Yukiaki Miyagawa

Abnormal neutrophil maturation in a neutrophil defect with morphologic abnormality and impaired function.

Neutrophils from a patient with recurrent pyogenic infections since infancy were found to have morphologic abnormalities and impaired functions. The neutrophils had an abnormal nuclear shape, no or few secondary granules, and no alkaline phosphatase activity. Primary granules were normal in number and structure, and were positive for peroxidase. Immature granulocytes were structurally normal. The neutrophils were impaired in chemotaxis and bactericidal capacity. The patients marrow cells formed increased numbers of granulocytic colonies of small size in culture. Her peripheral leukocytes produced elevated levels of CSA and adherent marrow cells did not inhibit colony formation. These data indicate an intrinsic neutrophil defect which allows normal proliferation of precursor cells, but results in abnormal morphogenesis and impaired function as the cells mature.

Induction of lymphokine-activated killer and natural killer cell activities from cryopreserved lymphocytes

Lymphokine‐activated killer (LAK) and natural killer (NK) cells were studied for their capacity to retain cytotoxicity after cryopreservation. LAK cells were generated by a 4‐day culture of lymphocytes with recombinant interleukin‐2 (rIL‐2). Cytotoxicity was measured by 51Cr‐release assay at effector:target ratios of 10:1 to 80:1. Cryopreserved LAK cells retained 58.8 to 87.4 percent of cytotoxicity, as compared with that in fresh control cells. Cryopreserved NK cell activity against K562 and Molt‐4 targets was 45.7 to 67.9 percent of the respective values of the fresh control cells. The responsiveness of NK cells to polyinosinic‐polycytidilic acid (poly I:C), interferon‐α (IFN‐α), or rIL‐2 remained intact. Activated NK cell activity after poly I:C or IFN‐α stimulation and that after rIL‐2 were, respectively, comparable to and higher than the endogenous NK cell activity of the fresh cells. The composition of lymphocyte subsets as determined by flow cytometry using monoclonal antibodies did not change after cryopreservation, indicating that cellular loss of the given subsets did not occur during the procedure. The retention of substantial levels of cytotoxicity in cryopreserved LAK and NK cells may make them promising candidates as cytotoxic effector cells.

Clinical significance of cytostasis activity of mononuclear cells in childhood acute lymphoblastic leukemia

Cytostasis activity of mononuclear cells (MNC) was studied in 32 children with acute lymphoblastic leukemia (ALL) with special reference to the relapse of the disease. MNC from ALL even in complete remission had decreased cytostasis activity. The cytostasis activity in ALL patients who relapsed within 3 months after the assay was significantly lower than that of the patients who continued to be in remission for more than 6 months after the assay. Serial assays in six patients demonstrated that a sudden decrease of the cytostasis activity occurred 4 to 8 weeks before the relapse when there were no apparent changes of the peripheral blood cell counts and bone marrow pictures. When relapsed, the cytostasis activity was markedly impaired. These results demonstrate that the assay of MNC cytostasis activity is a useful tool to predict or detect relapse of ALL at an earlier time. The cytostasis activity was still decreased after the cessation of chemotherapy for ALL, demonstrating a characteristic immunologic defect in ALL.

Decreased cytotoxic potential of fresh and recombinant interleukin 2-cultured large granular lymphocytes in childhood acute lymphoblastic leukemia.

Cytotoxic potential (cytotoxic efficiency and binding affinity) of large granular lymphocytes (LGL) in childhood acute lymphoblastic leukemia (ALL) patients was calculated by Michaelis-Mentens kinetic equation. Cytotoxic efficiency (Vmax) of fresh or 3-day recombinant interleukin 2 (IL-2)-cultured LGL, not of similarly cultured T cells, in on-therapy ALL patients was decreased (31% in fresh LGL, P less than 0.05 and 28% in cultured LGL, P less than 0.01) as compared with that in controls, although the binding affinity (Km) of these cells were not decreased. The cytotoxic efficiency and the binding affinity of IL-2-cultured LGL in off-therapy ALL patients was not decreased. These data indicate that the cytotoxic potential of LGL was selectively decreased in on-therapy ALL patients due to the impairment in the post-binding cytotoxic process, not due to the decrease in the binding affinity.

