Yan-Wen Zhou

Cellular immunosuppression in children with acute lymphoblastic leukemia: Effect of consolidation chemotherapy

SummaryThe present study was designed to evaluate the chemotherapy-induced cellular immunosuppression in 20 children with acute lymphoblastic leukemia (ALL) in remission and receiving maintenance chemotherapy. Peripheral blood was serially obtained from leukemic children during vincristine/cyclophosphamide/6-mercaptopurine/prednisone combined consolidation chemotherapy. The mean absolute number of peripheral blood lymphocytes as well as the mean absolute numbers of lymphocyte subsets (T cells, T cell subsets, B cells, and natural killer cells) from leukemic children before consolidation chemotherapy were all significantly lower than in control subjects; however, the percentages of lymphocyte subsets were similar in both groups. After consolidation chemotherapy, the percentages of CD4+ T lymphocytes and natural killer (NK) cells were significantly decreased and the percentages of monocytes and CD8+ T lymphocytes were significantly increased. Phytohemagglutinin- and 12-O-tetradecanoylphorbol-13-acetate-induced production of interleukin-2 (IL-2) and NK-cell-mediated cytotoxic activity by peripheral blood mononuclear cells (PBMC) were also substantially decreased in the post-therapy groups. NK activity correlated with the percentage of NK cells in PBMC. In contrast, OK432-induced production of tumor necrosis factor α (TNFα) and killer activity against NK-resistant target cells were significantly increased after therapy as compared with the pre-therapy and control groups. TNFα production correlated with the percentage of monocytes in PBMC. These results demonstrate that substantial quantitative and qualitative chemotherapy-induced abnormalities of the cellular immune system are present in the majority of patients treated with ALL. It is also suggested that the increased TNFα production by monocytes and the appearance of potent killing activity against NK-resistant targets might compensate for the defects of IL-2 production and NK activity during intensive consolidation chemotherapy.

Fas/APO-1 (CD95)-mediated cytotoxicity is responsible for the apoptotic cell death of leukaemic cells induced by interleukin-2-activated T cells.

Apoptotic cell death is induced by the cross‐linking of Fas/APO‐1 receptor (CD95) in acute myelogenous leukaemia (AML) cells. Since CD95 ligand (CD95L) is expressed on interleukin‐2 (IL‐2)‐activated T cells, we investigated the involvement of CD95‐CD95L pathway in T cell‐mediated cytotoxicity against AML cells. Activated CD8+ T cells could efficiently kill a parental CD95‐sensitive AML cell line, MML‐1 and, to a lesser extent, a CD95‐resistant clone, MML‐1R. Neither MML‐1 nor MML‐1R cells were killed by activated CD4+ T cells. The blocking monoclonal antibody (MoAb) against CD95, ZB4, caused a significant inhibition of T‐cell‐mediated cytotoxicity against MML‐1 cells, but not against MML‐1R cells. Interestingly, MML‐1 cells underwent the classic nuclear morphologic changes and DNA fragmentation characteristic of apoptosis when cultured with activated T cells. Enumeration of apoptotic and necrotic nuclei showed that both apoptosis and necrosis were induced in MML‐1 cells, whereas necrosis was exclusively observed in MML‐1R cells. Apoptosis of MML‐1 cells was completely blocked in the presence of ZB4 MoAb. Similarly, blocking by ZB4 MoAb significantly inhibited T‐cell‐mediated lysis of fresh AML cells in a CD95‐sensitive group, but not in a CD95‐refractory group. In addition CD8+ T cells expressed CD95L mRNA more abundantly than CD4+ T cells upon activation by IL‐2. These findings suggest that T‐cell‐mediated cytotoxicity against AML cells requires participation of CD95‐CD95L pathway for cytotoxic signal transduction leading to target apoptosis.

mRNA expression of Fas receptor (CD95)-associated proteins (Fas-associated phosphatase-1/FAP-1, Fas-associating protein with death domain/FADD, and receptor-interacting protein/RIP) in human leukaemia/lymphoma cell lines.

mRNA expression of Fas (CD95)‐associated proteins [Fas‐associating protein with death domain (FADD), receptor‐interacting protein (RIP), and Fas‐associated phosphatase‐1 (FAP‐1)] has been investigated in 26 Fas‐positive human leukaemia/lymphoma cell lines. Reverse transcriptase‐polymerase chain reaction analysis revealed that FADD and RIP mRNA were invariably expressed in both Fas‐sensitive and Fas‐insensitive cell lines. However, FAP‐1 mRNA was detected in only 11 of 26 cell lines. Interestingly 7/14 cell lines in the Fas‐sensitive group were positive for FAP‐1 mRNA expression. 8/12 cell lines in the Fas‐refractory group did not express FAP‐1 mRNA, but half of these cell lines were susceptible to tumour necrosis factor α‐induced growth inhibition. These findings suggest that the presence or absence of FAP‐1 mRNA expression did not always correlate with relative sensitivity of Fas‐mediated growth inhibition. Furthermore, it is assumed that leukaemia/lymphoma cells could possess structural or functional defects of Fas or Fas‐associated proteins resulting in the failure to trigger apoptotic cell death.

