Enhancement of the anti-leukemic activity of cytokine induced killer cells with an anti-CD19 chimeric receptor delivering a 4-1BB-zeta activating signal

OBJECTIVE: There is growing interest in the use of cytokine-induced killer (CIK) cells in cancer therapy. In this study, we sought to maximize the antileukemic activity of anti-CD19 receptor-modified CIK cells against B-lineage acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: CIK cells were transduced with retroviral vectors carrying different types of anti-CD19 chimeric receptors: anti-CD19-zeta, anti-CD19-DAP10, anti-CD19-4-1BB-zeta, and anti-CD19-CD28-zeta. A truncated form of the receptor was used as a control. Transduced CIK cells were then analyzed for their cytotoxic activity against ALL cells and for their capability to proliferate and to release cytokines after ALL encounter. RESULTS: CIK cells were efficiently transduced with all the anti-CD19 retroviral vectors. Anti-CD19 receptor expression conferred powerful killing activity against ALL cells. However, there were clear advantages when receptors containing the co-stimulatory molecules 4-1BB or CD28 were transduced. Such cells had significantly more potent cytotoxicity than cells expressing the anti-CD19-zeta or anti-CD19-DAP10. Moreover, the presence of 4-1BB or CD28 in the receptor increased the production of interleukin (IL)-2, tumor necrosis factor (TNF)-alpha, TNF-beta, IL-5, IL-6, and IL-8 elicited by coculture with ALL cells. Notably, anti-CD19-4-1BB-zeta CIK cells secreted particularly low levels of interleukin-10 and proliferated strongly after contact with ALL cells. CONCLUSIONS: Anti-CD19 chimeric receptors delivering primary and costimulatory signals render CIK cells powerfully cytotoxic against ALL cells and induce secretion of immunostimulatory cytokines and proliferation. These results support the testing of genetically modified CIK cells in clinical trials.     

Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity

Natural killer T (NKT) cells are important regulators of the immune system, but their trafficking machinery, including expression of chemokine receptors, has been poorly defined. Unlike other conventional T-cell populations, we show that most NKT cells express receptors for extralymphoid tissue or inflammation-related chemokines (CCR2, CCR5, and CXCR3), while few NKT cells express lymphoid tissue-homing chemokine receptors (CCR7 and CXCR5). A population with homing potential for lymph nodes (L selectin(+) CCR7(+)) exists only within a small subset of CD4 NKT cells. We show differential expression of chemokine receptors among NKT cell subsets: CCR4 is mainly expressed by a high cytokine (interleukin-4/interleukin-2)-producing (CD4) NKT subset, while CCR1, CCR6, and CXCR6 are preferentially expressed by the low cytokine-producing CD8 and CD4(-)CD8(-) subsets. In line with this, TARC/CCL17 (a CCR4 ligand) induces preferential chemotaxis of the CD4 NKT subset, while chemotactic activities of LARC/CCL20 (a CCR6 ligand) and MIP-1 alpha/CCL3 (a CCR1 ligand) are focused on the CD8 and CD4(-)CD8(-) NKT cells. We conclude that, unlike conventional naive, memory, or effector T cells, the entire NKT cell population expresses nonlymphoid tissue homing chemokine receptors, yet NKT cell subsets differ considerably from each other by displaying distinct and reciprocal expression patterns of some chemokine receptors. Our results identify chemokine receptors that are potentially important for trafficking of human blood NKT cell subsets and reveal their function (cytokine production capacity)-dependent differential trafficking potentials.     

Enhanced killing of human B-cell lymphoma targets by combined use of cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies

We have investigated combining adoptive immunotherapy with cytokine-induced killer (CIK) cells and anti-CD20 monoclonal antibodies (mAb) GA101 or rituximab to optimize B-cell non-Hodgkin lymphoma (B-NHL) therapy. CIK cultures alone demonstrated significant cytotoxic activity against B-NHL cell lines or freshly isolated samples in either an autologous or allogeneic combination. This natural cytotoxicity (NC) was mainly due to the predominating CD3(+)CD56(+) CIK population (40%-75%) present in the cultures. The addition of anti-CD20 mAb GA101 or rituximab further increased cytotoxicity by 35% and 15%, respectively. This enhancement was mainly due to antibody-dependent cytotoxicity (ADCC) mediated by the 1%-10% NK cells contaminating CIK cultures. The addition of human serum (HS) inhibited NK-cell activation induced by rituximab, but not activation induced by GA101.Overall lysis in presence of serum, even of a resistant B-NHL cell line, was significantly increased by 100 μg/mL of rituximab, but even more so by GA101, with respect to CIK cultures alone. This was due to the combined action of complement-mediated cytotoxicity (CDC), ADCC, and CIK-mediated NC. These data suggest that rituximab, and even more so GA101, could be used in vivo to enhance CIK therapeutic activity in B-NHL.     

