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Levels of transforming growth factor-beta (TGF-beta) activity in the conditioned medium of blood lymphocytes of twelve patients with aplastic anemia (AA), nine patients with myelodysplastic syndromes (MDS) and five normal volunteers were investigated. We were able to observe growth inhibitory activity on porcine endothelial cells only after acidification of the materials. The growth inhibitory activity is neutralized by anti-TGF-beta antibody. It indicates that TGF-beta exists as a latent form in the conditioned medium. On the basis of growth inhibition assay, the mean level of TGF-beta production of MDS patients was estimated to be 188 +/- 199 pg/1 x 10(7) cells and that of normal volunteers was 668 +/- 314 pg/1 x 10(7) cells. In contrast, the lymphocytes of almost all of the AA patients failed to produce detectable amounts of TGF-beta. No correlation between TGF-beta levels and peripheral blood parameters could be detected. Stimulation of lymphocytes by phytohemagglutinin is known to increase the production of TGF-beta. Induction of TGF-beta production was also observed in AA (45% of normal controls). Possible roles of the decreased production of TGF-beta in the pathogenesis of AA were discussed.

Granular lymphocytes (GLs) in patients with GL-proliferative disorders (GLPDs) are known to express the interleukin-2 receptor (IL-2R) beta chain (p70-75) constitutively and to proliferate in response to stimulation with IL-2 via the beta chain. In this report, we found that the anti-CD3 monoclonal antibody (MoAb) OKT3 could induce the proliferation of GLs from patients with T-cell lineage GLPDs (T-cell receptor-alpha beta+/CD3+16+), but not that of natural killer (NK) cell lineage GLs (T-cell receptor-alpha beta-/CD3-16+). In contrast, the anti-CD16 MoAb 3G8 that reacts with NK-lineage GLs could induce the proliferation of these GLs but not that of GLs with a T-cell phenotype. Furthermore, the anti-CD16 MoAbs CLB FcR gran1 (VD2) and OK-NK, which react with both T- and NK-lineage GLs, induced the proliferation of GLs with both T- and and NK-cell phenotypes. The proliferative response induced via the CD3 or IgG Fc receptor III (Fc gamma RIII: CD16) pathway was shown to be associated with the IL-2-dependent autocrine pathway by various findings, including the induction of endogenous IL-2 production, the coexpression of the IL-2R alpha chain (p55) and the IL-2R beta chain, and the inhibition of GL proliferation by anti-IL-2 or anti-IL-2R MoAb. These results suggest that GL proliferation is mediated at least partly through the IL-2-dependent autocrine pathway, and that the TCR/CD3 complex in T-cell phenotype GLs and the Fc gamma RIII in both T- and NK-cell phenotype GLs play a role in their activation in GLPDs.

The use of granulocyte colony-stimulating factor (G-CSF) after chemotherapy for acute myeloblastic leukemia (AML) has been reported. However, there is a drawback in that G-CSF may stimulate the proliferation of AML progenitors. To determine the parameter(s) indicative of responsiveness of AML blasts to G-CSF, various surface phenotypes of blasts were examined in relation to the blast colony formation stimulated by G-CSF in 39 AML patients. A correlation was found only with transferrin receptor positivity among the various phenotypes studied. The population mean of percentages of transferrin receptor-positive blasts in the group responding to G-CSF in vitro was significantly higher than that of blasts in the group not responding to G-CSF. A further correlation was found between transferrin receptor positivity and the number of G-CSF receptors on the blasts; that is, blasts expressing more G-CSF receptors have greater transferrin receptor positivity. In our previous study, we observed that blasts with a large number of G-CSF receptors produce more colonies in response to G-CSF. These results indicated that blasts expressing more transferrin receptors have a larger number of G-CSF receptors and may show more active proliferation in response to G-CSF. Therefore, the proliferative response of blasts to G-CSF can be predicted by examining transferrin receptor positivity. The clinical use of G-CSF in AML patients may be recommended when the patient's blasts have a low level of transferrin receptor expression. The measurement of transferrin receptors on blasts, instead of the rather complicated G-CSF receptor determination, would be a useful indicator for the safer application of G-CSF in AML patients.

We describe a case of multiple myeloma associated with myelofibrosis. This patient had hepatosplenomegaly, moderate anemia with anisocytosis and nucleated red blood cells, and Bence-Jones protein (kappa) in the urine. A bone marrow biopsy showed extensive marrow fibrosis and proliferation of numerous immature plasma cells containing kappa light chain in the cytoplasm. Melphalan-prednisolone therapy not only facilitated the disappearance of the immature plasma cells but also resulted in an improvement of myelofibrosis in the bone marrow. The immature plasma cell proliferation and marrow fibrosis in the bone marrow were seen again after interruption of chemotherapy. Therefore, this myelofibrosis may be secondary to the coexistent multiple myeloma.

