Cytotoxic potential of anti-CD7 immunotoxin (WT1-ricin A) to purge ex vivo malignant T cells in bone marrow

With the perspective of bone marrow purging in autologous transplantation, we investigated the cytotoxicity of the anti-T cell immunotoxin (IT) WT1-ricin A (anti-CD7) to malignant T cells obtained from patients with T cell acute lymphoblastic leukaemia or lymphoma. The cytotoxic efficacy of IT was based on the extent of protein synthesis inhibition. Cytotoxicity of IT to malignant T cells showed a dependency on antigen density comparable to the T cell lines GH1, CEM, Jurkat, HSB-2 and HPB-ALL and was enhanced considerably in the presence of 6 mM ammonium chloride. The ultimate proof of cell kill can only be obtained from clonogenic assays; however, culturing of malignant T cells was not feasible. Therefore these assays were performed with the cell line CEM that expresses comparable amounts of CD7 antigen as malignant T cells of most patients. More than 6-logs of CEM appeared to be eliminated after incubation with 10(-8) M WT1-ricin A. Immunotoxins are only effective after entering the target cell. The pattern of internalization of the IT was determined by means of 125I-WT1. After internalization the CD7 antigen was re-expressed on the cell membrane. This enables a long incubation period resulting in an increased elimination of malignant T cells. Even after 16 h the IT was still accumulated intracellularly. This pattern of continuous uptake of IT was reflected in a gradually increasing cytotoxicity with incubation time. Effective bone marrow purging can be carried out without adverse effects on progenitor cells with 10(-8) M WT1-ricin A. At that concentration the antibody binding capacity was saturated. We showed that the protein synthesis inhibition in malignant T cells by WT1-ricin A is comparable to the inhibition in T cell lines and that high amounts of CEM cells can be killed. These data suggest that cell lines can be used to test the efficacy of IT to malignant T cells. WT1-ricin A appears to be very potent for the purging of autologous bone marrow from patients with T cell malignancies.     

Ricin binding and protein synthesis inhibition in human hematopoietic cell lines

Previous studies showed that human blood cells exhibited varying sensitivities to ricin. To investigate the basis for these differences, ricin binding to human hematopoietic cell lines was assessed and correlated with in vitro ricin sensitivities. Resistant mutants were also isolated and characterized. Ricin binding to CEM cells was rapid, time-dependent, and blocked by unlabeled ricin, but not albumin; ricin binding approached saturation at 3 mumol/L. Scatchard analyses showed multiple classes of binding sites, with maximum and minimum Kd values estimated at 1.5 x 10(-8) mol/L and 2.5 x 10(-7) mol/L. At 4 degrees C, membrane-bound ricin dissociated slowly from the cell surface in the presence of unlabeled ricin, but greater than 95% of the surface-bound ricin was removed with 0.1 mol/L lactose. At 37 degrees C, ricin dissociated from the cell surface with biphasic kinetics. Ricin uptake at 37 degrees C increased linearly for 15 to 30 minutes and plateaued at levels representing 12% to 29% of the amount of ricin bound at 4 degrees C, depending on the cell line. Ricin binding at 4 degrees C varied two- to fivefold among hematopoietic cell lines and was reduced approximately tenfold by incubation with lactose. When compared with parent CEM cells, ricin-resistant CEM variants showed a greater than 95% reduction in ricin binding and showed no detectable binding with lactose added. However, these cells were as sensitive as parent CEM cells to an anti-T-cell ricin immunoconjugate. For all cells examined, there was a close correlation (r = +.9) between ricin bound per cell and in vitro ricin sensitivity. Human hematopoietic cells show widely varying ricin binding, indicating major differences in the carbohydrate content or structure of surface glycoproteins and glycolipids. These variations are probably the major determinant of nonspecific toxicity of ricin immunoconjugates.     