Two pathways of exocytosis of cytoplasmic granule contents and target cell killing by cytokine-induced CD3+ CD56+ killer cells

Cytokine-induced killer (CIK) cells are non-major histocompatibility complex-restricted cytotoxic cells generated by incubation of peripheral blood lymphocytes with anti-CD3 monoclonal antibody (MoAb), interleukin-2 (IL-2), IL-1, and interferon-gamma. Cells with the greatest effector function in CIK cultures coexpress CD3 and CD56 surface molecules. CIK cell cytotoxicity can be blocked by MoAbs directed against the cell surface protein leukocyte function associated antigen-1 but not by anti-CD3 MoAbs. CIK cells undergo release of cytoplasmic cytotoxic granule contents to the extracellular space upon stimulation with anti-CD3 MoAbs or susceptible target cells. Maximal granule release was observed from the CD3+ CD56+ subset of effector cells. The cytoplasmic granule contents are lytic to target cells. Treatment of the effector cells with a cell-permeable analog of cyclic adenosine monophosphate (cAMP) inhibited anti-CD3 MoAb and target cell- induced degranulation and cytotoxicity of CIK cells. The immunosuppressive drugs cyclosporin (CsA) and FK506 inhibited anti-CD3- mediated degranulation, but did not affect cytotoxicity of CIK cells against tumor target cells. In addition, degranulation induced by target cells was unaffected by CsA and FK506. Our results indicate that two mechanisms of cytoplasmic granule release are operative in the CD3+ CD56+ killer cells; however, cytotoxicity proceeds through a cAMP- sensitive, CsA- and FK506-insensitive pathway triggered by yet-to-be- identified target cell surface molecules.     

Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production

T cells with natural killer cell phenotype and function (NKT cells) have been described in both human and murine tissues. In this study, culture conditions were developed that resulted in the expansion of CD8(+) NKT cells from bone marrow, thymus, and spleen by the timed addition of interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and anti-CD3 monoclonal antibody. After 14 to 21 days in culture, dramatic expansion of CD3(+), CD8(+), alphabetaT-cell receptor(+) T cells resulted with approximately 20% to 50% of the cells also expressing the NK markers NK1.1 and DX5. The CD8(+) NKT cells demonstrated lytic activity against several tumor target cells with more than 90% lysis by day 14 to day 21 of culture. Cytotoxicity was observed against both syngeneic and allogeneic tumor cell targets with the greatest lytic activity by the cells expressing either NK1.1 or DX5. The expanded CD8(+) NKT cells produce T(H)1-type cytokines with high levels of IFN-gamma and tumor necrosis factor alpha. Expansion of the CD8(+) NKT cells was independent of CD1d. Ly49 molecules were expressed on only a minority of cells. A single injection of expanded CD8(+) NKT cells was capable of protecting syngeneic animals from an otherwise lethal dose of Bcl1 leukemia cells. Expanded CD8(+) NKT cells produced far less graft-versus-host disease (GVHD) than splenocytes across major histocompatibility barriers, even when 10 times the number of CD8(+) NKT cells as compared to splenocytes were injected. This reduction in GVHD was related to IFN-gamma production since cells expanded from IFN-gamma knock-out animals caused acute lethal GVHD, whereas cells expanded from animals defective in fas ligand, fas, IL-2, and perforin did not. These data indicate that CD8(+) NKT cells expanded in this fashion could be useful for preserving graft-versus-leukemia activity without causing GVHD.     

