Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy

OBJECTIVE: Cytokine-induced killer (CIK) cells are ex vivo expanded cells enriched in CD3(+)CD56(+) natural killer T (NKT) cells with major histocompatibility-unrestricted cytotoxicity against several tumoral targets, except B-lineage acute lymphoblastic leukemia (B-ALL). We redirected CIK cells cytotoxicity toward B-ALL with a chimeric receptor specific for the CD19 antigen and then explored if modified-CIK cells maintain the same chemotactic properties of freshly isolated NKT cells, whose trafficking machinery reflects their preferential localization into the sites of B-ALL infiltration. MATERIAL AND METHODS: CIK cells were expanded ex vivo for 21 days and analyzed for expression of adhesion molecules and chemokine receptors regulating adhesion and homing toward leukemia-infiltrated tissues. CIK cells were then transduced with the anti-CD19-zeta-internal ribosomal entry site-green fluorescent protein retroviral vector and characterized for their cytotoxicity against B-ALL cells in a (51)Cr-release assay and for their trafficking properties, including chemotactic activity, adhesion and transendothelial migration, and metalloproteases-dependent invasion of Matrigel. RESULTS: Similarly to freshly isolated NKT cells, CD49d and CD11a were highly expressed on CIK cells. Moreover, CIK cells expressed CXCR4, CCR6, and CCR7 (mean expression 72%, 60%, and 32%, respectively), presenting chemotactic activity toward their respective ligands. Anti-CD19 chimeric receptor-modified CIK cells became cytotoxic against B-ALL cells (mean lysis, 60%) and showed, after exposure to a CXCL12 gradient, high capacity to adhere and transmigrate through endothelial cells and to invade Matrigel. CONCLUSION: The potential capacity to localize into leukemia-infiltrated tissues of anti-CD19 chimeric receptor-redirected CIK cells, together with their ability to efficiently kill B-ALL cells, suggests that modified-CIK cells represent a valuable tool for 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.     

Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function

Recent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+ T cells using either Kb-zeta or Kb-CD28-zeta receptors had similar functional activities, although the CD28-zeta receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-zeta chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+ T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance.     

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.     

Anti-CD3 + interleukin-2 stimulation of marrow and blood: comparison of proliferation and cytotoxicity.

The proliferation and in vitro cytolytic activity of interleukin-2 (IL-2)-activated and anti-CD3 + IL-2-stimulated marrow mononuclear cells (MMC) and peripheral blood mononuclear cells (PBMC) were studied. Samples from 8 normal donors, 15 patients with acute lymphoblastic leukemia (ALL), and 7 patients with non-Hodgkin's lymphoma (NHL) in remission were cultured in IL-2 (100 U/mL) or IL-2 (100 U/mL) plus anti-CD3 (10 ng/mL). MMC as well as PBMC samples demonstrated significant synergy between IL-2 and anti-CD3 in the promotion of proliferation as measured by 3H thymidine incorporation on day 5 (P less than .001) or fold increase in cell number on day 14. Cryopreserved marrow specimens had equally rapid proliferation as fresh MMC when cultured in the presence of anti-CD3 + IL-2. Anti-CD3 concentrations of 3, 11, 33, and 100 ng/mL augmented proliferation similarly in the presence of IL-2 (0.1 to 100 U/mL). Mean fold increases in cell number of both marrow- and blood-derived cultures after 14 days were significantly higher for anti-CD3 + IL-2-stimulated cultures compared with cultures stimulated with IL-2 only (50- to 200-fold increase in cell number; P = .01). Comparison of remission MMC and PBMC from ALL and NHL patients with normal controls showed equivalent growth rates of activated cultures at 7, 14, and 21 days. Marrow purging with immunotoxin anti-CD19 pokeweed antiviral protein plus 4HC had no significant effect on proliferation of anti-CD3 + IL-2-stimulated MMC cultures in patients with ALL. Cytolytic activity of IL-2- and IL-2 + anti-CD3-activated PBMC and MMC cultures was assessed in 51Cr release assays using K562 (natural killer ([NK]-sensitive), Daudi (Burkitt's lymphoma-, NK-resistant), and Nalm-6 (ALL-, lymphokine-activated killer [LAK]-resistant) cell lines and cryopreserved ALL blasts. Cytolytic activity on a per-cell basis (percent cytotoxicity at an effector:target ratio of 30:1) was similar in IL-2-activated PBMC- and MMC-derived cultures from ALL patients. MMC activated with anti-CD3 plus IL-2 killed Daudi significantly less well than IL-2-activated cultures on days 12 and 19 (P = .03); no significant differences were observed in lysis of LAK-resistant Nalm-6 or cryopreserved ALL blast targets. Dose response of anti-CD3 augmentation of Daudi and Nalm-6 killing was different in IL-2- and IL-2 + anti-CD3-stimulated cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of 4-1BB (CD137) in the functional activation of cord blood CD28(-)CD8(+) T cells

