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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.

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 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.

Studies were undertaken to determine whether leukemia and lymphoma cells would be lysed by autologous and allogeneic lymphokine-activated killer (LAK) cells. Peripheral blood mononuclear cells (PBMC) from patients and normal donors were cultured for five days, 2 weeks, and 4 weeks with medium containing 2,500 units of recombinant interleukin 2 (IL-2) per mL, and their cytotoxicity was assayed by a five-hour 51Cr- release test. Of primary tumors isolated from patients with acute nonlymphoblastic leukemia, acute lymphoblastic leukemia, and non- Hodgkin's lymphoma, tumors of 37 out of 40 patients tested were shown to be susceptible to normal donors' LAK, and tumors of 18 of 20 patients tested were shown to be susceptible to autologous LAK. LAK cultured for longer periods showed a tendency to have lower cytotoxicity. LAK had also low, but significant, levels of cytotoxicity for nonmalignant target cells. Because PBMC expanded in IL-2-containing medium consisted mainly of OKT3-positive pan T cells, OKT8-positive suppressor/cytotoxic cells, and Leu-11-positive natural killer (NK) cells, and treatment with OKT3 and Leu-11 monoclonal antibodies (mAb) reduced LAK activity for autologous and allogeneic tumor cells, both T and NK cells appeared to be effector cells for LAK activity. Mechanisms of target-cell recognition in the LAK system seem to be different from those in alloreactive cytotoxic T lymphocytes (CTL) based on the results that, while cytotoxicity of alloreactive CTL was inhibited by the treatment of effector cells with mAb, OKT3, and OKT8, and by the treatment of target cells with a mAb that reacts with HLA class I antigen, LAK activity was not inhibited by the above treatment. When chromosomes of IL-2-expanded PBMC in nine patients and two normal individuals were analyzed, PBMC from one patient showed chromosomes of clonal abnormalities, and PBMC from five donors showed those of nonclonal abnormalities.

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.

We investigated the effect of recombinant human interleukin-11 (rhIL- 11) on human megakaryocytopoiesis. Nonadherent and T-cell-depleted human bone marrow (BM) mononuclear cells were cultured in a serum-free agar culture system. rhIL-11 alone did not stimulate the growth of human megakaryocyte colonies. However, when rhIL-11 was combined with optimal or suboptimal doses of rhIL-3, the number and size of the megakaryocyte colonies increased. The same results were obtained when highly purified BM CD34-positive cells were used as target cells. Next, we investigated the effect of rhIL-11 on the ploidy of megakaryocytes. The ploidy distribution of individual cells in megakaryocyte colonies obtained by rhIL-11 in combination with rhIL-3 was significantly shifted towards higher values. Furthermore, when highly purified CD41- positive BM cells were cultured in the presence of rhIL-11, the ploidy distribution was shifted towards higher values. This effect was not suppressed by anti-IL-6 antibody. These results suggest that rhIL-11 acts directly as a megakaryocyte potentiator and may play a role in regulating human megakaryocytopoiesis.

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 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.

A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4–8–16+ and WT31-. By two-color immunofluorescence staining, CD3+4- 8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin- dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

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.

Studies were undertaken to determine whether leukemia and lymphoma cells would be lysed by autologous and allogeneic interferon (IFN) activated peripheral blood mononuclear cells (PBMC). PBMC from healthy donors and from patients were cultured with and without 500 U of highly purified human fibroblast IFN/ml for 24 hr, and then their cytotoxic activity was assayed by a 5-hr 51Cr-release test. Of primary tumor cells isolated from patients, the cells of 5 of 15 patients with acute nonlymphocytic leukemia (ANLL), 5 of 9 patients with acute lymphocytic leukemia (ALL), 2 of 3 patients with chronic phase chronic myelogenous leukemia (CML), 2 of 3 patients with blastic phase CML, 1 patient with hairy cell leukemia, and 6 patients with diffuse non-Hodgkin's lymphoma were sensitive to IFN-activated PBMC of healthy donors, whereas the cells of 3 of the ANLL patients, 2 of the ALL patients, and 3 of the lymphoma patients were sensitive to unstimulated PBMC. Of the ANLL cells tested, myeloblasts, promyelocytes, and monoblasts were sensitive to either unstimulated or IFN-activated PBMC. Compared with the ANLL cells, the lymphoma cells were statistically significantly sensitive to activated effector cells (p less than 0.025). On the basis of the unlabeled target competition test and the recovery of cytotoxic cells within the fractions enriched in natural killer (NK) cells, NK cells appeared to mediate the above unstimulated and IFN-boosted cytotoxicity. In experiments using autologous effector-target cells from 11 patients, the addition of 500 U of IFN/ml enhanced the lytic activity of PBMC against autologous lymphoma cells in 1 patient, and higher concentrations of IFN, i.e., 2500 or 3500 U/ml, enhanced their cytotoxic activity against autologous leukemia or lymphoma cells in 4 of 8 patients. These data indicate that IFN-activated allogeneic PBMC are able to lyse both myeloid and lymphoid tumor cells, whereas higher concentrations of IFN are required to enhance lytic activity against autologous tumor cells.

