL 1) was found to stimulate established human bone marrow stromal layers in long-term culture to produce colony-stimulating activity (CSA). Maximal concentrations in the culture medium were reached 24 hours after a single IL 1 pulse. The effect could be neutralized by a specific rabbit anti-IL 1 antiserum. Stromal layers, once stimulated by IL 1, continued to release CSA into the culture medium in the absence of exogenous IL 1. A second IL 1 pulse induced CSA release in an identical manner, as did the primary stimulation, indicating that the CSA released was actively produced. Using specific immunologic assays, both granulocyte colony-stimulating factor (G-CSF) and macrophage CSF (M-CSF) could be identified in the culture supernatants, and production of both factors was inducible by IL 1. Shortly after initiation of the long-term marrow cultures "spontaneous" G-CSF and M-CSF release occurred. The release of G-CSF diminished following addition of the anti-IL 1 antiserum, indicating that endogenous production of IL 1 by stromal cells had contributed to this effect. These results further support the role of IL 1 as an important modulator of CSF production by cells of the hematopoietic microenvironment.

Lymphokine-activated killer cells selectively kill tumor cells in bone marrow without compromising bone marrow stem cell function in vitro.

Although it has been demonstrated that lymphokine-activated killer (LAK) cells kill tumor cells in a selective way without being toxic to a variety of normal cells, contradictory results exist about the possible toxicity of natural killer (NK) and LAK cells for hematopoietic progenitor cells. Therefore, the cytolytic activity and growth inhibitory effects of LAK cells on normal bone marrow progenitor cells and the ability of LAK cells to eliminate neoplastic hematopoietic cells from populations of bone marrow cells in vitro was studied. The results of these experiments show the following: (1) LAK cells have little cytolytic activity against normal bone marrow cells; (2) normal bone marrow cells fail to cold target compete for the killing of the hematopoietic tumor cell lines K562 and HL60 or freshly frozen acute myelocytic leukemia (AML) blast cells by LAK cells; (3) LAK cells inhibit the growth of K562 and HL60 to more than 90% in clonogenic assays; (4) LAK cells have no inhibitory effect on hematopoietic progenitor growth in CFU-GM (colony-forming unit-granulocytes, macrophages), CFU-E (colony-forming unit-erythrocytes), BFU-E (burst-forming units-erythrocytes), or CFU-GEMM (colony-forming unit-granulocytes, erythrocytes, macrophages, megakaryocytes) assays. These results indicate that LAK cells have low toxicity for normal bone marrow and that LAK activity against tumor cells is not adversely affected by the presence of normal bone marrow cells. The differences in cytolysis and growth inhibition of neoplastic hematopoietic cells and hematopoietic progenitor cells by LAK cells in vitro could create a therapeutic index that might allow the use of LAK cells for cleansing of the autologous bone marrow graft and for adjuvant therapy in combination with autologous bone marrow transplantation without compromising the reconstitution of the bone marrow in the host.

Generation of donor-derived antileukemic cytotoxic T-lymphocyte responses for treatment of relapsed leukemia after allogeneic HLA-identical bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) has been associated with an antileukemic effect, the graft-versus-leukemia (GVL) reactivity. Since T-cell depletion of the bone marrow graft performed to reduce the incidence and severity of graft-versus-host disease (GVHD) after BMT has been associated with an increase risk of relapse, the GVL reactivity has been attributed to the T lymphocytes from the graft. Previously, we demonstrated that leukemia-reactive cytotoxic T-lymphocyte (CTL) lines and clones could be generated from the peripheral blood of HLA-genotypically identical siblings of patients with leukemia by stimulation of the donor cells with irradiated leukemic cells from the patients. HLA class I as well as class II restricted CTL clones could be generated that recognized the leukemic cells. Some clones recognized the leukemic cells from the patient, but not the interleukin (IL)-2-stimulated lymphocytes from the same individual. To explore the possibility of clinically using donor-derived CTL lines directed against the leukemic cells from patients who relapsed after allogeneic BMT, a pilot study was performed using eight donor-recipient combinations. In seven of eight combinations donor-derived CTL lines could be generated that showed specific lysis of the leukemic cells from the patient. In five of these cases, the CTL lines showed reactivity with the leukemic cells, but not with the IL-2-stimulated lymphocytes from the same individual. In two cases, the CTL lines were cytotoxic for the IL-2-stimulated lymphoblasts as well as the leukemic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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.