In 12 patients with granular lymphocyte-proliferative disorders (GLPD), we studied the capacity of patient peripheral blood mononuclear cells (PBMC) to promote or suppress polyclonal IgG synthesis by normal non-T cells in pokeweed mitogen-containing medium using an enzyme-linked immunosorbent assay. During the experiments we found a patient whose PBMC possessed contrasuppressor function. The patient was a 27-year-old female with anaemia and lymphocytosis of CD3+CD8+ granular lymphocytes (GL). Reconstitution experiments using normal donor non-T cells and CD4+ and CD8+ T cells showed that addition of the patient's CD8+ cells abrogated the suppressor cell function of normal CD8+ T cells. The patient's PBMCs were CD3+, CD8+, Ia+, and Vicia villosa lectin-adherent characteristics which are consistent with those of normal blood contrasuppressor T cells. The T cell receptor beta and gamma genes were found to be monoclonally rearranged. Ultrastructurally, this patient's GLs exhibited clusters of dense cytoplasmic bodies, which were not detected in the GL of other patients with GLPDs. These results indicate that the clonal proliferation of contrasuppressor T lymphocytes had occurred in this patient.

An anti-CD3 Fab' x anti-CD10 Fab' bispecific hybrid F(ab')2 antibody (Ab) was generated. This bispecific Ab had a molecular mass of 100 to 110 Kd, and the capacity to react with both CD3+ T cells and CD10+ acute lymphoblastic leukemia (ALL) cells. We studied whether cytotoxic T lymphocytes (CTLs) could lyse patient CD10+ ALL cells after addition of the bispecific Ab. As effector CTLs, interleukin-2 (IL-2)-stimulated peripheral blood mononuclear cells (PBMCs) and CTL clones were used. When IL-2-stimulated PBMCs were assayed for cytotoxicity to 61Cr-labeled CD10+ ALL cells, their activity was shown to be markedly enhanced by the addition of the bispecific Ab. Most of the CTL clones established lacked cytotoxicity for CD10+ ALL cells, but addition of the bispecific Ab induced a significant level of cytotoxicity. CTLs derived from ALL patients also showed significant cytotoxicity for autologous CD10+ ALL cells after addition of the bispecific Ab. However, this Ab did not affect the cytotoxicity of CTLs when CD10- leukemic cells were used as the targets. These findings suggest that the bispecific Ab can be used for immunotherapy in patients with CD10+ ALL.

We investigated the proliferative effects of interleukins 1-7 (IL-1 to -7) on leukemic cells from 10 patients with T-lineage acute lymphoblastic leukemia (T-ALL). Five patients had CD7+4-8-acute leukemia, one had CD5+ acute leukemia, and four had CD1+ acute leukemia. To examine the proliferative effect of each interleukin, 3H-TdR incorporation method was used. In the presence of IL-1, no increase in 3H-TdR incorporation was observed for any of the T-ALL cells. With IL-2, 3H-TdR incorporation increased in cells from 5 out of 10 T-ALL patients, including those with CD7+4-8-, CD5+, and CD1+ acute leukemia. In the presence of IL-3 or IL-6, 3H-TdR incorporation increased in cells from 2 out of 5 patients with CD7+4-8- acute leukemia. However, CD5+ or CD1+ acute leukemia cells were not stimulated by IL-3 or IL-6. With IL-4, 3H-TdR incorporation was increased in the cells from 2 out of 5 patients with CD7+4-8- acute leukemia and in the cells of 2 of those with CD1+ acute leukemia. IL-5 increased the 3H-TdR incorporation by cells from 2 out of 5 patients with CD7+4-8- acute leukemia and 1 patient with CD1+ acute leukemia. IL-7 increased 3H-TdR incorporation in cells from all five CD7+4-8- acute leukemia and 2 of those with CD5+ or CD1+ leukemia. No synergistic effect was found when IL-7 and other cytokines were added to cells from the 3 patients with CD7+4-8- acute leukemia who were tested.

The number and the affinity of granulocyte colony-stimulating factor (G-CSF) receptors expressed by blast cells in acute myeloblastic leukaemia (AML) were determined using radiolabelled recombinant human G-CSF (rhG-CSF). Eighteen of 20 patients demonstrated specific binding, and Scatchard analysis revealed a single class of high affinity (Kd 15-130 pM) G-CSF receptors on the AML blasts. The number of G-CSF receptors varied from 55 to 1,200 per cell (mean 278). In the remaining two patients, specific binding was not observed. The number of G-CSF receptors did not differ significantly between various AML subtypes, but the mean receptor number was the highest on type M2 blasts. A chemical cross-linking study revealed that the G-CSF receptor has an approximate molecular weight of 140,000. Autoradiography showed heterogeneity of the distribution of G-CSF receptors on the AML blasts obtained from a single patient. The number of colonies stimulated by the addition of rhG-CSF varied from 0 to 566 per dish, and blast colony formation was observed in eight of 20 patients. The population mean of G-CSF receptor number expressed by blasts that formed colonies on stimulation with rhG-CSF was significantly higher than that on blasts which did not form colonies. These results suggest that a proliferative response of AML blasts to G-CSF may be predicted when the blasts express a large number of G-CSF receptors. Accordingly, it may be safer to restrict the clinical use of G-CSF to AML patients who have blasts with a low G-CSF receptor expression and no response to G-CSF in blast colony assay.