Amantadine potentiates T lymphocyte killing by an anti-pan-T cell (CD5) ricin A-chain immunotoxin

The studies described in this report demonstrate that 1-adamantanamine hydrochloride (amantadine) is a potent enhancer of the cytotoxic activity of the anti-pan-T lymphocyte (CD5) T101 monoclonal antibody conjugated to purified ricin A-chain (T101-immunotoxin; T101-IT). We also demonstrate that T101-IT in the presence of amantadine does not induce immunotoxin-mediated cytotoxicity in nontarget cells such as human marrow hematopoietic progenitor cells. These results provide further knowledge for the improvement of ex vivo purification of human bone marrow from normal or leukemic T cells prior to allogeneic or autologous stem cell transplantation, respectively. Furthermore, since amantadine has long been employed safely in human therapy, its use in conjunction with immunotoxins might be exploited in vivo.     

Use of multiple T cell-directed intact ricin immunotoxins for autologous bone marrow transplantation

The monoclonal antibodies (MoAb) T101, G3.7, 35.1, and TA-1 were conjugated to intact ricin using a thioether linkage. These MoAb detect, respectively, the CD5[gp67], CD7[p41], CD2[p50], and [gp95, 170] determinants that are found in the vast majority of cases of T cell acute lymphocytic leukemia (T-ALL). The resulting immunotoxins (ITs) and an equimolar mixture of these ITs were evaluated as potential purgative reagents for autologous transplantation in T-ALL. Leukemic cell lines were used to compare the kinetics of protein synthesis inactivation mediated by each IT. The cells were treated with IT in the presence of lactose in order to block the native binding of ricin. The observed rates of protein synthesis inactivation correlated with target antigen expression detected by fluorescence-activated cell sorter analysis. Of the four ITs, T101-ricin (T101-R) exhibited the fastest rate of inactivation, followed in order by G3.7-ricin, TA-1-ricin, and 35.1-ricin. At concentrations greater than 300 ng/mL, a cocktail containing an equimolar amount of all four ITs (referred to as the four- IT cocktail) exhibited kinetics that were as fast or faster than those of T101-R. The long-term cytotoxic effects of individual ITs and the four-IT cocktail were evaluated using a sensitive clonogenic assay. Each IT was specifically cytotoxic and inhibited 1 to 4 logs of clonogenic leukemic cells at doses (300 to 600 ng/mL) that can be used clinically. The four-IT cocktail was highly cytotoxic; a concentration of 300 ng/mL inhibited greater than 4 logs of leukemic cells while sparing the majority of committed (CFU-GM, CFU-E) and pluripotent (CFU- GEMM) hematopoietic stem cells. The determination of both short-term kinetics of protein synthesis inactivation and longer-term inhibition of clonogenic growth allowed new insight into cell killing by IT. Our results suggest that ITs continue to act on clonogenic target cells for a period of three to five days. Interestingly, the four-IT cocktail was not as potent against clonogenic leukemic cells as T101-R alone, although it exhibited kinetics of protein synthesis inhibition that were as fast as those of T101-R alone. This finding suggests that internalized ITs may differ in the length of time they remain active within the cell. Our results also demonstrate the importance of using several different assays to evaluate IT reagents.     

Autologous transplantation of bone marrow purged in vitro with anti-CD7- (WT1-) ricin A immunotoxin in T-cell lymphoblastic leukemia and lymphoma

Seven patients with high-risk acute T-cell lymphoblastic leukemia (T- ALL) and six with T cell lymphoma (T-LL) were treated with autologous bone marrow transplantation (ABMT) after in vitro purging of their bone marrow with WT1 (CD7)-ricin A-chain immunotoxin. CD7 expression on the tumor cells showed large variations between the individual patients and was highly related to the specific cytotoxicity of WT1-ricin A. Incubation of bone marrow with up to 10(-8)mol/L WT1-ricin A in the presence of 6 mmol/L NH4Cl did not compromise the growth potential of the hematopoietic progenitors CFU-GM, CFU-GEMM, and BFU-E. Hematologic engraftment (greater than 10(9) leukocytes/L) occurred within a normal time period (median, 17 days). Seven patients are alive and in complete remission (CR) at 48+, 44+, 40+, 26+, 11+, 7+, and 6+ months after ABMT. Four patients relapsed within 6 months after ABMT. Two of them had the lowest CD7 expression on their tumor cells, the other two were transplanted in CR2 and CR3. Two patients died from transplantation related infections. The immunologic reconstitution was delayed, although the numbers of T cells reached normal levels within 1 month. The number of CD7+ cells remained low up to 1 year after transplantation. The T4/T8-ratio was decreased for at least 6 months. The T-cell response to mitogens recovered to normal levels after 1 year. This study shows that ABMT with WT1-ricin A purged bone marrow in high-risk T-cell malignancies results in a complete hematopoietic and a delayed immunologic reconstitution. The actuarial relapse free survival is 61% at 3 years.     