Role in growth regulation of cytokines and cytokine receptors in acute lymphoblastic leukaemia expressing myeloid markers

Summary. The biological roles of cytokines and cytokine receptors were examined in acute lymphoblastic leukaemia cells expressing myeloid antigens (My+ ALL). Interleukin‐3 (IL‐3), granulocyte‐colony stimulating factor (G‐CSF), granulocyte‐macrophage‐CSF (GM‐CSF), and low molecular‐weight B‐cell growth factor (LW‐BCGF) could induce DNA synthesis in certain cases of My+ ALL. Whereas in My− ALL the stimulatory effects were shown only with LW‐BCGF. Acute myelogenous leukaemia (AML) cells were activated with multiple cytokines including interleukin‐1 (IL‐1), IL‐3, G‐CSF and GM‐CSF. Specific receptors for IL‐1 and IL‐3 were strongly expressed on both My+ and My− ALL cells. These receptors, however, were weakly detectable on AML cells. Additionally we studied the production of tumour necrosis factor‐α and IL‐1 by leukaemic blasts and found that distinct amounts of both cytokines were released from My+ ALL cells and AML cells, but not from My− ALL cells.

Inhibition of interleukin-2 receptor (CD25) expression induced on T cells from children with acute lymphoblastic leukemia

Abstract Peripheral blood lymphocytes obtained from children with acute lymphoblastic leukemia (ALL) at onset were studied for the expression of interleukin-2 (IL-2) receptor α-chain (CD25) by two-color flow-cytometric analysis. Stimulated with anti-CD3 monoclonal antibody (mAb) alone, CD25 expression was significantly suppressed in CD4+ T cells from 27 of 48 (56.3%) cases and in CD8+ T cells from 29 of 48 (60.4%) cases. When stimulated with anti-CD3 mAb plus phorbol 12-myristate 13-acetate (PMA), CD25 expression was clearly restored in certain cases of ALL. When PMA plus ionomycin were used for stimulation of T cells, CD25 was inducible in a majority of cases. Interestingly CD25 expression upon anti-CD3 mAb stimulation was recovered after complete remission had been achieved. These observations suggest the presence in ALL children at onset of an in vitro defect in the signal transduction pathway of the T-cell-receptor/CD3 complex, resulting in inefficient CD25 expression. However, immune-staining analysis indicated that protein kinase C was normally translocated from the cytosol fraction to the cell membrane fraction. The mobilization of cytoplasmic free calcium is also normal.

Hyperimmunoglobulinemia D following Cancer Chemotherapy

Five classes of serum immunoglobulin levels were investigated in 107 children with malignant or benign tumors. Hyperimmunoglobulinemia D (hyper-IgD) was observed in 31 of 82 children who were in complete remission off chemotherapy with a median follow-up of 4.5 years after cessation of chemotherapy. On the other hand, hyper-IgD was not found among 12 children with malignant or benign tumors treated with chemotherapy and a low incidence of hyper-IgD was observed during chemotherapy (1 of 13 cases). The result indicates that hyper-IgD is not uncommon in children off chemotherapy, suggesting that dysregulation of IgD synthesis persists long after cessation of antineoplastic drugs.

T cell subsets In peripheral blood and cerebrospinal fluid from children with aseptic meningitis

T cell subsets in peripheral blood (PB) and cerebrospinal fluid (CSF) obtained from patients with aseptic meningitis were studied using quantitative two‐color fluorescence analysis with a flow cytometer. The percentage of HLA‐DR+/CD3+ lymphocytes (activated T cells) in CSF was significantly increased in the recovery phase when compared to the acute phase, while no significant change in the activated T cells in PB was observed. More interestingly, CD4+ T lymphocytes in CSF were increased in the acute phase and subsequently decreased in the recovery phase. Instead, CD8+ T lymphocytes gradually accumulated into the CSF in the recovery phase, resulting in a successive decrease in the CD4/CD8 ratio. On the other hand, the CD4/CD8 ratio in PB remained normal during the course of aseptic meningitis. The present results suggest that T lymphocytes (CD4+ subset in the acute phase and CD8+ in the recovery phase) could be infiltrated and further activated at the site of inflammation, possibly in the subarachnoid space in the patients with aseptic meningitis.