A 28-year-old male was admitted to our hospital because of hepatosplenomegaly and granular lymphocytosis. His peripheral leukocyte count was 3,000/microliters with 43% of granular lymphocytes (GL). These GLs were immunologically phenotyped as CD2+CD3-CD4-CD8-CD16+CD56+HLA-DR+ and were found that TcR genes coding beta and gamma chains were not rearranged. Chromosomal analysis of his GLs stimulated with IL-2 showed 47 XY, +8. This patient was diagnosed as a granular lymphocyte leukemia of natural killer cell type. Blood chemistry showed elevation of serum GOT, GPT and LDH values. The fever persisted until administration of prednisolone was initiated. But 40 days after, high fever appeared again and the liver and spleen were extremely enlarged. Combined chemotherapy was then started but resulted in no effects. He died of hepatic failure on the 77th day from admission. 47 XY, +8, that has been reported in acute non-lymphocytic leukemia and myelodysplastic syndrome, may be related to the pathogenesis in some cases of granular lymphocyte leukemia.

A new serum-free assay system for leukemic colony formation (leukemic colony-forming units, L-CFU) was established, and, utilizing this system, the colony-promoting activities of recombinant human colony-stimulating factors (rhCSFs) and the effectiveness of CSFs on cellular self-renewal capacity were investigated. The serum-free assay system included deionized bovine serum albumin (1%), cholesterol (7.8 micrograms/ml), and ASF 101 medium. The plating efficiencies obtained by culturing with phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) ranged from 0.01% to 1.35% in this system. Spontaneous colonies were observed in 9 out of 13 cases studied. Recombinant human granulocyte CSF (rhG-CSF), rh granulocyte-macrophage CSF (rhGM-CSF), rh interleukin 3 (rhIL-3), and rh interleukin 1 (rhIL-1) stimulated colony formation in 10, 9, and 6 out of 13, and 5 out of 11 cases, respectively. The magnitude of stimulation by each CSF ranged from 6% to 145%, 21% to 200%, 0% to 1229%, and 14% to 182% of PHA-LCM, respectively. In eight cases, blast colony assays in serum-free and serum-containing cultures were simultaneously performed. The magnitudes of responsiveness of each CSF differed in the two assay systems; this indicated some effect of fetal calf serum. The value of self-renewal capacity was also examined and compared with that in serum-containing culture. Self-renewal capacity could be maintained with a serum-free culture system. It showed marked variation from case to case, and there was no correlation between the primary colony formation and the self-renewal capacity in both culture systems. Taken together, the development of a completely serum-free culture system was found to be efficient as evaluated by a L-CFU colony assay.

We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody (MoAb), designated Mik-beta 1, which has been recently developed. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis. These results indicate that flow cytometric analysis using MoAbs, anti-Tac, and Mik-beta 1 is useful for detecting the expression of the IL-2R chains.

Endoscopic appearance of the gastrointestinal tract of a patient with severe hemorrhagic enteric graft-vs.-host disease (GVHD) is presented. A 29-year-old man with chronic myelogenous leukemia suffered from severe enteric GVHD after allogeneic bone marrow transplantation. Endoscopy showed hemorrhagic ulceration of the upper jejunum, terminal ileum, and colon at the onset of melena. Sections of biopsies were compatible with acute GVHD. Repeat endoscopy showed gradual healing of the lesions after steroid pulse and antilymphocyte globulin therapy, but the patient died of cytomegalovirus pneumonitis 14 months later. Autopsy revealed submucosal fibrosis of the small intestine and colon.

We previously demonstrated that human granulocyte-macrophage colony (granulocyte-macrophage colony-forming units, CFU-GM) formation was inhibited by interleukin 2 (IL-2)-treated lymphocytes and their conditioned medium (CM). In the present study, the mechanism of this suppression was investigated. When anti-interferon (IFN)-gamma antibody or anti-tumor necrosis factor (TNF)-alpha antibody was added to CFU-GM agar culture with IL-2-treated lymphocytes or their CM, the inhibition of CFU-GM colony formation was partially abrogated, whereas anti-TNF-beta antibody did not abolish the inhibitory effects. When anti-IFN-gamma and anti-TNF-alpha antibodies were added simultaneously, full recovery of colony formation was observed. In the CM of IL-2-treated lymphocytes, detectable levels of IFN-gamma (81 +/- 15 U/ml) and TNF (3.1 +/- 1.1 U/ml) were found. Addition of IFN-gamma and TNF-alpha at these concentrations to the agar culture inhibited CFU-GM colony formation. Taken together, these results indicate that inhibition of human CFU-GM colony formation by IL-2-treated lymphocytes and their CM is mediated by IFN-gamma and TNF-alpha generated from IL-2-treated lymphocytes.