Optimal elimination of leukemic T cells from human bone marrow with T101-ricin A-chain immunotoxin

In view of bone marrow purging before autologous transplantation in T cell malignancies, an anti-human T cell immunotoxin (IT) has been prepared by coupling ricin A-chain to the monoclonal antibody T101 that binds the T1 differentiation antigen expressed by T lymphocytes as well as by T cell-derived hematologic malignancies. Using a sensitive and reliable clonogenic assay, optimal conditions were defined for the elimination of clonogenic human T leukemic cells among bone marrow cells. Maximal cytoreduction was obtained with IT at a dose of 2 micrograms/mL in the presence of 10 mmol/L NH4Cl. This treatment led to the reduction of more than six orders of magnitude of T101-positive clonogenic leukemic cells, with no harm to T101-negative cells. Moreover, we observed no toxicity of IT to human hematopoietic stem cells (CFU-GEMMT) derived from bone marrow of healthy volunteers. Thus, pretreatment of bone marrow samples with IT plus NH4Cl offers a safe, simple, reliable, and highly efficient means to eliminate undesirable leukemic T cells from the graft.     

Anti-Thy 1.2 monoclonal antibody linked to ricin is a potent cell-type-specific toxin.

The cell-type specificity of the toxin ricin, which ordinarily binds, enters, and kills cells through receptors containing galactose, has been altered by covalently binding it to a monoclonal antibody and by reversibly binding it to lactose. The antibody, a monoclonal rat IgG2b directed against the Thy 1.2 antigen, provides ricin with a new binding site for the murine thymus cell surface. Addition of lactose saturates the galactose-binding site on ricin and inhibits ricin from binding and killing cells via the galactose-containing receptors. The antibody-ricin hybrid protein, anti-Thy 1.2-ricin, formed with a thioether linkage, has been purified by size exclusion and affinity chromatography. When assayed by inhibition of protein synthesis of EL-4 cells, which express the Thy 1.2 antigen anti-Thy 1.2-ricin is equally as toxic as ricin on a molar basis. The hybrid protein toxicity is unchanged in the presence of 100 mM lactose, whereas unmodified ricin toxicity is reduced to 1% of its toxicity in the absence of lactose. This demonstrates the altered receptor specificity of the ricin hybrid. The cell-type specificity of the anti-Thy 1.2-ricin inhibition of protein synthesis correlates with the presence of the Thy 1.2 antigen. Anti-Thy 1.2-ricin at 4 microgram/ml in the presence of lactose inhibits protein synthesis within 3.5 hr by 60-80% in EL-4 cells but does not affect Thy 1.1 alloantigen and HeLa cells that lack the Thy 1 antigen. Anti-Thy 1.2-ricin in the presence of lactose selectively kills EL-4 cells at concentrations that do not kill AKR-K36 cells. This selectivity, expressed as the ratio of anti-Thy 1.2-ricin concentrations required to kill 40% of both cell types, is 700. Ricin-monoclonal antibody hybrids of this type combine a high degree of cell-type selectivity and toxicity and may have pharmacologic utility as antitumor reagents.     