The CD28(-) subset of CD8(+) T cells is associated with cytotoxic T lymphocyte (CTL) effector function. We investigated a potential role for 4-1BB, a costimulatory molecule structurally related to members of the tumor necrosis factor (TNF) receptor family, in the generation and functional activation of CD28(-) CTLs by using human cord blood (CB) cells composed exclusively of naive CD8(+) T cells with few or no CD28(-) CTLs. The 4-1BB was induced preferentially on the CB CD28(-)CD8(+) T cells when CD28 down-regulation was induced by interleukin 15 (IL-15) and IL-12 stimulation. Anti-4-1BB costimulation induced dramatic phenotypic changes in the CD28(-) CTLs, including restoration of CD28 expression as well as that of memory markers such as CD45RO and CC chemokine receptor 6 (CCR6). Anti-4-1BB costimulation also promoted long-term survival of CD28(-) CTLs, which were sensitive to activation-induced cell death upon anti-CD3 stimulation. The memory-type CD28(+) CTLs induced by anti-4-1BB costimulation acquired a greatly enhanced content of granzyme B, a cytolytic mediator, and enhanced cytotoxic activity as compared with CD28(-) CTLs. Strong cytotoxicity of memory-type CTLs to a 4-1BB ligand-expressing Epstein-Barr virus (EBV)-transformed B-cell line was almost completely abrogated by 4-1BB-Fc, a soluble form of 4-1BB, suggesting involvement of 4-1BB in cytolytic processes. Taken all together, our results suggest that 4-1BB plays a role in the differentiation of effector memory CTLs.     

Treatment of acute lymphoblastic leukaemia with the second generation of CD19 CAR‐T containing either CD28 or 4‐1BB

T cells modified with anti‐CD19 chimeric antigen receptor (CAR) containing either CD28 or 4‐1BB (also termed TNFRSF9, CD137) costimulatory signalling have shown great potential in the treatment of acute lymphoblastic leukaemia (ALL). However, the difference between CD28 and 4‐1BB costimulatory signalling in CAR‐T treatment has not been well elucidated in clinical trials. In this study, we treated 10 relapsed or refractory ALL patients with the second generation CD19 CAR‐T. The first 5 patients were treated with CD28‐CAR and the other 5 patients were treated with 4‐1BB CAR‐T. All the 10 patients were response‐evaluable. Three patients achieved complete remission and 1 patient with extramedullary disease achieved partial response after CD28‐CAR‐T treatment. In the 4‐1BB CAR‐T treatment group, 3 patients achieved complete remission. Furthermore, FLT‐3 ligand (FLT3LG) was highly correlated with response time and may serve as a prognosis factor. No severe adverse events were observed in these 10 treated patients. Our study showed that both CD28 CAR‐T and 4‐1BB CAR‐T both worked for response but they differed in response pattern (peak reaction time, reaction lasting time and reaction degree), adverse events, cytokine secretion and immune‐suppressive factor level.     

B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.     

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.     