The role of CD3 and CD8 Ag in CD16-mediated CTL triggering was studied in TCR-alpha beta+ and TCR-gamma delta+ granular lymphocytes (GL). In TCR-alpha beta+/CD3+4-8+16+ GL obtained from patients with GL-proliferative disorders, antibody-dependent cellular cytotoxicity was inhibited by anti-CD3 and anti-CD8 mAb. Anti-CD3 mAb also inhibited antibody-dependent cellular cytotoxicity activity of TCR-gamma delta+/CD3+4-8-16+ GL from a patient and that of TCR-gamma delta+/CD3+4-8+/-16+ T cell clones established from patients with proliferating TCR-gamma delta+ GL. In TCR-gamma delta+ T cell clones, cytotoxicity against Fc gamma R+ targets was induced by stimulation of CD16 Ag with anti-CD16 mAb, and such cytotoxicity was also inhibited by anti-CD3 mAb. These results indicate that CD3 and CD8 molecules play a regulatory role in CD16-mediated CTL triggering.

The effect of erythropoietin (Epo) on colony formation by blast progenitors in acute myeloblastic leukaemia other than erythroleukaemia was studied using a blast colony assay. Epo alone did not induce colony formation, but when it was used together with phytohaemagglutinin-stimulated leucocyte-conditioned medium (PHA-LCM) the number of leukaemic colonies significantly increased in nine out of 12 cases studies. Preincubation of Epo with anti-Epo antibody completely abolished this enhancement, indicating that the increase in colony numbers was caused by Epo itself. Cell surface phenotype analysis of colonies produced by Epo plus PHA-LCM showed no increase in percentages of erythroid and megakaryocyte lineages. The addition of Epo also increased the self-renewal capacity of leukaemic blast cells. Fresh leukaemic cells did not express Epo receptors, but they were induced after incubation with PHA-LCM. The present study thus showed that the proliferative response to Epo is not restricted only to the erythroid lineage, but also extends to AML blast cells other than those in erythroleukaemia in the presence of colony stimulating factors.

For the disease of the granular lymphocytes (GL) that contain azurophilic granules to proliferate in the peripheral blood, which is often complicated by anemia or neutropenia and which generally develop into chronic disease, the new designation of granular lymphocyte-proliferative disorders (GLPD) is being proposed. This disease include T-GL having CD3 antigen which forms a complex with T cell Ag receptor (TCR-alpha beta), and NK-GL which is CD3-negative but CD16- or NKH-1-positive, having non-MHC-restricted cytotoxicity. The two cases presented here demonstrate the characteristics of T-GL or NK-GL, however, while one case with NK-GL showed spontaneous decrease of GL, improvement of neutropenia and anemia without any treatment, the other case with T-GL became intractable and required repeated blood transfusion. On such differences in the clinical development of the disease, we have conducted investigations including a review of the literature to see whether there is any correlation between the characteristics of the proliferating cells and pathologic conditions of the disease, and whether this disease is indeed tumorous.

The gamma delta T-cell receptor-positive (gamma delta TCR+) lymphocytes were markedly expanded up to 68% of peripheral blood lymphocytes in a case with type I autoimmune polyglandular syndrome and pure red blood cell aplasia (PRCA). The gamma delta TCR+ cells showed CD4 negative, 16% dim-CD8 positive and 10% to 46% human leukocyte antigen-D-related (HLA-DR) positive, and exhibited no monoclonality as assessed by the patterns of TCR gene rearrangements. Functional studies revealed that the proliferative responses of the patient's peripheral blood mononuclear cells (PBMC) were severely depressed to candida antigen, alloantigens, and autoantigens (non-T cells). The gamma delta TCR+ cells had no suppressive effect on the proliferative response of the alpha beta TCR+ cells to candida. The patient's PBMC, isolated gamma delta TCR+ cells but not alpha beta TCR+ cells, exhibited non-major histocompatibility complex (MHC)-restricted cytotoxicity. Furthermore, the patient's PBMC and isolated gamma delta TCR+ cells inhibited burst-forming units-erythroid (BFU-E), but not colony-forming units/granulocyte-macrophage (CFU-GM). Supernatants derived from the patient's T cells similarly inhibited BFU-E but not CFU-GM. The clinical course of the patient also showed a close correlation between the decreased number of total lymphocyte counts, especially HLA-DR + gamma delta TCR+ cell counts, and recovery from PRCA. These observations suggest that the gamma delta TCR+ cells might be functional in vivo and involved in the pathogenesis of PRCA in this patient.