Generation of cytokine-induced killer cells from leukaemic samples with in vitro cytotoxicity against autologous and allogeneic leukaemic blasts

Cytokine-induced killer (CIK) cells are CD3(+)CD56(+) non-major histocompatibility complex (MHC)-restricted immune effector cells. The present report demonstrates that it was possible to expand CIK cells obtained at diagnosis from patients with acute leukaemia. The percentage of CD3(+)CD56(+) CIK cells generated following culture ranged between 7.6% and 65% (median of 35.3%) and these cells were able to kill the human natural killer target K562 cells. Although the same effector cells were able to lyse autologous acute myeloid leukaemia (AML) target cells, they were not able to lyse autologous acute lymphoblastic leukaemia target cells. Pre-absorption of the CIK effector cells by K562 cells did not completely abrogate the cytotoxicity of CIK cells against autologous blasts in 9 out of 12 samples tested. Moreover, it was observed that the cytotoxicity generated by the CIK effector cells against allogeneic leukaemic blasts was similar to that against autologous blasts. The present study suggests the potential application of CIK cells in the immunotherapy of AML, either in minimal disease state, as donor lymphocyte infusion in relapse post allogeneic transplant, or in cases of chemotherapy refractory leukaemia.     

Dual-functional capability of CD3+CD56+ CIK cells, a T-cell subset that acquires NK function and retains TCR-mediated specific cytotoxicity

CD3(+)CD56(+) cytokine-induced killer (CIK) cells display a potent cytolytic activity. The adhesion molecule lymphocyte function-associated antigen-1 plays a crucial role in binding as well as in cytolytic activity of CIK cells against tumor target cells expressing the corresponding ligands. CIK cells express activating natural killer (NK) receptors, including NKG2D, DNAX accessory molecule-1 (DNAM-1), and low levels of NKp30. Cell signaling not only through TCR/CD3 but also through NKG2D, DNAM-1, and NKp30 leads to CIK cell activation resulting in granule exocytosis, cytokine secretion, and cytotoxicity. Antibody blocking experiments showed that DNAM-1, NKG2D, and NKp30 are involved in the TCR-independent tumor cell recognition and killing. Anti-CMV-specific CIK cells could be expanded in standard CIK cultures and mediate both specific, MHC-restricted recognition and TCR-independent NK-like cytolytic activity against leukemic cell lines or fresh leukemic blasts. Antibody blocking of lymphocyte function-associated antigen-1 and DNAM-1 led to significant reduction of both CTL and NK-cell functions, whereas blocking of NKG2D and NKp30 only inhibited NK-like cytotoxicity. Their dual-effector function suggests that CIK cells, when used in a clinical setting, may control both neoplastic relapses and viral infections, 2 frequently associated complications in patients who received a transplant.     

T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect

Relapse of B-lineage acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (HSCT) commonly results from the failure of a graft-versus-leukemia (GVL) effect to eradicate minimal residual disease. Augmenting the GVL effect by the adoptive transfer of donor-derived B-ALL-specific T-cell clones is a conceptually attractive strategy to decrease relapse rates without exacerbating graft-versus-host disease (GVHD). Toward this end, we investigated whether a genetic engineering approach could render CD8(+) cytotoxic T lymphocytes (CTLs) specific for tumor cells that express the B-cell lineage cell surface molecule CD19. This was accomplished by the genetic modification of CTLs to express a chimeric immunoreceptor composed of a CD19-specific single-chain immunoglobulin extracellular targeting domain fused to a CD3-zeta intracellular signaling domain. CD19-redirected CTL clones display potent CD19-specific lytic activity and chimeric immunoreceptor-regulated cytokine production and proliferation. Because B-ALL cells can evade T-cell/natural killer- cell recognition by down-regulation of cell surface accessory molecules that participate in the formation of a functional immunologic synapse, we compared the CD19-specific effector function of genetically modified CD8(+) CTLs toward CD19(+) cells with disparate levels of intercellular adhesion molecule 1 (ICAM-1), leukocyte function-associated antigen 1 (LFA-1), and LFA-3. We observed that recognition of B-lineage tumor lines by CD19-specific CTLs was not impaired by low levels of ICAM-1, LFA-1, and LFA-3 cell surface expression, a functional attribute that is likely a consequence of our high-affinity CD19-specific chimeric immunoreceptor. Furthermore, the CD19-specific CTLs could lyse primary B-ALL blasts. These preclinical observations form the basis for implementing clinical trials using donor-derived CD19-specific T-cell clones to treat or prevent relapse of B-ALL after allogeneic HSCT.     