Eradication of lymphoma by CD8 T cells following anti-CD40 monoclonal antibody therapy is critically dependent on CD27 costimulation

Growing evidence points to the potential of agonistic anti-CD40 mAbs as adjuvants for vaccination against cancer. These appear to act by maturing dendritic cells (DCs) and allowing them to prime CD8 cytotoxic T lymphocytes (CTLs). Although it is well established that optimal T-cell priming requires costimulation via B7:CD28, recent studies emphasize the contribution of TNF receptors to this process. To understand how anti-CD40 mAbs trigger effective antitumor immunity, we investigated the role of TNFR superfamily members CD27 and 4-1BB in the generation of this immunity and showed that, although partially dependent on 4-1BB:4-1BBL engagement, it is completely reliant on CD27:CD70 interactions. Importantly, blocking CD70, and to some extent 4-1BBL, during anti-CD40 treatment prevented accumulation of tumor-reactive T cells and subsequent tumor protection. However, it did not influence changes in DC number, phenotype, nor the activity of CTLs once immunity was established. We conclude that CD27:CD70 and 4-1BB:4-1BBL interactions are needed for DC-driven accumulation of antitumor CTLs following anti-CD40 mAb treatment. Finally, in support of the critical role for CD70:CD27, we show for the first time that agonistic anti-CD27 mAbs given without a DC maturation signal completely protect tumor-bearing mice and provide a highly potent reagent for boosting antitumor T-cell immunity.     

Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection

To study human immunodeficiency virus (HIV)-specific cellular immunity in vivo, we transferred syngeneic lymphocytes after ex vivo expansion and transduction with a chimeric receptor gene (CD4/CD3-zeta) between identical twins discordant for HIV infection. Single and multiple infusions of 10(10) genetically modified CD8(+) T cells resulted in peak fractions in the circulation of approximately 10(4) to 10(5) modified cells/10(6) mononuclear cells at 24 to 48 hours, followed by 2- to 3-log declines by 8 weeks. In an effort to provide longer high-level persistence of the transferred cells and possibly enhance anti-HIV activity, we administered a second series of infusions in which both CD4(+ )and CD8(+) T cells were engineered to express the chimeric receptor and were costimulated ex vivo with beads coated with anti-CD3 and anti-CD28. Sustained fractions of approximately 10(3) to 10(4) modified cells/10(6) total CD4(+) or CD8(+) cells persisted for at least 1 year. Assessment of in vivo trafficking of the transferred cells by lymphoid tissue biopsies revealed the presence of modified cells in proportions equivalent to or below those in the circulation. The cell infusions were well tolerated and were not associated with substantive immunologic or virologic changes. Thus, adoptive transfer of genetically modified HIV-antigen-specific T cells was safe. Sustained survival in the circulation was achieved when modified CD4(+ )and CD8(+) T cells were infused together after ex vivo costimulation, indicating the important role played by antigen-specific CD4(+) T cells in providing "help" to cytotoxic effectors. (Blood. 2000;96:467-474)     

Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo.

OBJECTIVE. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. METHODS. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. RESULTS. IL-2-induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2-dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. CONCLUSION. Anti-CD5 has an inhibitory effect on in vivo-activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to "switching-off" of T cell activation in the joints.     

Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chimeric envelope glycoproteins.

Interleukin-2 (IL-2) is a cytokine that induces the proliferation of certain IL-2 receptor expressing quiescent cells. Human IL-2 was fused to the amino-terminus of amphotropic murine leukemia virus (MLV) envelope glycoproteins. Retroviral vectors were pseudotyped with both the IL-2 chimeric envelope and the wild-type amphotropic MLV envelope. The chimeric IL-2 glycoproteins were incorporated on retroviral vectors and the IL-2-displaying vector particles could bind specifically to cell surface IL-2 receptors. In addition, the IL-2-displaying vectors could infect proliferating cells through amphotropic receptors irrespective of whether the cells expressed the IL-2 receptor. IL-2-displaying vector particles could also transiently stimulate the cell cycle entry and proliferation of several IL-2-dependent cell lines. Finally, retroviral vectors displaying IL-2 could efficiently transduce G0/G1-arrested cells expressing the IL-2 receptor at a 34-fold higher efficiency compared with vectors with unmodified envelopes. This new strategy, whereby C-type retroviral vector particles display a ligand that activates the cell cycle of the target cells at the time of virus entry, may represent an alternative to lentivirus-derived retroviral vectors for the infection of quiescent cells. In addition, upon infection of an heterogeneous population of nonproliferating cells, MLV-retroviral vectors that display cytokines/growth factors will allow the transgene of interest to be integrated specifically in quiescent cells expressing the corresponding cytokine/growth factor receptor.     

