Dendritic Cells Therapy with Cytokine-Induced Killer Cells and Activated Cytotoxic T Cells Attenuated Th2 Bias Immune Response.

THE AIM OF THIS STUDY: The purpose of this study is to investigate whether the DC cells combined with CIK cells (DC/CIK) and DC activated cytotoxic T cells (DC-ACT) treatment can promote antitumor response and change the immune indicators by targeting the heterogeneous tumor cell populations at a system level. METHODS: In this study, 112 patients with cancer were assigned to the DC/CIK treatment and 116 patients received the DC-ACT therapy. We detected the lymphocyte subsets and other immune indicators pre- and post-treatment to evaluate the changes of patient's immunity and compare the differences in immune status between two adoptive cellular immunotherapies. RESULTS: DC/CIK therapy elevated the percentage of CD3+ HLA-DR+ T cells, NK cells and several serological cytokines such as IL-2, IL-6 after cell infusion (p < .05). DC-ACT therapy could increase the total CD3 + T cells, CD8 + T cells, CD3+ HLA-DR+ cells and IL-12 cytokines after cell infusion (p < .05). The levels of IL-4/IFN-γ, IL-4/IL-12 and IL-6/IL-12 were reduced significantly in the DC-ACT group compared with DC/CIK group. These observations suggested that DC-ACT therapy has more dominance to induce Th1 cytokine response instead of skewing toward the Th2 cytokine profile based on the immunomodulatory properties. CONCLUSIONS: These results indicated that DC, CIK, and DC-ACT cells exert anti-tumor activity through the different pathways. Thus, this work may provide valuable insights into the clinical curative effect evaluation of immunocyte therapy and the design of combined immunotherapeutic strategies for malignant tumors.

Efficient killing of radioresistant breast cancer cells by cytokine-induced killer cells.

Recurrence of breast cancer after radiotherapy may be partly explained by the presence of radioresistant cells. Thus, it would be desirable to develop an effective therapy against radioresistant cells. In this study, we demonstrated the intense antitumor activity of cytokine-induced killer cells against MCF-7 and radioresistant MCF-7 cells, as revealed by cytokine-induced killer-mediated cytotoxicity, tumor cell proliferation, and tumor invasion. Radioresistant MCF-7 cells were more susceptible to cytokine-induced killer cell killing. The stronger cytotoxicity of cytokine-induced killer cells against radioresistant MCF-7 cells was dependent on the expression of major histocompatibility complex class I polypeptide-related sequence A/B on radioresistant MCF-7 cells after exposure of cytokine-induced killer cells to sensitized targets. In addition, we demonstrated that cytokine-induced killer cell treatment sensitized breast cancer cells to chemotherapy via the downregulation of TK1, TYMS, and MDR1. These results indicate that cytokine-induced killer cell treatment in combination with radiotherapy and/or chemotherapy may induce synergistic antitumor activities and represent a novel strategy for breast cancer.

[Heparin-treated dendritic cells promote Th0 to Th1 differentiation via the Toll-like receptor 3 in peripheral blood monocytes of patients with chronic hepatitis B].

OBJECTIVE: To investigate the mechanisms underlying the ability ofheparin-treated dendritic cells (DCs) to promote Th0 to Th1 differentiation in chronic hepatitis B (CHB). METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from CHB patients and cultured in RPMI-1640 with recombinant GM-CSF and IL-4 with or without heparin to obtain DCs for study. The levels of Toll-like receptors (TLRs) on the DCs were measured using FACS and qPCR techniques.DC subsets with high expression of TLRs were selected for analysis of functional changes by treatment with the corresponding TLR-siRNA. The CD4+ T cell subpopulation was purified from peripheral blood by Dynal immunomagnetic beads, and then the production of IL-12 by DCs in the presence of poly(I:C) or R848 and ofIFN and IL-4 by Th cells co-cultured with DCs was evaluated by ELISA. The t-test was used for statistical analysis. RESULTS: TLR3 expression, and not expression of TLR 7 or TLR8,was significantly increased in heparin-treated DCs as compared to levels detected in the DCs without heparin treatment (t =2.849,P less than 0.05;t =3.027,P less than 0.05). The level of IL-12 produced by heparin-treated DCs stimulated with poly(I:C) was obviously higher than that produced by DCs without heparin treatment and stimulated with poly(I: C) (t =8.68,P less than 0.01) or with R848 (t =19.01,P less than 0.01). However, the IL-12 production by TLR3-siRNA transfected-DCs was significantly reduced (t =31.49, P less than 0.01).When Th cells from allogenic patients with CHB were co-cultured with the TLR3-siRNA transfectedDCs, the frequency ofCD4+ IFN+ cells was significantly reduced (1.64+/-0.57% vs.6.31+/-0.88%,P less than 0.01),as was the capability of Thl to generate IFNg (t =20.83,Pless than 0.01). CONCLUSION: Heparin may have up-regulated the TLR3 expression level of DCs, and sequentially promoted Th0 to Th1 differentiation.

Bispecific antibody activated T cells: A newly developed T cells with enhanced proliferation ability and cytotoxicity.