By Northern blot analysis using a cDNA clone of the perforin gene, we studied the levels of perforin mRNA in peripheral blood mononuclear cells from 11 cases of granular lymphocyte-proliferative disorders (GLPDs). The granular lymphocytes studied were characterized by morphologic, immunophenotypic, and immunogenotypic analyses. Cytolytic functions of the lymphocytes assayed included nonmajor histocompatibility complex-requiring cytotoxicity, anti-CD3-redirected cytotoxicity, antibody-dependent cellular cytotoxicity, and lectin-dependent cellular cytotoxicity. The results showed that in lymphocytes with strong cytolytic functions high levels of perforin mRNA existed, whereas in lymphocytes with weak or undetectable levels of cytolytic functions, low levels of perforin mRNA existed. Because the levels of perforin mRNA correlated with those of cytolytic functions, perforin is probably a mediator in cytolytic functions of granular lymphocytes in patients with GLPDs. When the lymphocytes were cultured for 1 day, however, the levels of cytolytic activity were increased, and those of perforin mRNA were decreased. Therefore, we cannot rule out the possibility that factors other than perforin protein are involved in the cytolytic functions of granular lymphocytes.

The effect of interleukin-5 (IL-5) on blast colony formation of acute myeloblastic leukaemia (AML) cells was studied. IL-5 showed a weak but significant colony stimulatory effect in one out of six cases studied. IL-5 induced eosinophilic colony growth in this case, and chromosomal and histochemical analyses strongly suggested that these eosinophilic colonies were derived from leukaemic blast progenitor cells. To our knowledge, this is the first report that IL-5 induces eosinophilic colonies on fresh AML cells. A possible induction of differentiation of AML blast cells by IL-5 is suggested.

Five patients with refractory anemia were treated with high-dose methylprednisolone. An immediate and long-lasting hematological improvement without serious side effects was achieved in two of the patients, although chromosomal abnormalities persisted in both. The clinical course and laboratory data of these two patients are described. Restoration of normal hematopoiesis was achieved regardless of the enhancement of colony formation of granulocyte progenitor cells by the simultaneous addition of hydrocortisone in vitro. The same treatment was given to six patients who had refractory anemia with an excess of myeloblasts (RAEB), but no improvement was observed in any of these patients. It appears that high-dose methylprednisolone can be valuable in the treatment of refractory anemia, but is not useful for RAEB.

A case (41 year old man) of idiopathic hypereosinophilic syndrome is presented. Typical eosinophilic endocarditis with restrictive cardiac failure and ventricular thrombosis was demonstrated by various clinical investigations, including cardiac pool scan and biopsy. The patient died after 22 months, and an autopsy revealed disseminated eosinophilic infiltration in the pancardium of the whole heart, aorta, main pulmonary artery, liver, and spleen.

The colony stimulating activities (CSAs) of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant interleukin-3 (rIL-3) on blast cells from various types of acute myeloblastic leukemia (AML) patients were studied using an in vitro blast colony assay. The stimulatory magnitude of rGM-CSF was highest in type M4 blasts but almost non-existent in type M1 blasts. Some preferential stimulatory action of rG-CSF was observed in type M2, M4 blasts and in overt leukemia in patients with refractory anemia with excess of blasts. On the other hand, the stimulatory activity of rIL-3 did not differ much with the AML subtype. Exposure to rGM-CSF, rG-CSF or rIL-3 did not alter the cellular phenotype or morphology of the leukemic blasts or the self-renewal capacity of blast progenitors. Simultaneous addition of rGM-CSF, rG-CSF and rIL-3, in various combinations, produced variable results and a simple additive effect was seen in 40% of the cases studied and either a significantly larger or smaller value in the remaining cases. Recombinant GM-CSF, rG-CSF and rIL-3 when added together did not reconstitute CSA of phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM).

We investigated the effects of recombinant human interleukins 1 to 6 (rIL-1 to -6) on the proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). The 3H-TdR incorporation in the presence of various cytokines was examined in cells from 14 patients: 12 with B-lineage ALL, 1 with T-lineage ALL and 1 with biphenotypic leukemia. In B-lineage ALL, a significant increase in 3H-TdR incorporation was observed in 5/12 cases (42%) in the presence of rIL-1 alpha, in 10/12 cases (83%) with rIL-2, in 9/12 cases (75%) with rIL-3, in 3/6 cases (50%) with rIL-4, in 4/6 cases (67%) with rIL-5, and in 4/12 cases (33%) with rIL-6. The mean stimulation index of the cells showing a positive response was 1.74 for rIL-1 alpha, 3.40 for rIL-2, 2.55 for rIL-3, 1.86 for IL-4, 1.56 for rIL-5, and 2.97 for rIL-6. T-lineage ALL cells were stimulated only in the presence of rIL-2, and biphenotypic leukemia cells were not stimulated by any of the cytokines tested.