Granular lymphocytes have been characterized as cells with azurophilic granules in the cytoplasm. Patients with increased numbers of granular lymphocytes are designated as granular lymphocyte-proliferative disorders (GLPDs). A variety of haematological abnormalities are associated with T-cell-lineage GLPD. Among these, pure red cell aplasia is frequent, and adequate therapy is required. Seven patients with pure red cell aplasia, or a related condition complicating T-cell-lineage GLPD, were entered into this study. Cyclophosphamide was initiated at a daily oral dose of 100 mg. After 2 weeks the dose was reduced to 50 mg/d, and maintained at that dose. Cyclophosphamide was administered until the lymphocyte count was <1 x109 l and T-cell receptor-beta gene analysis was used to monitor the response to treatment. All the patients were successfully treated, irrespective of their former treatment. Clinical remission was associated with the disappearance of the abnormal granular lymphocyte clone, as detected by Southern blot hybridization analysis. Therapeutic responses began after 8 weeks, and clinical complete remissions were obtained after 6 months. Oral cyclophosphamide monotherapy can successfully treat the pure red cell aplasia associated with T-cell-lineage GLPD.

The cytokine interleukin-11 (IL-11) promotes normal human megakaryocytopoiesis in vitro. However, its role in abnormal megakaryocytopoiesis is not well known. Accordingly, we studied its effects on human megakaryoblastic cell lines CMK and Meg-J. IL-11 stimulated the proliferation of CMK and Meg-J in a dose-dependent manner with maximal growth being achieved at the concentration of 50 and 500 ng/mL, respectively. The growth of the cells was inhibited by anti-IL-11 antibody and IL-11 antisense oligonucleotides. IL-11 transcripts were detected in these two cell lines using a reverse transcriptase-polymerase chain reaction assay. These findings indicate that IL-11 might be an autocrine growth factor for megakaryoblastic cells. IL-11 transcripts also existed in other leukemia cell lines: HL- 60, U937, and K562. However, the growth of these cells was not stimulated by IL-11, and was not inhibited by IL-11 antisense oligonucleotides. These results suggested that IL-11 might regulate malignant cells of the megakaryocytic lineage, in part by an autocrine loop.

Some patients with granular lymphocyte-proliferative disorders (GLPD) have been reported to have an aggressive clinical course with a poor prognosis and to be refractory to chemotherapy. In this study, expression of multidrug resistance P-glycoprotein on peripheral blood mononuclear cells (PBMC) of 11 patients with GLPD was examined by staining with MRK 16, a monoclonal antibody that binds to an external epitope of P-glycoprotein, and with the dye rhodamine, a known substance to be excreted from the cells through P-glycoprotein. Among those tested, the PBMC of six of eight patients with T-cell-type GLPD as well as those of three of three patients with natural killer cell- type GLPD expressed P-glycoprotein significantly. Furthermore, PBMC of two of two patients were also poorly stained with the dye rhodamine, and the treatment of PBMC with either verapamil or quinidine, multidrug resistance-reversing agents, led to their increased staining, suggesting that these PBMC actively release chemotherapeutic agents.

An anti-CD3 Fab' x anti-CD13 Fab' bispecific antibody (BsAb) was generated. This BsAb reacted with both CD3+ T cells and CD13+ acute myeloid leukemia (AML) cells. We investigated whether cytokine- stimulated peripheral blood mononuclear cells (PBMC) could lyse patient AML cells after addition of the BsAb. When interleukin-2 (IL-2)- stimulated PBMC were assayed for their cytotoxicity against 51Cr- labeled allogeneic and autologous CD13+ AML cells, their activity was markedly enhanced by the addition of the BsAb. PBMC stimulated with IL- 2 plus anti-CD3 monoclonal antibody (MoAb) showed higher proliferative ability and higher cytotoxicity if this was expressed as lytic units per culture. IL-7-stimulated PBMC also exhibited enhanced cytotoxicity against CD13+ AML cells after addition of the BsAb. Ultrastructurally, CD13+ AML cells incubated with IL-2 plus anti-CD3 MoAb-stimulated PBMC and the BsAb showed apoptotic morphologic changes. A colony assay for AML blast progenitors showed that the colony formation of CD13+ AML cells was inhibited by the addition of autologous IL-2 plus anti-CD3 MoAb-stimulated PBMC, and that this inhibition was further enhanced by the addition of the BsAb. A colony assay for normal bone marrow progenitor cells showed that the addition of autologous IL-2 plus anti- CD3 MoAb-stimulated PBMC and the BsAb inhibited the formation of granulocyte-macrophage colonies and mixed-cell colonies. However, the degree of inhibition was smaller than that for the AML blast colonies. Taken together, these findings suggest that this BsAb may be useful for ex vivo purging of CD13+ AML cells in autologous bone marrow transplantation.