Functional Significance of Adhesion Molecules in Fas-Dependent Apoptotic Cell Death Induced by Interleukin-2-Activated T Cells

We investigated the functional significance of the adhesion molecules CD2 and lymphocyte function-associated antigen-1 (LFA-1: CD11a/CD18) in Fas-Fas ligand (FasL) death pathway. Interleukin-2-activated T cells expressed a large amount of FasL protein and could efficiently kill a Fas-sensitive leukemic cell line, MML-1. The major part (over 80%) of MML-1 cell death was Fas-dependent. Antibodies to CD2 and CD11a/CD18 completely inhibited MML-1 target cell lysis, whereas effector to target cell binding was partially reduced or not affected at all. These results suggest that effector/target interaction via CD2/CD58 and LFA-1/CD54 systems would be essential for triggering target cell death. More interestingly, there is the discordance in the ability of anti-CD2, and particularly anti-LFA-1 antibodies, to block Fas-dependent cell death versus effector to target conjugate formation. This suggests some non-adhesive role for CD2 and LFA-1 in induction of Fas-dependent cell death. Although these antibodies were capable of inhibiting T cell proliferative response, there was no significant reduction of FasL or granzyme B expression. Thus, the signaling pathway for growth inhibition via CD2 and LFA-1 could not be linked to signaling for FasL and granzyme B expression.

Shedding of CD9 antigen by bone marrow cells from patients with acute lymphoblastic leukaemia

The levels of soluble CD9 antigen released into spent medium from bone marrow (BM) cells were assayed using a unique enzyme‐linked immunosorbent assay. We demonstrated that a considerable amount of soluble CD9 antigen was consistently detected in the spent medium from CD9+ leukaemic blasts, but little from normal or regenerating BM cells. The sensitivity and specificity of CD9 antigen assay were tested. First, BM cells taken from patients in complete morphologic remission (CR) were studied, and the distinct level of CD9 antigen was detected in 16·3% of the cases. Second, the outcome of leukaemic patients with significant shedding of CD9 antigen from BM cells while in CR was investigated: all patients have developed systemic relapse within 5–24 (median 15·4) weeks. In contrast, 24/34 patients without the increase of CD9 antigen levels are still in CR (follow‐up 19–149 weeks, median 56·2 weeks). Only 10 patients in this group have relapsed so far. No false‐positive results were detected with this sensitive assay for soluble CD9 antigen, and the introduction of appropriately planned prospective studies is justified on the basis of these observations.

2‘,5’‐oligoadenylate synthetase activity and T cell subset in the cerebrospinal fluid and peripheral blood of aseptic meningitis

2′,5′‐oligoadenylate synthetase activity, which is assumed to be induced by interferon, is reported to be one of the useful markers reflecting interferon activity. The enzyme activity of patients with aseptic meningitis and febrile convulsion were compared in order to evaluate interferon activity as one of the local immuno‐defense mechanisms of aseptic meningitis. The surface antigen of mononuclear cells in cerebrospinal fluid and peripheral blood of some patients with aseptic meningitis was also measured. The enzyme activity of patients with aseptic meningitis was 191.4 pmol/dL in the cerebrospinal fluid and 395.8 pmol/dL in the serum during the acute phase, while that of patients with febrile convulsion was 45.2 pmol/dL in the cerebrospinal fluid and 326.0 pmol/dL in the serum. The enzyme activity of the former patients significantly decreased during the recovery phase in both the cerebrospinal fluid and serum. CD3 positive cells in the peripheral blood were 56.3% of the total mononuclear cells during the acute phase and 65.2% during the recovery phase, whereas in the cerebrospinal fluid mononuclear cells, they were 87.1 and 85.5%, respectively. During the acute phase, CD4 positive cells were the predominant T lymphocyte subset in the cerebrospinal fluid cells, while CD8 positive cells were predominant during the recovery phase. The relative proportions of CD4 positive and CD8 positive cells during the acute and recovery phase in the cerebrospinal fluid mononuclear cells were quite high compared to the recovery phase, although that ratio of peripheral blood mononuclear cells was not changed throughout the course. It was concluded that T lymphocytes and increased 2′,5′‐oligoadenylate synthetase activity in the cerebrospinal fluid may be one of the important components in the local inflammatory process independent of the systemic host defense mechanism in aseptic meningitis.