GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Secondary lymphoid tissue chemokine mediates T cell-dependent antitumor responses in vivo

Secondary lymphoid tissue chemokine (SLC, also referred to as Exodus 2 or 6Ckine) is a recently identified high endothelial-derived CC chemokine. The ability of SLC to chemoattract both Th1 lymphocytes and dendritic cells formed the rationale to evaluate this chemokine in cancer immunotherapy. Intratumoral injection of recombinant SLC evidenced potent antitumor responses and led to complete tumor eradication in 40% of treated mice. SLC-mediated antitumor responses were lymphocyte dependent as evidenced by the fact that this therapy did not alter tumor growth in SCID mice. Studies performed in CD4 and CD8 knockout mice also revealed a requirement for both CD4 and CD8 lymphocyte subsets for SLC-mediated tumor regression. In immunocompetent mice, intratumoral SLC injection led to a significant increase in CD4 and CD8 T lymphocytes and dendritic cells, infiltrating both the tumor and the draining lymph nodes. These cell infiltrates were accompanied by the enhanced elaboration of Th1 cytokines and chemokines monokine induced by IFN-gamma and IFN-gamma-inducible protein 10 but a concomitant decrease in immunosuppressive cytokines at the tumor site. In response to irradiated autologous tumor, splenic and lymph node-derived cells from SLC-treated tumor-bearing mice secreted significantly more IFN-gamma, GM-CSF, and IL-12 and reduced levels of IL-10 than did diluent-treated tumor-bearing mice. After stimulation with irradiated autologous tumor, lymph node-derived lymphocytes from SLC-treated tumor-bearing mice demonstrated enhanced cytolytic capacity, suggesting the generation of systemic immune responses. These findings provide a strong rationale for further evaluation of SLC in tumor immunity and its use in cancer immunotherapy.     

Phase 1 clinical trial of irradiated autologous melanoma cells adenovirally transduced with human GM-CSF gene

The objective of this study was to determine the safety and antitumor activity of an autologous GM-CSF-secreting melanoma cell vaccine that was engineered ex vivo with recombinant replication-incompetent adenovirus harboring a human GM-CSF gene (Adv/hGM-CSF). Melanoma samples were surgically obtained from 30 patients (15 female and 15 male, ages ranging from 23 to 87) and were processed for vaccine preparation. Due to stringent eligibility criteria, 9 out of 30 patients were enrolled in the phase 1 clinical trial (FDA IND7677). Melanoma cell lines established from surgical specimens of 9 patients were transduced with Adv/hGM-CSF (MOI of 100) and subsequently irradiated at 35 Gy. These cell lines secreted human GM-CSF in vitro at an average rate of 80-424 ng/10(6) cells/24 h. All patients were intradermally and subcutaneously injected at several sites with irradiated autologous melanoma cells (2x10(6)-1x10(7) in 300 microl saline), 2-10 times, at intervals of 4-8 weeks. None of the patients vaccinated showed any serious adverse systemic response. Three patients (nos.1, 6 and 7) demonstrated local reaction (erythema) to the vaccination. Tumor-specific CTL assays performed in the absence of K562 cells showed that the levels of CTLs in peripheral blood of 5 patients increased following vaccination, whereas those in one patient declined. Levels of CTLs assayed in the presence of K562 cells were considerably lower than those assayed in the absence of K562 cells, but were also found to increase following vaccination in the peripheral blood of 6 patients. A patient who had been vaccinated 10 times (patient 1) responded to the vaccination by apparent reduction in size of metastatic tumor in the lung. Immunohistochemical examination of the vaccination sites of patient 1, biopsied after the 3rd and 4th vaccination. showed that the vaccination sites responded with infiltration of inflammatory cells, such as T cells (CD3+, CD8+), macrophages and dendritic cells (CD83+), for a period up to about 8 days. These data suggest that repeated vaccinations with irradiated autologous GM-CSF-producing tumor cells were well tolerated by patients and led to the activation of an antitumor immune response in some patients.     