Cell co-operation in the generation of secondary cytotoxic responses was studied by selectively sensitizing lymphocytes in mixed lymphocyte culture (MLC) across I or D region difference and by combining the primed lymphocytes in the secondary MLC. Secondary cytotoxic responses were induced in D-region-primed lymphocytes by restimulation with the original priming D-region antigens, by co-culturing with the I-region-primed lymphocytes in the presence of the priming I-region antigens, or by cell-free supernatants obtained 24 h after the restimulation of D-region-primed lymphocytes and I-region-primed lymphocytes, The active MLC supernatants produced by both I-region-primed and D-region-primed cells also induced accelerated proliferative responses in D-region-primed lymphocytes. Heat-treatment or ultraviolet irradiation of the stimulator cells eliminated the capacity of the cells to induce the production of CTL-helper factor in I-region-primed and D-region-primed lymphocytes. It was concluded that both I-region-primed and D-region-primed lymphocytes produce a cell-free factor which induces proliferation and secondary cytotoxicity in D-region-primed lymphocytes. The possible participation of D-region reactive helper T cells and D-region reactive cytotoxic T cells in the cytotoxic responses to D-region antigens in the absence of I-region difference is discussed.

Medullasin, a new serine protease found in bone marrow cells, increased markedly human natural killer cell activity. Whereas the natural killer cell activity measured immediately after the treatment with medullasin remained almost on the same level as the control, an incubation at 37 degrees C for several hours increased markedly the natural killer cell activity of the lymphocytes treated with medullasin. Enhancement of the natural cytotoxicity was caused by the treatment with physiologic concentrations of the protease (5-20 micrograms/ml). Inhibitors of medullasin such as phenylmethylsulfonyl fluoride and elastatinal prevented the activation of natural cytotoxicity. Depletion of lymphocytes bearing Fc receptors for IgG abolished the enhancement of natural killer cell activity by medullasin. Interferon activity was not detected in the supernatant of lymphocyte cultures stimulated with medullasin. The medullasin enhanced further the natural killer cell activity of lymphocytes stimulated with interferon. Medullasin activity was detected neither in unstimulated nor stimulated (by concanavalin A or phytohemagglutinin) human lymphocytes. The protease was released easily from human mature granulocytes into culture medium. It is considered from these results that the level of human natural killer cell activity is regulated by medullasin released by mature granulocytes.

To determine whether vindesine receptors are present in human leukaemic cells, K562 cells (established from chronic myelogenous leukaemia in blastic crisis) were incubated with 3H-vindesine. Binding of 3H-vindesine increased with incubation time and with increase in number of K562 cells. However, when excessive amounts of nonradioactive vindesine were added, the 3H-vindesine was displaced. Binding of 3H-vindesine was only inhibited by vinblastine, vincristine and vindesine. These results suggest that K562 cells have receptors for vindesine and that these receptors are common to vinca alkaloids. Scatchard analysis showed that the number of vindesine receptors differed according to the kind of cells tested. K562 and a T-cell leukaemia-derived cell line, MOLT-4, had more receptors than an acute promyelocytic leukaemia-derived cell line, HL-60, and normal blood lymphocytes. The degree of vindesine affinity to receptors did not differ markedly among the above-mentioned cells.

The effect of recombinant interferons (rIFNs) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units, L-CFU) from the peripheral blood of patients with acute myeloblastic leukemia (AML) was examined. rIFN-alpha, rIFN-beta, and rIFN-gamma inhibited L-CFU in a dose-dependent manner. When 3000 U/ml rIFN-alpha, -beta, and -gamma were added, L-CFU were suppressed to 20%, 12%, and 40% of the control cultures, respectively. The concentrations of rIFN-alpha, -beta, and -gamma required for 50% inhibition of colony formation were 63, 29, and 250 U/ml, respectively. The self-renewal capacity of L-CFU was inhibited by rIFNs in a dose-dependent manner, the degree of inhibition being the same for all three rIFNs. These rIFNs also inhibited normal granulocyte-macrophage progenitors to a similar degree as L-CFU. Taken together, these in vitro studies indicate that these rIFNs may be efficacious in the treatment of AML, though development of granulocytopenia may be observed as a complication of IFN therapy.

Although protein-losing enteropathy can be associated with a variety of disorders, only three cases have been described in association with systemic lupus erythematosus. In the case described herein, protein-losing enteropathy was the only clinical manifestation of systemic lupus erythematosus. Small intestinal biopsy revealed edema and mild mononuclear cell infiltration in lamina propria mucosae and no evidence of lymphangiectasia. X-ray studies of the gastrointestinal tract were normal. Protein-losing enteropathy responded to high-dose corticosteroid therapy. Protein-losing enteropathy should be suspected as a possible cause of unexplained hypoalbuminemia in systemic lupus erythematosus.

Human megakaryocyte colony formation was observed in serum-free agar cultures with a medium containing deionized bovine serum albumin, iron-saturated transferrin, 2-mercaptoethanol, and recombinant human interleukin 3 (IL-3). Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet glycoprotein IIb/IIIa. Colony numbers reached a peak at day 14, with a mean of 47 megakaryocyte colonies (range 23-104) per 2 x 10(5) bone marrow mononuclear cells. Recombinant human IL-3 stimulated the growth of megakaryocyte progenitors. Similar results were obtained using nonadherent, T-cell-depleted mononuclear cells. These findings suggest that IL-3 stimulates the growth of megakaryocyte progenitors directly without activation of accessory cells, or the requirement of factors present in the serum or plasma. This serum-free culture system may be useful for studying the effects of purified natural and recombinant biological regulators on human megakaryocyte progenitors.