An immunotoxin with therapeutic potential in T cell leukemia: WT1-ricin A

A conjugate of the monoclonal antibody WT1 and ricin A-chain was studied for its suitability for purging marrow of leukemic T cells for autologous transplantation in T cell acute lymphocytic leukemia (T- ALL). The conjugate was powerfully cytotoxic to the human T-ALL cell line, GH1, which expresses the WT1 antigen at a high density. Treatment of the cells with the conjugate at 10(-11) M reduced their rate of protein synthesis by 50%, and the inclusion of 6 mM ammonium chloride in the cultures enhanced the potency of cytotoxic effect by 10-100- fold. Clonogenic assays indicated that less than 0.1% of GH1 cells survived 3-hr exposure to the conjugate in ammonium chloride. WT1 alone did not react with multipotent (CFU-GEMM) hematopoietic progenitors in normal human bone marrow, as measured by fluorescence-activated cell sorting. Under conditions giving maximal killing of GH1 cells, there was no toxicity to multipotential progenitors in normal human marrow.     

Differential cytotoxicity of an ether lipid on lymphoma and bone marrow cells and its role in purging malignant lymphoid cells from remission bone marrow contaminated with tumor cells.

Four human clonogenic malignant lymphoid cell lines (CEM, Su-DHL-4, Li-A, and Raji) as well as normal human bone marrow stem cell progenitor cells were investigated for clonal in vitro growth before and after incubation with the ether lipid ET-18-OCH3 for various times (1, 4, and 18 h) and at increasing concentrations of the drug (25, 50, 75, and 100 micrograms/ml). The clonal growth of the malignant lymphoid cell lines was inversely correlated with concentrations and times of drug incubation. The antineoplastic effect of ET-18-OCH3 was further amplified by subsequent cryopreservation. In a situation of 4-h exposure to less than or equal to 50 micrograms/ml ET-18-OCH3 and subsequent cryopreservation, in which greater than 50% of the normal human bone marrow progenitor cells survived, 1-3 logs of the malignant lymphoblastoid cells were killed, indicating a potential value of this drug for bone marrow purging in lymphoid malignancy. In order to simulate the situation of autologous bone marrow transplantation (ABMT) in complete remission of the disease, we contaminated normal human bone marrow cells with malignant CEM or Su-DHL-4 lymphoid cells at a ratio of 100:1. Results show that 4 h of incubation with 75 micrograms/ml ET-18-OCH3 and subsequent cryopreservation can eliminate 2-3 logs of clonogenic cells of the malignant lymphoblastoid cell lines under conditions that allow recovery of greater than 50% of the normal human hematopoietic progenitors.     

Autologous bone marrow transplantation with marrow decontaminated by immunotoxin T 101 in the treatment of leukemia and lymphoma: first clinical observations

Four patients with T-cell malignancies of poor prognosis (three with non-Hodgkin's lymphoma and one with acute lymphoblastic leukemia) received the following consolidation therapy for complete or partial remission: cyclophosphamide (120 mg/kg) plus total-body irradiation, followed by reinfusion of cryopreserved autologous marrow previously purged in vitro by immunotoxin T 101 (SR 41322). This immunotoxin is made of the murine monoclonal T 101 antibody coupled to chain A of ricin. The doses of immunotoxin used were 10(-9) and 10(-8) M, and the durations of incubation were 4 and 20 hours at 37 degrees C. Recovery of progenitors CFUc and BFUe was total following incubation with immunotoxin T 101, but diminished after cryopreservation (15%-80% for CFUc, 33%-47% for BFUe), suggesting an increased fragility of the incubated progenitors to freezing. In every case, hematopoietic recovery occurred within normal time periods, with a wbc count greater than 10(9)/L and a platelet count greater than 50 X 10(9)/L on Day 22 (range, 15-31) and Day 21 (range, 22-47), respectively, demonstrating the feasibility of autologous bone marrow transplantation with marrow pretreated by immunotoxin. However, the slow recovery of lymphocytes and the development of severe infections in two patients may indicate that an in-depth study of immunological reconstitution after in vitro treatment of the marrow with immunotoxin T 101 is necessary.     