Radiation combines with immune checkpoint blockade to enhance T cell priming in a murine model of poorly immunogenic pancreatic cancer

Radiation has been a pillar of cancer therapy for decades. The effects of radiation on the anti-tumour immune response are variable across studies and have not been explicitly defined in poorly immunogenic tumour types. Here, we employed combination checkpoint blockade immunotherapy with stereotactic body radiation therapy and examined the effect on tumour growth and immune infiltrates in subcutaneous and orthotopic mouse models of pancreatic cancer. Although immune checkpoint blockade and radiation were ineffective alone, their combination produced a modest growth delay in both irradiated and non-irradiated tumours that corresponded with significant increases in CD8+ T cells, CD4+ T cells and tumour-specific T cells as identified by IFNγ ELISpot. We conclude that radiation enhances priming of tumour-specific T cells in poorly immunogenic tumours and that the frequency of these T cells can be further increased by combination with immune checkpoint blockade.     

CD34+-derived CD11c+ + + BDCA-1+ + CD123+ + DC: expansion of a phenotypically undescribed myeloid DC1 population for use in adoptive immunotherapy

BACKGROUND: DC are commonly defined as HLA-DR+/Lin- cells that can be CD11c+ + + CD123+/ -, termed DC1/myeloid DC that induce a Th1 response, or CD11c- CD123+ + +, termed DC2/lymphoid DC that induce a Th2 response. However, significant heterogeneity within DC preparations is apparent and supports the existence of several distinct DC subpopulations. This study aimed to expand and characterize CD34+ DC for use in immunotherapy. METHODS: CD34+ cells were seeded at 1 x 10(5)/mL and expanded for 14 days in RPMI + 10% autologous plasma supplemented with GM-CSF, IL-4, Flt-3L and SCF. Maturation was induced with TNF-alpha and PGE2 for 2 days. DC were analyzed morphologically, phenotypically with a panel of MAb to lineage and DC markers, and functionally in MLR, T-cell assays and T-cell cytokine secretion by ELISA. RESULTS: Significant cellular expansion was observed: 60+/-5 x 10(6) DC from 1 x 10(6) CD34+ cells (n=28). Phenotypically DC were characterized as HLA-DR+ +, CD11c+ + +, CD80+ +, CD83+, CD86+ +, CD123+ +, CD15+ +, CD33+ +, BDCA-1+ +, CD4+ and Lin-. DC displayed potent allostimulatory capacity and efficient presentation of KLH and tetanus toxin. DC-primed T cells secreted IFN-gamma (Th1); however, no detectable IL-4 (Th2) was noted. DISCUSSION: We present features of CD34+ DC that have not been previously described. The CD34+ DC generated represent a population of myeloid DC functioning as DC1 but phenotypically expressing markers characteristic of both DC1 and DC2. This novel DC population is capable of inducing naive T-cell responses and can be expanded to clinically useful numbers. CD34+-derived DC represent attractive candidates for use in adoptive T-cell immunotherapy.     

EBV-induced molecule 1 ligand chemokine (ELC/CCL19) promotes IFN-gamma-dependent antitumor responses in a lung cancer model

The antitumor efficacy of EBV-induced molecule 1 ligand CC chemokine (ELC/CCL19) was evaluated in a murine lung cancer model. The ability of ELC/CCL19 to chemoattract both dendritic cells and T lymphocytes formed the rationale for this study. Compared with diluent-treated tumor-bearing mice, intratumoral injection of recombinant ELC/CCL19 led to significant systemic reduction in tumor volumes (p < 0.01). ELC/CCL19-treated mice exhibited an increased influx of CD4 and CD8 T cell subsets as well as dendritic cells at the tumor sites. These cell infiltrates were accompanied by increases in IFN-gamma, MIG/CXCL9, IP-10/CXCL10, GM-CSF, and IL-12 but a concomitant decrease in the immunosuppressive molecules PGE(2) and TGFbeta. Transfer of T lymphocytes from ELC/CCL19 treated tumor-bearing mice conferred the antitumor therapeutic efficacy of ELC/CCL19 to naive mice. ELC/CCL19 treated tumor-bearing mice showed enhanced frequency of tumor specific T lymphocytes secreting IFN-gamma. In vivo depletion of IFN-gamma, MIG/CXCL9, or IP-10/CXCL10 significantly reduced the antitumor efficacy of ELC/CCL19. These findings provide a strong rationale for further evaluation of ELC/CCL19 in tumor immunity and its use in cancer immunotherapy.     

