Xao-Li Zhang

Intracellular cytokine profile of T cells from children with acute lymphoblastic leukemia

Purpose: During an ongoing immune response, cytokines produced by T helper types 1 (Th1) and 2 (Th2) together with T cytotoxic types 1 (Tc1) and 2 (Tc2) are critical to the effectiveness of that response. Dysregulated expansion of one or the other subset may contribute to the impaired function of the T-cell-mediated immune system in cancer patients. In the present study we have investigated whether such dysregulation might exist in children with acute lymphoblastic leukemia (ALL). Methods: We analyzed 61 blood samples from 45 children with B cell precursor ALL and 16 healthy children. Interleukin(IL)-2, IL-4, and interferon γ (IFNγ) production of their respective purified CD4+ and CD8+ T cells were assessed at the single-cell level by intracellular-cytokine-staining flow cytometry. Results: At the time of diagnosis, IL-2-producing cell populations in CD4+ and CD8+ T cells were reduced below the normal range in 31 of 44 (70.5%) and 23 of 38 (60.5%) cases respectively. Similarly, IFNγ-producing cell populations in CD4+ and CD8+ T cells decreased in 17 of 44 (38.6%) and 18 of 38 (47.4%) cases respectively. Conversely cell populations capable of IL-4 production in CD4+ and CD8+ T cell subsets were increased in 13 of 30 (43.3%) and 15 of 30 (50.0%) cases respectively. Therefore, the Th1-to-Th2 and Tc1-to-Tc2 ratios (1.6 ± 2.2 and 7.7 ± 6.7 respectively) were significantly lower in peripheral blood T cells of ALL patients (n = 30) than those (6.0 ± 2.9 and 20.1 ± 10.3 respectively) in 15 healthy controls (P < 0.0001). Although both CD45RA+/CD4+ and CD45RA+/CD8+ cells significantly increased in 43 ALL patients (P < 0.05), there existed no apparent correlation between CD45 isoform expression and cytokine (IL-2 and IFNγ) production. Interestingly, the ability to produce both IL-2 and IFNγ was recovered in 8 cases examined, after complete remission had been achieved. Conclusion: These observations suggest that, in both CD4+ and CD8+ T cells of ALL patients, there is a dysregulation in the functionality of Th1 (Tc1) and Th2 (Tc2) cells with a gross reduction of Th1 (Tc1) cell populations and an expansion in Th2 (Tc2).

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.

Immunological unresponsiveness and apoptotic cell death of T cells in measles virus infection

The phenotypic alterations associated with T cells during measles virus infection have been demonstrated and an attempt has been made to show programmed cell death (PCD) of T cells activated in vivo. During the acute phase of illness, activated T cells increased rapidly. Memory T cells (CD45RO+), especially CD8+ memory T cells also tend to increase. During the recovery phase, CD8+ T cells declined rapidly, and naive (CD45RA+) T cells increased in numbers. The anti‐CD3 monoclonal antibody‐induced expression of interleukin‐2 receptor (CD25) was suppressed. However, the addition of phorbol 12‐myristate 13‐acetate (PMA) caused the significant recovery of CD25 expression. In addition, PCD of activated T cells from measles patients was induced in culture. After triggering of the T cell receptor‐CD3 complex, cells became more susceptible to PCD. Interestingly, the addition of PMA could inhibit PCD of activated T cells. Taken together, these data suggest unresponsiveness and activation‐induced cell death of T cells during the primary response to measles virus antigens, depending on the activation status of protein kinase C.

CD3-Mediated T-Cell Activation is Inhibited by Anti-CD44 Monoclonal Antibodies Directed to the Hyaluronan-Binding Region

The CD44 molecule has been shown to play a role in T cell adhesion and activation. We have investigated the ability of five anti-CD44 monoclonal antibodies (MoAb) including 15C6, 18A3, BU75 (Ancell), J173 (Immunotech), and L178 (Becton Dickinson) to regulate T cell activation. Three MoAb: 15C6, BU75, and J173 were found to selectively inhibit DNA synthesis, interleukin-2 (IL-2) receptor expression, and G1-->S transition of the cell cycle in T cells stimulated with anti-CD3 MoAb. None of anti-CD44 MoAb had influence on T cell proliferation induced by IL-2 or phorbol 12-myristate 13-acetate plus ionomycin. Inhibition of the CD3 pathway by anti-CD44 MoAb occurred by binding of MoAb directly to T cells without the involvement of monocytes or Fc receptors. In addition, the inhibitory anti-CD44 MoAb clearly suppressed intracellular calcium mobilization in T cells stimulated with anti-CD3 MoAb. Interestingly, the ability of anti-CD44 MoAb to inhibit T cell activation was well correlated with their capability to block the binding of hyaluronan (HA) to CD44 molecules. These results suggest that anti-CD44 MoAb directed to HA-binding site could selectively inhibit CD3-mediated T cell activation. Furthermore, CD44-mediated inhibitory signals would be linked to the blocking of early CD3-mediated signal transduction.

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.

Expression of homing-associated cell adhesion molecule (H-CAM/CD44) on human CD34+ hematopoietic progenitor cells

We investigated the expression of CD44 molecule on CD34+ hematopoietic progenitor cells. Significantly lower expression of CD44 was observed on bone marrow (BM) CD34+ cells compared with circulating CD34+ cells in cord blood and peripheral blood. Using fluorescence-activated cell sorting, human CD34+ BM cells were fractionated into CD44+ and CD44- populations. Immunofluorescence analysis revealed that the majority of CD34+CD44- cells expressed B-lymphocyte-associated CD10 and CD19 antigens, whereas only a part of CD34+CD44+ cells were positive for CD19. Myeloid and erythroid progenitor cells were found predominantly in CD34+ CD44+ cell fractions. In short-term suspension cultures, cell proliferation and G1-->S transition in the cell cycle were enhanced in CD34+CD44+ cells. In contrast, a large part of CD34+CD44- cells underwent apoptotic cell death. Although co-culture with BM stromal cells could partially prevent CD34+CD44- cells from undergoing apoptosis, significant increase of apoptotic cells was consistently observed. Furthermore, CD34+CD44- cells plated on BM stromal cells could differentiate into CD34-CD44-CD10-CD19+ cells. These findings suggest that CD34+CD44- cells expressing CD19 would represent unique B-lymphocyte-committed precursors in BM, which might undergo apoptotic cell death in the early steps of B-cell differentiation.