Protein A from Staphylococcus aureus is known to stimulate human lymphocytes. Using 3H-thymidine incorporation, virus plaque assay and induction of cytotoxic T lymphocytes (CTL), this study showed that soluble or insoluble protein A stimulated different lymphocyte subpopulations. Soluble protein A is highly mitogenic to T lymphocytes. In both 3H-thymidine incorporation and virus plaque assay, its maximum stimulation was as high as the stimulation by the nonspecific mitogens phytohemagglutinin and concanavalin A, and a higher CTL response than that induced by phytohemagglutinin or concanavalin A was induced. Mitogenic activity to B lymphocytes was negligible. S. aureus (Cowan I strain) is itself considered to be an insoluble form of protein A and is 3--4 times more mitogenic to B lymphocytes than pokeweed mitogen without any increase in virus plaque-forming cells. No mitogenicity was noted to T lymphocytes. Sepharose CL-4B-protein A, also known as insoluble protein A, stimulated both T and B lymphocytes effectively, but its mitogenicity to T lymphocytes was considered to be due to the soluble protein A released from the Sepharose CL-4B beads.

The effects of recombinant interferons (rIFNs) on colony formation by progenitor cells from the peripheral blood of patients with hairy cell leukemia (HCL) were examined. Results showed that both rIFN-alpha and -beta inhibited HCL colony formation in a dose-dependent manner, with the inhibitory effect of rIFN-alpha being similar in degree with that of rIFN-beta. The inhibitory effect of rIFN-gamma, however, was not uniform; of four patients, inhibition was observed in cells from two patients, but no effect was found in the cells of the other two. These results indicate that rIFN-alpha and -beta could be effective in the treatment of HCL, whereas rIFN-gamma may be of limited efficacy.

In vitro culture of human peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the expansion of lymphocytes including lymphokine-activated killer (LAK) cells. Using flow cytometry, studies were undertaken to determine the phenotype and LAK activity of each subset of lymphocytes expanded in vitro as a result of incubation for 2 weeks with 2500 U/ml of recombinant IL-2. Such expanded PBMC, when examined by two-color staining with various combinations of anti-CD3, 4, 8, 16, and NKH-1 monoclonal antibodies, consisted of the following six subgroups of cells: (1) CD3+4+8-, (2) CD3+4-8+, (3) CD3+4-8-, (4) CD3-16+NKH-1+, (5) CD3-16-NKH-1+, and (6) CD3-16-NKH-1-. Of the six subgroups, all five subgroups that could be tested, i.e., CD3+ T cells (CD3+4+8-, CD3+4-8+, CD3+4-8-), CD16+ natural killer (NK) cells (CD3-16+NKH-1+), and CD3-16-NKH-1- non-T non-NK cells, possessed LAK activity. Both NKH-1- as well as NKH-1+ T and non-T cells possessed LAK activity.

Adult T-cell leukemia (ATL) is characterized by peripheral lymph node enlargement, hepatosplenomegaly and skin lesions. The association of local mass lesions of other organs with ATL is extremely rare. This report describes a 57-year-old woman with chronic type ATL with associated local tumor masses in the nasal cavity, paranasal sinuses and larynx as well as skin infiltration. Histologic investigation of the skin lesion and nasal mucosa revealed non-Hodgkin lymphoma, diffuse, mixed type. Her chief complaints were progressive dyspnea and hoarseness. Leukemic cell masses in her upper respiratory tract caused narrowing of the airway, which was responsible for her complaints.

Recently, a streptococcal preparation, OK-432 has been used successfully as an immunopotentiator for immunotherapy in patients with malignant tumors in Japan. In this paper, we report that the administration of OK-432 augments the cytotoxic activity of peripheral blood lymphoid cells against a natural killer (NK) cell-sensitive erythroleukemic cell line, K562, in tumor patients. In patients before or after surgery, sufficient amounts of OK-432 strongly augmented the cytotoxic activity within 3 days after the initial administration of OK-432. Thereafter the levels of cytotoxicity declined rapidly. The administration of a lower dose of OK-432 gave a lower increase in cytotoxicity. Enhanced cytotoxicity occurred with the reintroduction of OK-432 but remained at lower levels of activity. Characterization and fractionation of OK-432-induced effector cells revealed that the augmented cytotoxicity seemed to be carried mainly by NK cells. A low titer of interferon was detected in 3 of 10 patients within 72 hr after the first inoculation of the agent. Furthermore, we discuss the potency of OK-432 for the induction of interferon in detail.