Cytokine-induced killer cells for cell therapy of acute myeloid leukemia: improvement of their immune activity by expression of CD33-specific chimeric receptors

Background

Cytokine-induced killer cells are ex vivo-expanded cells with potent antitumor activity. The infusion of cytokine-induced killer cells in patients with acute myeloid leukemia relapsing after allogeneic hematopoietic stem cell transplant is well tolerated, but limited clinical responses have been observed. To improve their effector functions against acute myeloid leukemia, we genetically modified cytokine-induced killer cells with chimeric receptors specific for the CD33 myeloid antigen.

Design and Methods

SFG-retroviral vectors coding for anti-CD33-ζ and anti-CD33-CD28-OX40-ζ chimeric receptors were used to transduce cytokine-induced killer cells. Transduced cells were characterized in vitro for their ability to lyse leukemic targets (4-hour ⁵¹chromium-release and 6-day co-cultures assays on human stromal mesenchymal cells), to proliferate (³H-thymidine-incorporation assay) and to secrete cytokines (flow cytomix assay) after contact with acute myeloid leukemia cells. Their activity against normal CD34⁺ hematopoietic progenitor cells was evaluated by analyzing the colony-forming unit capacity after co-incubation.

Results

Cytokine-induced killer cells were efficiently transduced with the anti-CD33 chimeric receptors, maintaining their native phenotype and functions and acquiring potent cytotoxicity (up to 80% lysis after 4-hour incubation) against different acute myeloid leukemia targets, as also confirmed in long-term killing experiments. Moreover, introduction of the anti-CD33 chimeric receptors was accompanied by prominent CD33-specific proliferative activity, with the release of high levels of immunostimulatory cytokines. The presence of CD28-OX40 in chimeric receptor endodomain was associated with a significant amelioration of the anti-leukemic activity of cytokine-induced killer cells. Importantly, even though the cytokine-induced killer cells transduced with anti-CD33 chimeric receptors showed toxicity against normal hematopoietic CD34⁺ progenitor cells, residual clonogenic activity was preserved.

Conclusions

Our results indicate that anti-CD33 chimeric receptors strongly enhance anti-leukemic cytokine-induced killer cell functions, suggesting that cytokine-induced killer cells transduced with these molecules might represent a promising optimized tool for acute myeloid leukemia immunotherapy.     

Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells

Natural killer (NK) cells hold promise for improving the therapeutic potential of allogeneic hematopoietic transplantation, but their effectiveness is limited by inhibitory HLA types. We sought to overcome this intrinsic resistance by transducing CD56+CD3- NK cells with chimeric receptors directed against CD19, a molecule widely expressed by malignant B cells. An abundance of NK cells for transduction was secured by culturing peripheral blood mononuclear cells with K562 cells expressing the NK-stimulatory molecules 4-1BB ligand and interleukin 15, which yielded a median greater than 1000-fold expansion of CD56+CD3- cells at 3 weeks of culture, without T-lymphocyte expansion. Expression of anti-CD19 receptors linked to CD3zeta overcame NK resistance and markedly enhanced NK-cell-mediated killing of leukemic cells. This result was significantly improved by adding the 4-1BB costimulatory molecule to the chimeric anti-CD19-CD3zeta receptor; the cytotoxicity produced by NK cells expressing this construct uniformly exceeded that of NK cells whose signaling receptors lacked 4-1BB, even when natural cytotoxicity was apparent. Addition of 4-1BB was also associated with increased cell activation and production of interferon gamma and granulocyte-macrophage colony-stimulating factor. Our findings indicate that enforced expression of signaling receptors by NK cells might circumvent inhibitory signals, providing a novel means to enhance the effectiveness of allogeneic stem cell transplantation.     