Adoptive cell therapy using ex vivo expanded lymphocytes has shown remarkable efficacy in tumor immunotherapy recently. Among various transfused immune cells, T lymphocytes are the most widely used since they are critical mediators of the immune system and have the capacity to kill tumor cells. However, there are drawbacks in the expanded T cells for transfusion including limited cytotoxicity, limited proliferation and lack of specificity. To improve the quality of these ex vivo expanded T cells, we have designed a new method to expand a group of T cells which are named bispecific antibodies activated T cells. It is the first time that such cells are induced by introducing the bispecific antibody drug (blinatumomab) and feeder cells (normal B cells and irradiated B cell originated lymphoma cells) to the traditional T cells culture system. Culture of freshly isolated human peripheral blood mononuclear cells in this newly designed cell culture system enabled these expanded T cells that (a) displayed a robust proliferation ability; (b) showed fully activated phenotype and enhanced cytokines production; (c) had a low proportion of CD4+CD25+ T regulatory cells and (d) exhibited strengthened cytotoxicity at relatively low effector: target ratios. This work further confirmed the feasibility of rapid induction and expansion of large amounts of human T cells in vitro by using bispecific antibodies and feeder cells. This strategy could also be used for other immune cells rapid expansion and help to improve the quality of these expanded immune cells for adoptive transfusion.

Increased cycles of DC/CIK immunotherapy decreases frequency of Tregs in patients with resected NSCLC.

Regulatory T cells (Tregs) suppress antitumor immune responses. Cycles of Dendritic cells (DC) vaccination combined with cytokine-induced killer (CIK) cells (DC/CIK) treatment were significantly related with good prognosis. Therefore, we investigated whether increased cycles of immunotherapy could decrease frequency of Tregs in patients with resected non-small cell lung cancer (NSCLC). Previous study from our laboratory has determined that the optimal cutoff point of the cycle count was 3cycles. We examined the levels of Tregs and the related cytokines by flow cytometric and cytokine analysis in these patients after more than (≥) 3cycles or less than (<) 3cycles of DC/CIK cell treatment. Significant reduction of Tregs frequency, Treg-generated cytokines level and recurrence rate were presented in patients received with ≥3cycles of DC/CIK cell treatment compared with patients with <3cycles of treatment. Interestingly, Tregs frequency and the related cytokines level were similar between patients suffered tumor recurrence and patients without recurrence in both groups. Together, our findings reveal that increased cycle count of DC/CIK cell immunotherapy contribute to decline of Tregs frequency and cancer recurrence rate in patients with resected NSCLC.

Gemcitabine treatment enhanced the anti-tumor effect of cytokine induced killer cells by depletion of CD4+CD25bri regulatory T cells.

Cytokine induced killer (CIK) cells have a powerful tumor cells killing activity both in vitro and in vivo and transfusion of these cells have become an adjuvant treatment for tumors. CIK cells are induced and amplified from peripheral blood mononuclear cells (PBMCs) with multiple cytokines. As CD4+CD25bri regulatory T cells can be also induced by high dose of interleukin 2 (IL-2) which is used for CIK cells amplification in the CIK cell culture system, the anti-tumor activity of CIK cells was suppressed to some extent. In order to overcome this unwanted suppressive factor, we found that low dose of gemcitabine could reduce the proportion of CD4+CD25bri regulatory T cells in the CIK cell culture system and significantly enhance the anti-tumor activity of CIK cells in vitro. The levels of interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) were also reduced significantly following the depletion of CD4+CD25bri regulatory T cells in gemcitabine treated CIK cell culture system. In vivo experiment showed that low dose of gemcitabine treated CIK cells significantly suppressed tumor growth and prolonged their lifespan in tumor-bearing nude mice, with the proportion of CD4+CD25bri regulatory T cells reduced. Meanwhile, we detected lower levels of IL-10, TGF-ß and a higher level of interferon-γ (IFN-γ) in tumor-bearing nude mice that received gemcitabine treated CIK cells transfusion than those in other groups. The possible mechanism involved in the enhanced anti-tumor activity in vivo was that gemcitabine treated CIK cells created a strengthened anti-tumor immune microenvironment with the changed levels of cytokines such as IL-10, TGF-ß and IFN-γ. These results suggested a strategy to improve the adoptive immune therapy in recent use by removing the suppressive factors and a more effective tumor treatment combining chemotherapy and immunotherapy.

Enhanced anti-colon cancer immune responses with modified eEF2-derived peptides.