Human myeloma stem cells were detected by their capacity to form colonies in culture. Cells separated from aspirated marrow were cultured for 10 days in semi-solid methylcellulose with medium conditioned by T lymphocytes stimulated by phytohaemagglutinin (PHA-TCM). The colonies formed consisted mostly of lymphoplasmacytoid cells or plasma cells, and the immunoglobulins in the patients' myeloma cells were demonstrated also in the cytoplasm of the colony cells. The number of colonies were proportional to the number of cells plated and to the concentration of PHA-TCM. When the proportion of proliferating colony-forming units of multiple myeloma (CFU-MM) was studied using the (3H)-dT-suicide technique, the high-specific-activity (3H)-dT killed 21-45% of the CFU-MM in 7 myeloma patients. According to a single dose of Co-y-irradiation, the mean doses for impairment of regeneration (Do) were 1.00 and 1.63 Gy in 2 cases, the extrapolation numbers being 1.6 and 2.0.

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 first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggresive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1 B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.

After depletion of monocytes, natural killer (NK) cells were partially purified from peripheral blood by Percoll density gradient sedimentation. The NK cells were then cultured for 1 d and assayed for their cytotoxicity against various types of normal and malignant target cells. All types of target cells tested were found to be susceptible to NK cells. The susceptible targets were autologous T and B lymphocytes, mitogen-induced T and B blasts, monocytes, large granular lymphocytes, autologous or allogeneic lymphoma and leukemia cells isolated from patients, and cultured cell lines, including those resistant to interferon-activated lymphocytes. Such a broad spectrum of cytotoxicity was demonstrated in 1 d of culture, and freshly prepared NK cells were not cytotoxic, or, if anything, were less cytotoxic. Monocytes and their supernatants, added throughout the course of culture, markedly inhibited the development of their cytotoxicity. These results may suggest that, although NK cells having ability to lyse autologous normal and malignant target cells are present in vivo, their lytic activity is regulated by coexisting monocytes.

1. During the process of cell death, rises in cytosolic Ca2+ concentration ([Ca2+]i) together with structural changes were investigated in target cells attacked by purified CD3‐,CD16+ human natural killer (NK) cells. 2. In the target cell line K562, a rapid [Ca2+]i rise to 1‐2 microM occurred a few minutes after NK cell‐target cell contact, immediately followed by leakage of the Ca2+ indicator dye fura‐2 from the cell. Cells were permeabilized, but their chromatin was not fragmented. The changes were basically consistent with those seen in necrosis induced by activated complement. 3. In the target cell line MOLT‐4, which expressed the apoptosis‐inducing surface antigen Fas much more strongly than K562, the majority of attacked cells displayed a [Ca2+]i rise to 0.7‐1 microM followed by a slow decline, often associated with diminishing [Ca2+]i oscillations. As a whole, [Ca2+]i remained higher than 150 nM for at least 1.5‐3 h (approximately 100 nM in control cells). 4. MOLT‐4 cells attacked by NK cells became bubble shaped within 20 min of the main [Ca2+]i rise reaching its peak, and then both the cell and chromatin were fragmented into small pieces. These findings were basically consistent with those in apoptosis induced by a monoclonal antibody against the surface antigen Fas. 5. NK cells induced both necrosis and apoptosis in cell lines insensitive to NK cells in the presence of an antibody against the major histocompatibility complex class I (antibody‐dependent cell‐mediated cytotoxicity, ADCC). The distinct Ca2+ responses patterns described above corresponded to necrosis or apoptosis in different cells stimulated by the common ADCC pathway. 6. Human NK cells were found to be capable of inducing necrosis (membrane damage) or apoptosis (nuclear damage) depending on the target cell types. The characteristic Ca2+ response profile was a good indicator for distinguishing between the modes of cell death induced by the cytotoxicity of the killer cells.