Immunotoxins containing saporin 6 and monoclonal antibodies recognizing plasma cell-associated antigens: effects on target cells and on normal myeloid precursors (CFU-GM)

Monoclonal antibodies 8A and 62B1, recognizing plasma cell-associated antigens, were covalently linked to saporin 6, a ribosome-inactivating protein similar to the A-chain of ricin. Both immunotoxins were tested on target human cell lines U266 and Raji, on non-target K562 cell line and on myeloid CFU-GM progenitors. The cloning efficiency and viability of target cells were strongly reduced by 8A-saporin 6 and 62B1-saporin 6 immunotoxins, with an ID50 up to 200,000-fold lower than free saporin 6, whilst the K562 non-target cell line was unaffected. Normal human myeloid precursors (CFU-GM) were inhibited by immunotoxins only to a limited extent. An application of this model for autologous bone marrow transplantation in multiple myeloma patients is proposed. Since no eradication of cloning target cells was achieved by a single immunotoxin, mixtures made with different antibodies could help to reach this goal.     

T-lymphocyte killing by T101-ricin A-chain immunotoxin: pH-dependent potentiation with lysosomotropic amines

To maximize T-lymphocyte killing with anti-pan-T-lymphocyte immunotoxin (IT), prepared by linking ricin A-chain to monoclonal antibody (MoAb) T101 (T101-RTA IT), we have established the nature and the extent of parameters that influence the sensitivity of T lymphocytes to the IT. We showed that peripheral blood T lymphocytes, which are much less susceptible than malignant T cells to the T101-RTA IT, could become highly sensitive to the IT when used in conjunction with NH4Cl. However, enhancement of the IT by NH4Cl only occurred when the pH rose above neutrality. This pH-sensitive process of IT activation by NH4Cl, which led to an all-or-nothing effect within an extremely narrow pH window of 0.7 pH unit width, is due to the fact that NH3 is the effective enhancing component of NH4Cl. We also showed that F(ab')2 or Fab containing IT were much more effective than those produced using the whole IgG counterpart. From these data, we defined a procedure for an optimal and specific elimination of T lymphocytes in vitro by treating them with (Fab)T101-RTA at 10(-8) mol/L at pH 7.8 in the presence of NH4Cl for two hours. This peripheral blood cell processing elicited an abrogation of three logs of functional T-cell response. Under the same conditions, there was no reduction in the number of marrow hematopoietic precursor granulocyte-macrophage colony-forming units (CFU-GM).     

Anti-CD38-blocked ricin: an immunotoxin for the treatment of multiple myeloma [see comments]

We report the development of a potent anti-CD38 immunotoxin capable of killing human myeloma and lymphoma cell lines. The immunotoxin is composed of an anti-CD38 antibody HB7 conjugated to a chemically modified ricin molecule wherein the binding sites of the B chain have been blocked by covalent attachment of affinity ligands (blocked ricin). Conjugation of blocked ricin to the HB7 antibody has minimal effect on the apparent affinity of the antibody and no effect on the ribosome-inactivating activity of the ricin A-chain moiety. Four to six logs of CD38+ tumor cell line kill was achieved at concentrations of HB7-blocked ricin in the range of 0.1 to 3 nmol/L. Low level of toxicity for normal bone marrow (BM) granulocyte-macrophage colony- forming units (CFU-GM), burst-forming units-erythroid (BFU-E), colony- forming units-granulocyte/erythroid/monocyte/macrophage (CFU-GEMM) cells was observed. Greater than two logs of CD38+ multiple myeloma cells were depleted from a 10-fold excess of normal BM mononuclear cells (BMMCs) after an exposure to HB7-blocked ricin under conditions (0.3 nmol/L) that were not very toxic for the normal BM precursors. HB7- blocked ricin was tested for its ability to inhibit protein synthesis in fresh patients' multiple myeloma cells and in normal BMMCs isolated from two healthy volunteers; tumor cells from four of five patients were 100-fold to 500-fold more sensitive to the inhibitory effect of HB7-blocked ricin than the normal BM cells. HB7 antibody does not activate normal resting peripheral blood lymphocytes, and HB7-blocked ricin is not cytotoxic toward these cells at concentrations of up to 1 nmol/L. The potent killing of antigen-bearing tumor cells coupled with a lack of effects on peripheral blood T cells or on hematopoietic progenitor cells suggests that HB7-blocked ricin may have clinical utility for the in vivo or in vitro purging of human multiple myeloma cells.     