Studies were undertaken to determine whether natural killer (NK) cells would lyse autologous and allogeneic lymphoma cells. When large granular lymphocytes, which are known to mediate NK activity, were enriched from peripheral blood and used as effector cells, they lysed autologous lymphoma cells of all of eight patients tested, and those of healthy donors lysed lymphoma cells of all of ten patients tested. The addition of interferon to the culture medium enhanced their cytotoxicity in three of the eight patients in the autologous effector-tumor system and in four of the ten patients in the above allogeneic system. On the basis of the unlabeled target competition test and the decrease in cytotoxicity with anti-NK antibody treatment, NK cells appeared to be the main cytotoxic effector cells for autologous and allogeneic lymphoma cells.

By analyzing surface antigens and cytolytic functions of proliferating large granular lymphocytes (LGLs), three types of T cell LGL lymphocytosis were delineated. The first, most commonly encountered type exhibited CD3+4-8+16+, WT31+ phenotype, low or undetectable non-major histocompatibility complex (MHC)-restricted cytotoxicity, and moderate to strong antibody-dependent cellular cytotoxicity (ADCC) and lectin-dependent cellular cytotoxicity (LDCC). Because these LGLs carried T cell antigen receptor (Ti) recognized by WT31 monoclonal antibody (MoAb), and treatment with anti-Ti, anti-CD3 MoAbs and phytohemagglutinin elicited non-MHC-restricted cytotoxicity, they may have developed from populations of in vivo primed cytotoxic T lymphocytes with unknown antigen specificity. The second, rare type of LGL lymphocytosis exhibited CD3+4-8-16+, WT31 phenotype, and strong non-MHC-restricted, ADCC and LDCC cytotoxicities. These cells were probably derived from the lymphocytes of the same phenotype found in small numbers in normal peripheral blood. Because anti-CD3 MoAb inhibited non-MHC-restricted cytotoxicity of the LGLs, a Ti not detected by WT31 MoAb, but putatively present seemed to serve as a specific receptor for target tumor cell recognition. The third type of LGL lymphocytosis showed CD3+4+8-16+, WT31+ phenotype, and lacked cytolytic activities and parallel tubular arrays. These LGLs probably evolved from cells with the same characteristics selectively located in the germinal centers of lymphoid tissues. Taken together, in patients with LGL lymphocytosis, T cell-associated antigens expressed on LGLs were shown to be involved in the regulation of LGL-mediated cytolytic activities. In addition, studies of surface antigens and the effects of MoAbs and lectins on cytolytic activities may be useful in clarifying the normal counterpart of LGLs from which leukemic or reactively proliferating LGLs originate.

A case of T-cell chronic lymphocytic leukemia is reported. The leukemic cells had the morphologic features of medium-sized, mature-looking lymphocytes, and had an affinity for the central nervous system. Cytochemically, they were positive for alpha-naphthyl acetate esterase and acid phosphatase. They formed E-rosettes (E+) and reacted with OKT11 but not with OKT3/Leu-4, OKT4/Leu-3, OKT8/Leu-2, or OKM1, and did not possess IgG-Fc receptors (Fc gamma R). Functionally, they did not respond to phytohemagglutinin or concanavalin A, were not natural killer cells or antibody-dependent as well as alloantigen-reactive killer cells. Furthermore, they did not possess a helper or suppressor T-cell function for immunoglobulin synthesis. Results of immunologic studies suggest that the leukemic cells were derived from a normal counterpart of a lymphocyte subset present as a minor component of the peripheral blood, namely an E+, OKT3-, OKM1-, Fc gamma R- subset, the function of which is not yet identified.

A case of paroxysmal nocturnal hemoglobinuria (PNH) which developed terminal transferase (TdT)-positive leukemia 5 years after the diagnosis of PNH was studied. Most of the leukemic cells were suggestively lymphoid by cytochemistry and electron microscopy, and TdT-positive by immunofluorescence studies. The development of acute lymphoblastic leukemia during the course of PNH suggests that in PNH the clonal abnormality may involve lymphoid cells as well as myeloid cells, thus raising the possibility of the disease being a disorder of the pluripotential stem cell.

Using spontaneously established autologous and allogeneic LCL as stimulator and target cells, we studied the generation of autoreactive cytotoxic T lymphocytes (CTL) in primary and secondary autologous MLC. In the primary autologous MLC, cytotoxic activity was detected on the 6th day and was found to be specific to autologous LCL. In the secondary autologous MLC, cytotoxic activity was detected earlier on the 3rd day, and was at a higher level than in the case of the primary autologous MLC. This cytotoxic activity was induced in both the primary and the secondary autologous MLC when T lymphocytes were used as responder cells. When autologous LCL-primed lymphocytes were used as responder cells in the secondary MLC, autologous LCL-specific CTL were not only generated by autologous LCL but also by allogeneic LCL, and the autologous LCL-specific CTL induced by allogeneic LCL was thought to be due to the nonspecific helper effect of allogeneic LCL-stimulation.