A case of WT31 -, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4—8 —16+ and WT31 —. By two-color immunofluorescence staining, CD3+4—8-cells were found to be WT31 —, and a small population of WT31 + cells expressed either CD4 or CD8. WT31-, CD3 + cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31 -reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-β and Ti-γ genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody-and lectin-dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

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.© 1988 by Grune & Stratton, Inc. 0006-4971/88/7102-0030$ 3.00/0

Proliferating cell nuclear antigen (PCNA) was detected in the peripheral blood mononuclear cells (PBMC) of patients with malignant lymphoma (ML). Twenty-one of 27 patients with ML had PCNA expressing PBMC (5.25 ± 4.75% cells), which tended to increase in the advanced clinical stage of ML. PCNA in PBMC extracts was detected in 11 of 16 patients (54.5 ± 41.9 ng/ml). The percentage of PCNA-positive cells correlated significantly with the concentration of PCNA in PBMC extracts (P < 0.005). Serum PCNA was detected in 6 of 16 patients (160.1 ± 141.1 ng/ml), but did not correlate with the number of PCNA-positive cells. In some cases, the concentration of serum PCNA increased after chemotherapy while the percent PCNA-positive cells decreased. Our finding indicate that detection of PCNA in PBMC appears to help monitoring the extent of disease in ML and the serum PCNA level may be used in therapeutic studies of lymphoma patients.

In a serum-free liquid culture, thrombopoietin (TPO) selectively stimulated the growth of megakaryocytic cells from CD34-positive cord blood cells. Using these cultured cells, we investigated cytokine production by human megakaryocytes. Day 10 megakaryocytes (2 × 105) secreted > 1000 pg/ml of interleukin (IL)-8, in contrast to small amounts of IL-1β and IL-6. A time-course study showed that the IL-8 production of megakaryocytes occurred at the late phase of the culture period. The megakaryocyte-conditioned medium had the chemotactic potential of polymorphonuclear leucocytes, which was abrogated by the addition of anti-IL-8 antibody, suggesting the secretion of biologically active IL-8. The combination of TPO and IL-1α was required for a significant augmentation of the IL-8 secretion. Direct evidence for IL-8 synthesis in megakaryocytes was provided by reverse transcription–polymerase chain reaction on purified CD41b+ cells and by the detection of intracellular IL-8 in CD41b+ cells. These results suggest that TPO stimulates not only the proliferation and differentiation of the progenitors capable of megakaryocytic lineage expression but also IL-8 release by the megakaryocytic cells with the aid of IL-1.

A human T-cell clone (TA-NB-2) that could lyse both autologous and allogeneic hepatocytes from chronic hepatitis patients with type non-A, non-B virus (NANB) was established. This clone produced CD3+ CD8+ cytotoxic T lymphocytes and expressed an antigen specific for alpha and beta subunits of T-cell receptor. The cytotoxic activity of the clone was abrogated by incubation with anti-CD3 monoclonal antibody. Anti-HLA monoclonal antibodies did not block the lysis of the target hepatocytes by TA-NB-2 cells. The cytotoxicity of TA-NB-2 clone against hepatocytes from patients with chronic NANB hepatitis was 39.8 +/- 13.2% (mean +/- SD; n = 17) (range, 14.2-60.5%), whereas that against hepatocytes from control patients with chronic type-B hepatitis, acute hepatitis B, acute hepatitis A, or alcoholic liver cirrhosis was 4.0 +/- 7.7% (n = 12) (range, -10.8 to 14.0%). The results suggest that TA-NB-2 cells specifically recognize a hepatitis NANB-related antigen expressed on hepatitis NANB-infected hepatocytes by T-cell receptor and that the recognition is not restricted by the major histocompatibility complex antigens. The results also suggest that most, if not all, cases of chronic hepatitis due to NANB are caused by one agent; TA-NB-2 clone may be useful as a tool to identify this particular hepatitis-related antigen.

Abstract:  A 50-year-old man was admitted suffering from severe anemia and renal dysfunction. He had been admitted for the first time at the age of 49, and was diagnosed with multicentric Castleman's disease (MCD) and secondary amyloidosis. At that time, marked erythroid hypoplasia was demonstrated by both aspiration and biopsy of bone marrow. A diagnosis of pure red-cell aplasia (PRCA) was made. Immunosuppressive agents improved his symptoms and laboratory data. We report here a very rare case of PRCA following MCD and amyloidosis, and with reference to the literature, we discuss the relation between MCD and related diseases.

The first case of human systemic infection by an Aspergillus flavus isolate demonstrated to produce aflatoxins in vitro and in vivo is described. The patient, a 41-year-old man with acute myelogenous leukaemia, developed a complication of suspected pulmonary Aspergillus infection during remission induction therapy. Antifungal chemotherapy brought about a considerable degree of improvement, but remission of the underlying disease was not attained. Bone marrow transplantation was also not effective. The patient showed recovery from neutropenia but died despite aggresive antifungal chemotherapy. The autopsy revealed lesions in the lungs, myocardium, kidneys, brain, thyroid gland and skin due to a suspected Aspergillus sp. A fungus isolated from the right lung and the skin lesions was identified as A. flavus. Aflatoxins B1 B2 and M1 were detected in culture filtrates of the isolated A. flavus, and in an extract of lung lesions. These aflatoxins are considered to have played an important role in damaging the immune system of the patient through their toxic effects.