化疗联合自体细胞因子诱导杀伤细胞治疗急性白血病的临床观察

目的评价化疗联合自体细胞因子诱导的杀伤细胞(CIK)治疗急性白血病(AL)的疗效。方法选择经化疗达完全缓解(CR)>6个月的AL患者41例。19例患者接受化疗联合自体CIK细胞治疗,22例接受同期单纯化疗作为对照组。CIK细胞治疗采用血细胞分离机大量采集患者的外周血单个核细胞,用抗CD3单抗、IL2、IFNγ培养10d,将细胞洗涤后经静脉于化疗后第一天回输给患者。结果CIK组19例患者化疗同时共接受52疗程CIK细胞治疗,每例患者平均接受2~3个疗程CIK细胞治疗。每疗程回输CIK细胞总数为(22~300)×109/L,平均(142±85)×109/L。4年持续CR(CCR)率CIK组为734%;单纯化疗组4年预期CCR率为273%。两者差异有统计学意义(P<0005)。接受≥3个疗程CIK治疗的10例患者至观察截止时均处于CCR,中位CCR期43个月(23~52个月);接受<3个疗程CIK治疗的患者4/9例复发。结论化疗联合自体CIK细胞治疗AL的CCR率明显优于单纯化疗;疗效与疗程有关,疗程≥3个患者的疗效优于疗程<3个的患者。     

CIK cells from patients with HCC possess strong cytotoxicity to multidrug-resistant cell line Bel-7402／R

ABM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo. METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2 000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhlFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68. RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry. CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.     

Intensive expansion of natural killer T cells in the early phase of hepatocyte regeneration after partial hepatectomy in mice and its association with sympathetic nerve activation

When C57BL/6 mice were partially hepatectomized (PHx), severe lymphocytosis was induced in the liver in the early phase of hepatocyte regeneration (4 to 12 hours after PHx). A major lymphocyte subset expanding in this organ was estimated to be natural killer 1.1(+) (NK1.1(+)) intermediate CD3 (CD3(int)) cells (i.e., NKT cells). CD3(int) cells are extrathymic T cells generated in situ in the liver. These changes were suppressed when mice with PHx were pretreated with a beta-adrenergicD antagonist (i.e., beta-blocker), propranolol (PPL). This might have been caused by sympathetic nerve stimulation during hepatocyte regeneration. An alpha-blocker showed a similar effect, although the magnitude of suppression was lower than that of the beta-blocker. We previously showed that NK and NKT cells express surface beta-adrenergic receptors and are activated in number by sympathetic nerve stimulation. In the present study, NK cytotoxicity mediated by liver lymphocytes obtained from mice with PHx decreased, whereas NKT cytotoxicity against syngeneic thymocytes increased. Purified CD3(int) cells were also found to be able to mediate NKT cytotoxicity against regenerating hepatocytes. These results suggest that sympathetic nerve stimulation after PHx results in subsequent activation of NKT cells and that these NKT cells might be associated with immunologic surveillance during hepatocyte regeneration.     

Expansion of natural killer cell receptor (CD94/NKG2A)-expressing cytolytic CD8 T cells and CD4+CD25+ regulatory T cells from the same cord blood unit

OBJECTIVE: Cord blood contains a significant number of precursor cells that differentiate to cytotoxic effector cells and immunoregulatory cells. We tried to expand inhibitory natural killer cell receptor CD94-expressing CD8 T cells with cytolytic activity and CD4(+)CD25(+) regulatory T cells from the same cord cell unit. METHODS: Cytotoxic CD94-expressing CD8 T cells were expanded from CD4-depleted cord blood using an immobilized anti-CD3 monoclonal antibody and a cytokine and also CD4(+)CD25(+) regulatory T cells were expanded from a CD4-enriched fraction derived from the same cord blood unit using anti-CD3/CD28 monoclonal antibody-coated Dynabeads and cytokines. RESULTS: We were able to obtain a more than 1000-fold expansion of CD94-expressing CD8 T cells and a more than 50-fold expansion of CD4(+)CD25(+) cells from the same cord blood unit. These expanded CD4(+)CD25(+) cells expressed FoxP3 mRNA at a level about 100-fold higher than that in isolated CD25(-) cells and could suppress allogeneic mixed lymphocyte culture by >80% (effector cells: CD4(+)CD25(+) cells = 2:1). Cytolytic activities of purified CD94-expressing cells detected by a 4-hour (51)Cr release assay against K562 were >60%. Coculture of CD94-expressing cells with expanded CD4(+)CD25(+) cells did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells. CONCLUSION: These expanded cytolytic CD94-expressing CD8 cells might be able to induce a graft-vs-leukemia effect without enhancing graft-vs-host disease, and CD4(+)CD25(+) cells might be able to suppress allogeneic responses, including graft-vs-host disease and graft rejection after cord blood transplantation.     