Eukaryotic elongation factor-2 (eEF2) is overexpressed in many human cancers and is an attractive target for cancer immunotherapy. The eEF2 derived polypeptides have been shown to be able to induce cytotoxic T lymphocytes from healthy donor. Here, we demonstrate the evidence indicating that modification of a segment of peptides from wild type eEF2-derived immunogenic peptides is able to further enhance its capacity of inducing antigen-specific cytotoxic T lymphocytes (CTLs) against colon cancer cells. Using peptide-MHC binding algorithms, potential HLA-A2.1-restricted epitopes capable of inducing specific CD8(+) CTLs were identified. By analyzing HLA-A2.1 affinity and immunogenicity, we further identified one novel immunogenic peptide, P739-747 (RLMEPIYLV), that elicited specific CTL responses in HLA-A2.1/K(b) transgenic mice and culture with peripheral blood lymphocytes from colon cancer patients. Furthermore, replacing certain amino acids (at positions 1, 3, 7) within the P739-747 sequence improved the immunogenicity against eEF2. Several analogs containing the auxiliary HLA-A*0201 anchor residues were able to stably bind to HLA-A*0201 and enhance CTL responses compared with the native sequence; two of them showed increased anti-tumor effects during the adoptive immunotherapy in vivo. Thus, these results support that modified immunogenic analogs are promising candidates for peptide-based cancer vaccination and immunotherapy.

Dendritic cell immunotherapy combined with cytokine-induced killer cells promotes skewing toward Th2 cytokine profile in patients with metastatic non-small cell lung cancer.

Dendritic cell (DC) vaccination and cytokine-induced killer (CIK) cell therapy (DC/CIK) have shown limited success in the treatment of advanced non-small cell lung cancer (NSCLC). To investigate the reason for this limited success, the effects of DC/CIK cell therapy on the immune responses of tumor-bearing patients and patients with resected NSCLC were evaluated. In the total 50 patients studied, the serum concentrations of the Th2 cytokines (IL-4 and IL-10) in tumor-bearing patients were significantly higher than those with resected NSCLC before immunotherapy. The post-therapy Th1 cytokine (IFN-γ) level in patients with resected NSCLC significantly increased from the pre-therapy level. In contrast, significantly enhanced post-therapy Th2 cytokine (IL-4 and IL-10) levels were found in tumor-bearing patients. The intracellular staining assay revealed that DC/CIK cell therapy increased the IFN-γ-producing T lymphocyte (CD8(+)IFN-γ(+)) frequency in patients with resected NSCLC, but these lymphocytes were not found in tumor-bearing patients. Furthermore, overproduction of vascular endothelial growth factor (VEGF) in tumor-bearing patients showed a statistically positive correlation with IL-4, suggesting that VEGF might be responsible for the predominance of serum Th2 cytokines. In a word, tumor-bearing patients developed a Th2-dominant status that could not be reversed toward Th1 following immunotherapy. A combined regiment of DC vaccination and CIK cell therapy with other treatments to overcome systemic Th2-dominant immune response might improve the current clinical benefit.

Response gene to complement 32 (RGC-32) expression on M2-polarized and tumor-associated macrophages is M-CSF-dependent and enhanced by tumor-derived IL-4.

Response gene to complement 32 (RGC-32) is a cell cycle regulator involved in the proliferation, differentiation and migration of cells and has also been implicated in angiogenesis. Here we show that RGC-32 expression in macrophages is induced by IL-4 and reduced by LPS, indicating a link between RGC-32 expression and M2 polarization. We demonstrated that the increased expression of RGC-32 is characteristic of alternatively activated macrophages, in which this protein suppresses the production of pro-inflammatory cytokine IL-6 and promotes the production of the anti-inflammatory mediator TGF-ß. Consistent with in vitro data, tumor-associated macrophages (TAMs) express high levels of RGC-32, and this expression is induced by tumor-derived ascitic fluid in an M-CSF- and/or IL-4-dependent manner. Collectively, these results establish RGC-32 as a marker for M2 macrophage polarization and indicate that this protein is a potential target for cancer immunotherapy, targeting tumor-associated macrophages.

Autologous tumor lysate-pulsed dendritic cell immunotherapy with cytokine-induced killer cells improves survival in gastric and colorectal cancer patients.

Gastric and colorectal cancers (GC and CRC) have poor prognosis and are resistant to chemo- and/or radiotherapy. In the present study, the prophylactic effects of dendritic cell (DC) vaccination are evaluated on disease progression and clinical benefits in a group of 54 GC and CRC patients treated with DC immunotherapy combined with cytokine-induced killer (CIK) cells after surgery with or without chemo-radiotherapy. DCs were prepared from the mononuclear cells isolated from patients using IL-2/GM-CSF and loaded with tumor antigens; CIK cells were prepared by incubating peripheral blood lymphocytes with IL-2, IFN-γ, and CD3 antibodies. The DC/CIK therapy started 3 days after low-dose chemotherapy and was repeated 3-5 times in 2 weeks as one cycle with a total of 188.3 ± 79.8 × 10(6) DCs and 58.8 ± 22.3 × 10(8) CIK cells. Cytokine levels in patients' sera before and after treatments were measured and the follow-up was conducted for 98 months to determine disease-free survival (DFS) and overall survival (OS). The results demonstrate that all cytokines tested were elevated with significantly higher levels of IFN-γ and IL-12 in both GC and CRC cohorts of DC/CIK treated patients. By Cox regression analysis, DC/CIK therapy reduced the risk of post-operative disease progression (p<0.01) with an increased OS (<0.01). These results demonstrate that in addition to chemo- and/or radiotherapy, DC/CIK immunotherapy is a potential effective approach in the control of tumor growth for post-operative GC and CRC patients.