类风湿关节炎患者外周血Th17细胞的检测及意义

目的 探讨Th17细胞在类风湿关节炎(RA)患者外周血中的水平及意义.方法 分离RA患者和健康者外周血单个核细胞(PBMC),免疫磁珠阴选CD4~+T细胞,用或不用非特异性刺激剂(A-CD3、A-CD28),然后加佛波酯/离子霉素(PMA/Ion),经固定,透膜处理进行细胞内染色,流式细胞术(FCM)检测CD4~+T细胞内白细胞介素(IL)-17~+/干扰素(IFN)-γ~+、IL-17~+/IL-6~+水平.分组:①健康对照组;②RA病情稳定组;③RA病情活动组.结果 免疫磁珠阴选CD4~+T细胞纯度>90%.RA病情活动组IL-17表达水平(1.54±0.41)显著高于RA病情稳定组(0.70±0.21,P<0.01),二者又显著高于健康对照组(0.42±0.12,P<0.01).用A-CD3、A-CD28、IL-23刺激后,CD4~+T细胞IL-17胞内表达水平较无刺激组显著增加(P<0.05).CD4~+T细胞IFN-γ表达水平呈现与IL-17表达相似的特点,而RA患者与健康对照组IL-6表达水平差异无统计学意义.RA患者IL-17胞内表达水平与IFN-γ、IL-6无显著相关.结论 RA患者外周血CD4~+T细胞中存在异常增高的Th17细胞,且其水平与病情活动相关,有望作为判断RA患者病情活动的指标之一.     

Inhibition of synovial fluid T cell proliferation by anti-CD5 monoclonal antibodies. A potential mechanism for their immunotherapeutic action in vivo

Objective. Monoclonal antibodies (MAb) directed against the T cell surface molecule CD5 are able to provide accessory stimulatory signals to resting T cells. The potential role of CD5 as an immunoregulatory molecule in inflammatory synovitis was examined. Methods. Synovial fluid and peripheral blood T cells of patients with active rheumatoid arthritis (RA) were purified and stimulated with interleukin-2 (IL-2), and the effect of MAb directed against CD5 on IL-2 responsiveness was examined. Results. IL-2—induced proliferation of synovial fluid T cells was strongly inhibited by anti-CD5 MAb, but not by anti-CD28 or anti-CD3 MAb. In RA peripheral blood T cells, MAb directed against CD5, CD3, and CD28 induced IL-2—dependent T cell growth, similar to findings in healthy controls. The difference in activity of anti-CD5 MAb on synovial fluid T cells compared with peripheral blood T cells was not due to different surface expression of CD5. Conclusion. Anti-CD5 has an inhibitory effect on in vivo—activated synovial fluid T cells. The disease-ameliorative effects of anti-CD5 immunotoxin treatment of RA may be partly due to “switching-off” of T cell activation in the joints.     