Human T-cell antigens defined by monoclonal antibodies. Absence of T65 on committed myeloid and erythroid progenitors

The complement-dependent cytotoxicity of monoclonal T-cell antibody (T101) for normal and abnormal hemopoietic progenitors was assessed. T101 demonstrated toxicity for normal T-colony-forming cells from peripheral blood and bone marrow. Cytotoxicity was absent for normal peripheral blood and bone marrow granulocytes/macrophage (CFU-C) and erythroid (BFU-E) progenitors. The antibody was also not toxic for peripheral blood blast progenitors from patients with acute myelogenous leukemia (AML). These studies indicate the absence of the antigen defined by T101 (T65) from normal progenitor cells and from blast progenitors in patients with AML. T101 may be used in the treatment of T-cell malignancies and in the prevention of graft-versus-host disease (GVHD) without damage to normal progenitor cells.     

Immunotoxins containing saporin linked to different CD2 monoclonal antibodies: in vitro evaluation

In this study we describe immunotoxins prepared with different CD2 monoclonal antibodies (mAbs) and a ribosome-inactivating protein, saporin. The CD2 immunotoxins were tested on different models. Anti-CD2–saporin conjugates inhibited protein synthesis by a neoplastic CD2+ cell line (SKW-3) and by an interleukin 2 dependent polyclonal CD2+ lymphoid cell culture (T lymphoblasts), with IC₅₀s ranging from 10^-13 m to 10^-11 m (as saporin). Similar results were obtained with proliferation inhibition tests (³H-thymidine incorporation) on phytohaemagglutinin (PHA) driven lymphoid cultures and on mixed lymphocyte culture activated lymphocytes. Moreover a CD2–ricin A chain conjugate was less effective than an analogous immunotoxin containing the same CD2 mAb and saporin in inhibiting lymphocyte proliferation induced by PHA (IC₅₀ approximately 10^-9 m as ricin A chain versus 10^-12 m as saporin). The conjugates were not toxic on bone marrow stem cells. These results suggest that CD2–saporin immunotoxins could represent an effective tool for CD2+ lymphomas or leukaemias, and for T-dependent immune disorders, such as transplanted organ rejection and graft-versus-host disease.     

T cell chronic lymphocytic leukemia: presence in bone marrow and peripheral blood of cells that suppress erythropoiesis in vitro.

The pathogenesis of the anemia associated with malignancy was investigated in a patient with T cell chronic lymphocytic leukemia. The plasma clot culture system was used as a measure in vitro of erythropoiesis. The patient's peripheral blood and marrow T lymphocytes obtained both before and after transfusion therapy suppressed erythroid colony formation by normal human bone marrow cells. Pretreatment of the patient's bone marrow T cells by antithymocyte globulin (ATG) and complement reversed this suppression. In addition, pretreatment of the patient's marrow cells with ATG and complement markedly augmented erythropoiesis in vitro. The expression of erythroid activity caused by the selective destruction of the suppressor T lymphocytes in the patient's bone marrow with ATG and the suppression of normal erythropoiesis by the patient's bone marrow and peripheral blood lymphocytes suggest that interaction between the malignant T cell and the erythropoietin-responsive stem cell is important in production of anemia in this patient.     

Specific toxicity of 2-chlorodeoxyadenosine toward resting and proliferating human lymphocytes