Big tumor regression induced by GM-CSF gene-modified 3LL tumor cells via facilitating DC maturation and deviation toward CD11c+CD8alpha+ subset

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a powerful immune-stimulating factor that helps to generate a systemic, strong, and long-lasting immune response. However, whether the transduction of GM-CSF to tumor cell results in tumor regression and optimizes local immune microenvironment remains to be investigated. In this study, using an experimental murine tumor model, we demonstrated that the in vivo growth of 3LL tumor cells modified with the GM-CSF gene (3LL-GM) was inhibited even when the tumor diameter was over 7 mm (big tumor), and mice inoculated with GM-CSF gene-modified 3LL cells survived over 90 days, whereas mice inoculated with control parental 3LL cells and 3LL cells transduced with control vector all succumbed to the tumor by day 17 after tumor inoculation. Further analysis showed that targeted expression of GM-CSF in 3LL tumor cells markedly enhanced the systemic antitumor effect, including specific lymphocytes proliferation, cytotoxicity against 3LL tumor, and increased production of IFN-gamma. GM-CSF gene-modified 3LL cells significantly protected the mice from the parental 3LL tumor challenge. More importantly, the percentage of dendritic cells (DCs) in tumor site was greatly increased and the DCs differentiated into CD11c(+)CD8alpha(+) cells, which were reported to be able to benefit the induction of CD8(+) cytotoxic T lymphocytes (CTLs) that contribute to tumor regression. Our research indicated that GM-CSF could optimize the immune microenvironment in the tumor site, which provides a potent approach for immunotherapy of tumors.     

DC-CIK治疗对晚期肝细胞癌患者免疫功能及循环肿瘤细胞的影响

目的:研究DC-CIK细胞治疗对晚期肝细胞癌患者甲胎蛋白、免疫功能及循环肿瘤细胞数的影响。方法:选取2013年至2015年我院收治的26例肝细胞癌并伴有复发或转移的患者,对其治疗前后甲胎蛋白、免疫功能及循环肿瘤细胞数的数据进行分析,比较DC-CIK细胞治疗前后甲胎蛋白、循环肿瘤细胞、免疫功能的变化。根据细胞治疗次数分为1次组及1次组,比较两组免疫功能的变化。结果:DC-CIK细胞治疗前甲胎蛋白为(603.32±155.78)ng/mL细胞回输后为(571.24±147.49)ng/mL差异无统计学意义(P0.05)。DC-CIK细胞治疗前及细胞回输后CTC检测阳性率分别为81.8%、36.4%治疗前及回输后循环肿瘤细胞数量分别为(8.36±10.642)、(1.55±2.464),细胞治疗前后比较差异均有统计学意义(P0.05)。细胞治疗前1天T细胞亚群CD3+、CD3~+CD4~+、CD3~+CD8~+、CD4/CD8分别为(66.05±15.31)%、(41.89±12.33)%、(23.15±8.05)%、(2.10±0.77),回输后分别为(69.69±12.91)%、(44.80±11.11)%、(23.13±7.12)%、(2.29±0.91)治疗前后比较差异无统计学意义(P0.05)。细胞治疗次数1次组和1次组免疫功能变化差异无统计学意义(P0.05)。结论:DC-CIK细胞治疗可减少肝细胞癌伴复发或转移患者循环肿瘤细胞的数量,但对甲胎蛋白和免疫功能无明显影响。     