Modulation of cord blood CD8+ T-cell effector differentiation by TGF-beta1 and 4-1BB costimulation

Transforming growth factor-beta1 (TGF-beta1), an immunosuppressive cytokine, inhibits cytotoxic T cell (CTL) immune responses. In contrast, 4-1BB (CD137), a costimulatory molecule in the tumor necrosis factor (TNF) receptor family, amplifies CTL-mediated antitumor immune responses. We investigated whether TGF-beta1 responses could be reversed by 4-1BB costimulation during in vitro differentiation of naive CD8+ T cells into effector CTL cells. TGF-beta1 potently suppressed CTL differentiation of human cord blood naive CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-beta1-mediated suppression of CTL differentiation was abrogated by 4-1BB costimulation but not by CD28 or another member in the TNF receptor family, CD30. 4-1BB costimulation suppressed Smad2 phosphorylation induced by TGF-beta1, suggesting that 4-1BB effects were at the level of TGF-beta1 signaling. 4-1BB effects on the TGF-beta1-mediated suppression were enhanced by interleukin 12 (IL-12) but counteracted by IL-4; 4-1BB expression was up- or down-regulated, respectively, by IL-12 and IL-4. IL-4 was more dominant than IL-12 when both cytokines were present during 4-1BB costimulation in the presence of TGF-beta1. This indicates critical roles for IL-4 and IL-12 in regulating 4-1BB effects on TGF-beta1-mediated suppression.     

Retargeting of natural killer-cell cytolytic activity to ErbB2-expressing cancer cells results in efficient and selective tumor cell destruction

The continuously growing natural killer (NK) cell line NK-92 is highly cytotoxic against malignant cells of various origins without affecting normal human cells. Based on this selectivity, the potential of NK-92 cells for adoptive therapy is currently being investigated in phase I clinical studies. To further enhance the antitumoral activity of NK-92 cells and expand the range of tumor entities suitable for NK-92-based therapies, here by transduction with a retroviral vector we have generated genetically modified NK-92 cells expressing a chimeric antigen receptor specific for the tumor-associated ErbB2 (HER2/neu) antigen, which is overexpressed by many tumors of epithelial origin. The chimeric antigen receptor consists of the ErbB2-specific scFv(FRP5) antibody fragment, a flexible hinge region derived from CD8, and transmembrane and intracellular regions of the CD3 zeta chain. Transduced NK-92-scFv(FRP5)-zeta cells express high levels of the fusion protein on the cell surface as determined by fluorescence-activated cell-scanning (FACS) analysis. In europium release assays, no difference in cytotoxic activity of NK-92 and NK-92-scFv(FRP5)-zeta cells toward ErbB2-negative targets was found. However, even at low effector-to-target ratios, NK-92-scFv(FRP5)-zeta cells specifically and efficiently lysed established and primary ErbB2-expressing tumor cells that were completely resistant to cytolytic activity of parental NK-92 cells. These results demonstrate that efficient retargeting of NK-92 cytotoxicity can be achieved and might allow the generation of potent cell-based therapeutics for the treatment of ErbB2-expressing malignancies.     

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。     

Enhanced cytotoxicity of IL-24 gene-modified dendritic cells co-cultured with cytokine-induced killer cells to hepatocellular carcinoma cells

As a novel human cytokine found recently, IL-24 could selectively kill tumor cells by multiple ways. Dendritic cells (DCs) are the major antigen-presenting cells. Recent studies have revealed that IL-24 can promote the antigen-presenting function of DCs. In this study, we evaluated the antitumor effect and mechanism of co-cultured cytokine-induced killer (CIK) cells and autologous DCs modified with IL-24 gene on hepatocellular carcinoma (HCC) cells. DCs and CIK cells were prepared routinely from human peripheral blood mononuclear cells. Recombinant adenovirus AdVGFP/IL-24 (Ad-IL24) was constructed expressing IL-24. IL-24 gene was transduced into DCs via Ad-IL24, the cells obtained were named DC-IL-24. We demonstrated that the expression rates of CD80, CD83, CD1a, HLA-DR, CD40, CXCR4 on DC-IL-24 were significantly increased compared with those of the control group. DC-IL-24 produced markedly higher levels of IL-24, IL-12 and TNF-α as compared with those of DCs. On comparison with non-transfected DCs co-cultured with CIK cells, transfected DCs co-cultured with CIK cells had a significant higher lytic activity against SMMC7721 cells, a HCC cell line.