2-Chlorodeoxyadenosine (CdA), an adenosine-deaminase-resistant purine deoxynucleoside, is markedly toxic toward human T-lymphoblastoid cell lines in vitro and is an effective agent against L1210 leukemia in vivo. The present studies have examined the toxicity, and in some cases, metabolism, of CdA in (1) multiple established human cell lines of varying phenotype, (2) leukemia and lymphoma cells taken directly from patients, (3) normal bone marrow cells, and (4) normal peripheral blood lymphocytes. Nanomolar concentrations of CdA blocked the proliferation of lymphoblastoid cell lines with a high ratio of deoxycytidine kinase to deoxynucleotidase. The drug had virtually no effect on the growth of cell lines derived from solid tissues. The CdA inhibited the spontaneous uptake of tritiated thymidine by many T and non-T, non-B acute lymphoblastic leukemia cell specimens at concentrations less than or equal to 5 nM. The same concentrations did not impair either thymidine uptake or granulocyte-monocyte colony formation by normal bone marrow cells. In common with deoxyadenosine, but unlike several other agents affecting purine and purine metabolism, CdA was lethal to resting normal T lymphocytes and to slowly dividing malignant T cells. In both resting and proliferating lymphocytes, the CdA was phosphorylated by deoxycytidine kinase and entered a rapidly turning over nucleotide pool. Dividing lymphocytes also incorporated abundant CdA into DNA. The selective toxicity of CdA toward both dividing and resting lymphocytes may render the drug useful as an immunosuppressive or antileukemic agent.     

Granulocyte/macrophage progenitor cells from peripheral blood and bone marrow differ in their response to prostaglandin E1

Prostaglandins of the E series (PGE) inhibit proliferation of normal bone marrow granulocyte/macrophage progenitors (CFU-GM). Circulating CFU-GM are known to differ from marrow CFU-GM in many characteristics, and in the present study, we compared the effect of PGE1 on circulating and bone marrow progenitors in normals and in patients with chronic myelogenous leukemia (CML). PGE1 caused a dose-dependent inhibition of normal marrow CFU-GM. Circulating CFU-GM were inhibited only at concentrations of 10(-5) mol/L or greater, and progenitor proliferation was, in fact, significantly stimulated at PGE1 concentrations between 10(-8) and 10(-6) mol/L. Bone marrow CFU-GM from patients with CML were inhibited in a manner similar to that of normal bone marrow. Circulating cells from patients with CML were, however, less sensitive to PGE1 inhibition than CML bone marrow cells and demonstrated a pattern intermediate between normal circulating and normal marrow progenitors. These studies suggest that peripheral blood and bone marrow contain different progenitor cell populations.     

Cytogenetics and Granulopoietic Effects of Bone Marrow Fibroblastic Cells in Fanconi's Anaemia

S ummary. To determine if abnormalities exist in the bone marrow stroma in Fanconi's anaemia, we studied the cytogenetic composition of in vitro bone marrow fibroblastic cells (FC) from a patient with this disorder and compared it to those obtained from skin fibroblasts, peripheral blood lymphocytes, and direct bone marrow preparations. The presence of granulocytic progenitors in bone marrow and T lymphocyte colonies in peripheral blood was also determined in addition to the ability of this patient's FC to stimulate normal granulocytic progenitors. We found that the FC had far fewer chromosomal abnormalities and stimulated normally the growth of granulocyte colonies. Granulocyte progenitors were not found, but T lymphocyte colonies were abundant. These results support the concept that a defect in haematopoietic stromal elements is not responsible for the aplasia developing in the disorder.     

Monoclonal rat anti-human lymphocyte antibody Campath-1 binds to T and B lymphocytes but effectively lyses only T cells

Binding and human complement-mediated T and B lymphocyte lysis were investigated in bone marrow samples obtained from 15 normal donors and 12 patients with a variety of malignant disorders undergoing marrow cryopreservation prior to autologous bone marrow transplantation. All marrow samples were obtained during remission except for one patient with neuroblastoma. The mononuclear cell fractions were collected and the distribution of B cell restricted markers (surface Ig and GP-70) and T cell surface markers (Leu-1 and rosettes with sheep red blood cells) were studied before and after marrow purging with Campath-1, a monoclonal rat anti-human lymphocyte antibody, and autologous serum as complement. Effective lysis of T lymphocytes (greater than 99.5%) was documented in all cases. However, although effective binding of Campath-1 to B lymphocytes was uniform, no effective lysis occurred. The data suggest that effective lysis of mature T lymphocytes can be accomplished while leaving normal B lymphocytes intact.