Expansion of CD1d-restricted NKT cells in patients with primary HIV-1 infection treated with interleukin-2

Innate CD1d-restricted natural killer T (NKT) cells are infected and lost in HIV-1-infected patients, and this could contribute to HIV-1 pathogenesis because NKT cells play an important role in directing both adaptive and innate immunity. Administration of interleukin-2 (IL-2) to HIV-1-infected patients leads to substantial and sustained CD4+ T-cell expansion, involving both naive and memory cells. We investigated whether IL-2 treatment could restore the NKT cell compartment in patients with primary HIV-1 infection. We show that IL-2 combined with effective antiretroviral therapy (ART) resulted in significant expansion of CD1d-restricted NKT cells. Expansion occurred in both the CD4- and CD4+ subsets of NKT cells, and expanded cells expressed the CD161 maturation marker while expression of the HIV coreceptor CCR5 was reduced. These data indicate that IL-2 treatment in combination with effective ART is beneficial for the restoration of innate NKT cell immunity in patients with primary HIV-1 infection.     

Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells.

BACKGROUND/AIMS: Previous studies have shown that hepatic natural killer (NK) cells, also called pit cells, have a higher cytotoxicity against certain tumor cells and have a higher expression of the cell adhesion molecule CD11a as compared with blood NK cells. We further investigated the involvement of the adhesion molecules, reported to be involved in target cell killing by blood NK cells, in pit cell-mediated colon carcinoma cell killing. METHODS: 51Cr-release and DNA fragmentation were used to quantify target cell lysis and apoptosis, respectively. Adhesion of pit cells to CC531s monolayers was quantitated. RESULTS: Flow cytometric analysis showed that pit cells expressed CD2, CD11a, CD18 and CD54. CC531s cells expressed only CD54. Treatment of freshly isolated pit cells with monoclonal antibodies (mAbs) to CD11a and CD18 inhibited not only the pit cell-mediated CC531s cytolysis but also the pit cell-induced apoptosis of CC531s cells. The combination of mAbs to CD11a, CD18 and CD54 further increased the inhibition of pit cell-mediated CC531s cytolysis and apoptosis. Anti-CD2 mAb did not affect these processes. The binding of pit cells to CC531s cells was also inhibited by anti-CD11a, and CD18 mAbs, but not by anti-CD2 mAb. Anti-CD54 mAb reduced the target cell killing and the binding only slightly. CONCLUSIONS: These results indicate that CD11a/CD18 (LFA-1) present on pit cells plays an important role in pit cell-mediated target cell adhesion, lysis and apoptosis. This finding might explain why pit cells, which have a higher expression of LFA-1 as compared to blood NK cells, are more cytotoxic against tumor cells as compared to blood NK cells.     

Expression and coreceptor activity of STRL33/Bonzo on primary peripheral blood lymphocytes

CCR5 and CXCR4 are the major coreceptors that mediate human immunodeficiency virus 1 (HIV-1) infection, while most simian immunodeficiency virus (SIV) isolates use CCR5. A number of alternative coreceptors can also mediate infection of some virus strains in vitro, although little is known about their in vivo relevance. Therefore, we characterized the expression pattern and coreceptor activity of one of these alternative coreceptors, STRL33/Bonzo, using a newly developed monoclonal antibody. In addition to being highly expressed (approximately 1000-7000 STRL33 ABS [antibody binding sites]) on specific subsets of natural killer cells (CD3(-)/CD16(-/low)/CD56(+) and CD3(-)/CD16(low)/CD56(-)) and CD19(+) B lymphocytes (approximately 300-5000 STRL33 ABS), STRL33 was expressed at levels sufficient to support virus infection on freshly isolated, truly naive CD4(+)/CD45RA(+)/CD62L(+) cells (6000-11 000 ABS). STRL33 expression on peripheral blood mononuclear cells (PBMCs) was increased by mitogenic stimulation (OKT3/IL-2 [interleukin-2] had a greater effect than phytohemaglutinin (PHA)/IL-2), but it was dramatically decreased upon Ficoll purification. Infection of CCR5(-) human peripheral blood lymphocytes (PBLs) showed that 2 different SIV envelope (Env) proteins mediated entry into STRL33(+) cells. More importantly, the preferential infection of STRL33(+) cells in CCR5(-) PBLs by an R5/X4/STRL33 HIV-1 maternal isolate in the presence of a potent CXCR4 antagonist (AMD3100) suggests that STRL33 can be used as a coreceptor by HIV-1 on primary cells. Rhesus macaque (rh) STRL33 was used less efficiently than human STRL33 by the majority of SIV Env proteins tested despite similar levels of expression, thereby making it less likely that STRL33 is a relevant coreceptor in the rhesus macaque system. In summary, the expression pattern and coreceptor activity of STRL33 suggest its involvement in trafficking of tumor-infiltrating lymphocytes and indicate that STRL33 may be a relevant coreceptor in vivo.     