Effect of Radiotherapy on Natural Killer Activity in Childhood Acute Lymphoblastic Leukemia and Lymphoma

Natural killer (NK) activity was increased after 24 Gy prophylactic cranial irradiation in childhood acute lymphoblastic leukemia (ALL), although there was no change in the percentage and the number of CD16 (Leu 11)(+) cells. NK activity as well as the percentage and the number of CD16(+) cells was not decreased in malignant lymphoma children treated with high doses of cervical irradiation (20 Gy <). A single dose of 20 Gy irradiation to lymphocytes augmented NK activity 1.4-fold in vitro in healthy individuals. Radiosensitive concanavalin A-induced suppressor T cells did not play a role in the suppression of NK activity, indicating that the enhanced NK activity induced by irradiation was not due to irradiation-induced impairments of suppressor T cells. The supernatants from irradiated lymphocytes slightly enhanced NK activity. In conclusion, the augmentation of NK activity by irradiation seems to be due to its direct effect on NK cells, not via NK suppressor cells but perhaps humoral factors play some role in this respect.

Production of high levels of interleukin 1-like activity by the SPI-802 human leukemia cell line with E receptors

The SPI-802 human leukemia cell line, which possesses E receptors and used to have natural killer activity, has been demonstrated to produce high levels of interleukin 1 (IL-1)-like activity. SPI-802 supernatants prepared in 1% serum-containing cultures with lipopolysaccharide stimulation, like similarly prepared adherent-cell-derived IL-1, enhanced phytohemagglutinin-induced mouse thymocyte proliferation. When adherent-cell IL-1 gave 50% maximum activity at a reciprocal dilution of 20, SPI-802 supernatant gave it at 200, indicating the production of high levels of IL-1-like activity by the cell line. SPI-802 supernatant promoted the production of interleukin 2 (IL-2) by the Jurkat-F1884 T-cell line: Levels of IL-2 activity obtained with 15% SPI-802 supernatant were almost equivalent to those obtained with 50% adherent-cell IL-1 as estimated by the maximum proliferation of IL-2-dependent cytotoxic T cells. SPI-802 supernatant by itself exhibited no IL-2 activity. Major IL-1-like activity of SPI-802 supernatant was present in fractions from AcA54 columns corresponding to Mr 12,000-20,000 and 60,000-70,000 and resolved on isoelectrofocusing into two distinct species with pI values of 5.0 and 7.0, being consistent with the results of adherent-cell IL-1. The SPI-802 cell line having E receptors is an ideal source of a soluble factor with the biological and biochemical characteristics of human IL-1.

In Vitro Assay for Responsiveness of Lymphocytes to Transfer Factor by a New Leukocyte Migration Inhibitory Test

Transfer factor (TF) causes nonimmune lymphocytes to produce leukocyte migration inhibitory factor (LMIF) in the presence of purified protein derivative (PPD). The activity of TF was measured by leukocyte migration inhibitory test (LMIT). The LMIT was a modification of the conventional agarose droplet method. To express the activity of LMIF quantitatively and simply, LMIF titer was introduced. The LMIF titer was obtained from the combination of two factors, LMIF dilution and cell migration diameter, and therefore this made the LMIT much more sensitive as compared to the conventional LMIT. The responsiveness of lymphocytes from acute lymphoblastic leukemia (ALL) and from cell‐mediated immunodeficiency in children to TF was assayed by LMIT. In ALL, the lymphocyte responsiveness was poor in relapse but improved with remission. The responsiveness was remarkably well in 3 patients with cell‐mediated immunodeficiency. This method appears useful for the in vitro evaluation of responsiveness of lymphocytes to TF.

Functional Analyses of Cord IgM: Cytotoxic Antibody Against Common Killer T Cells

We have analysed cytotoxic antibody against T lymphocytes (TLFA) in cord IgM. TLFA IgM bound to T lymphocytes as determined by the radio immuno assay. TLFA IgM inhibited lymphocyte blastogenesis induced by Con A and PHA but did not inhibit that induced by PWM in the presence of complement. Based on these results, further studies were done to determine which function(S) of Con A‐ and PHA‐induced lymphocytes could be inhibited by TLFA IgM. TLFA IgM inhibited Con A‐induced T cell activity to suppress PWM‐induced antibody formation and autologous mixed lymphocyte culture. In addition, TLFA IgM markedly inhibited PHA‐mitogen factor induced killer T cell colony formation and their activity as determined by the colony assay and lymphocyte‐mediated cytolysis using K562 and Daudi cell lines respectively. TLFA appears to play a role in protecting a fetus from immunologic rejection by the mother, by killing maternal killer cells which may trasfer across the placenta and harm the fetal tissues.