Decrease of CD56(+)T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma

CD56(+)T cells and CD56(+)natural killer (NK) cells are abundant in the human liver. The aim of this study was the further characterization of these cells in the liver with or without hepatitis C virus (HCV) infection. Liver mononuclear cells (MNC) were isolated from liver specimens obtained from the patients during abdominal surgery. In addition to a flow cytometric analysis, liver MNC and PBMC were cultured with the immobilized anti-CD3 Ab, IL-2, or a combination of IL-2 and IL-12 and their IFN-gamma production and the antitumor cytotoxicity were assessed. The liver MNC of HCV (-) patients contained 20% CD56(+)T cells whereas the same proportions decreased to 11% in chronic hepatitis livers and to 5% in cirrhotic livers. The proportion of NK cells also decreased in the cirrhotic livers. On the other hand, the populations of these cells in PBMC did not significantly differ among patient groups. The IFN-gamma production and the cytotoxicity against K562 cells, Raji cells, and a hepatocellular carcinoma, HuH-7 cells, greatly decreased in the cirrhotic liver MNC. In contrast, the cytotoxicity in PBMC did not significantly differ among the patient groups and was lower than that in the liver MNC of HCV (-) patients. CD56(+)T cells and NK cells but not regular T cells purified from liver MNC cultured with cytokines showed potent cytotoxicities against HuH-7 cells. These results suggest that a decreased number of CD56(+)T cells and NK cells in cirrhotic livers may be related to their susceptibility to hepatocellular carcinoma.     

The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism

Antibodies against CD20 can activate complement and induce antibody-dependent cellular cytotoxicity (ADCC) in B lymphocytes. In B-cell lines, such antibodies also induce apoptosis. In this study, the expression and function of CD20 on B-cell chronic lymphocytic leukemia (B-CLL) cells were analyzed. Flow cytometric analysis demonstrated that B-CLL cells express CD20 with a fluorescence intensity that is significantly weaker than that of normal CD5(+) and CD5(-) B cells and that of malignant CD5(-) low-grade non-Hodgkin lymphoma cells. A small population of cells from healthy donors that have an expression pattern of CD5 and CD20 identical to that of B-CLL cells were identified, and this population was confirmed to be of T lineage, not B lineage. Culture of freshly isolated B-CLL cells in the presence of the chimeric anti-CD20 antibody rituximab and a cross-linking F(ab)(2) fragment, resulted in dose- and time-dependent induction of apoptosis. The induction of apoptosis occurred under conditions in which the influence of complement activation and ADCC was negligible. Cross-linking of rituximab induced strong and sustained phosphorylation of the 3 mitogen activated protein (MAP) kinases c-Jun NH2-terminal protein kinase, extracellular signal-regulated kinase, and p38. Introduction of the p38 inhibitor SB203580 into the system completely blocked signaling downstream of p38, as evidenced by the absence of MAPKAP K2 activity, and significantly reduced the degree of anti-CD20-induced apoptosis. These results demonstrate that cross-linking of rituximab bound to CD20 on freshly isolated B-CLL cells induces apoptosis through a signaling pathway that is dependent on p38 MAP-kinase activation.     

Human mucosal T-cell cytotoxicity

Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CD3-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effectors do not have Fc receptors for immunoglobulin G, and are therefore distinct from T gamma cells, which mediate antibody-dependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7-. In contrast, peripheral blood anti-CD3-T effectors are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As anti-CD3-triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.