Basic fibroblast growth factor (B-FGF) induces early- (CFU-s) and late-stage hematopoietic progenitor cell colony formation (CFU-gm, CFU-meg, and BFU-e) by synergizing with GM-CSF, Meg-CSF, and erythropoietin, and is a radioprotective agent in vitro

Basic fibroblastic growth factor (B-FGF) is a hormone-like protein which belongs to a class of heparin-binding growth factors. B-FGF is synthesized and released to circulate in the blood where it can be recognized by target cells through specific high-affinity plasma membrane receptors. B-FGF is known to be a potent mitogen for a number of specific cell types. We report data which demonstrates B-FGF can influence noncommited and specific lineage-derived hematopoietic progenitors when incubated in vitro. When combined with adherent cell-depleted normal murine marrow cells, B-FGF increased the number of both day 9 and day 12 spleen colony-forming units (CFU-s) from lethally irradiated animals. However, day 12-derived CFU-s were more sensitive to B-FGF, since optimal CFU-s production was observed at 10 ng/ml vs. 100 ng/ml for day 9 CFU-s (p less than 0.05). In adherent cell-depleted murine and human marrow cultures, the addition of B-FGF possessed synergistic activity in combination with the optimal concentration of GM-CSF for CFU-gm at a dose of 10 ng/ml which was inhibited in the presence of protamine sulfate (LD50 dose, 100 mu gm/ml), an inhibitor of B-FGF mitogenic activity, or in the presence of heparin (LD50 dose, 100 U/ml), an effective B-FGF binding agent. B-FGF also expressed synergistic activity in the presence of optimal concentrations of erythropoietin and Meg-CSF for murine and human BFU-e, and murine CFU-meg. No in vitro colony formation was observed when cells were cultured in the presence of B-FGF, but in the absence of the specific hematopoietic growth factor. Finally, B-FGF was also shown to be an effective radioprotective agent in vitro. Murine and human CFU-gm exposed to increasing doses of radiation (0.5 to 5 Gy) combined with GM-CSF and increasing doses of B-FGF (0.1 to 100 ng/ml) produced less radiation-induced toxicity compared to cultures containing GM-CSF alone. This data demonstrates B-FGF influences early- and late-stage hematopoietic progenitors, possesses synergistic activity with hematopoietic growth factors, and is a radioprotective agent in vitro. These results suggest B-FGF must be considered as a member of the family of molecules capable of influencing hematopoiesis in vitro.     

Mononuclear cell subpopulations and infiltrating lymphocytes in erythema dyschromicum perstans and vitiligo

Erythema dyschromicum perstans (EDP) and vitiligo are two cutaneous pigmentary dermatoses of unknown etiology. In the present study, the leukocyte infiltrates in the affected skin of EDP and vitiligo patients were studied using the avidin-biotin (ABC) immunoperoxidase technique and monoclonal antibodies which recognise the following mononuclear cell subgroups: T-suppressor/cytotoxic (CD8-Leu-2), T-helper (CD4 = OKT4), T-suppressor + macrophages (Leu-15), Pan T (CD3 = Leu-4), macrophages (Leu-M3) and Langerhans cells (CD1 = Leu-6), and other cellular markers such as Ia antigens and the Interleukin-2 receptor (CD25 = TAC). The immunocytochemical analysis showed a selective accumulation of CD3+, CD8+, Leu-15-, T-cytotoxic cells in the epidermis of both EDP and early lesions of vitiligo. In addition, an increase in the number of epidermal Langerhans cells (CD1+) was observed in some cases of EDP and vitiligo. The CD4/CD8 ratios in affected and uninvolved skin for both disorders were not significantly different, although values lower than unity were only observed in the infiltrates of affected skin. Ia antigen positivity was observed in the dendritic cells of the dermis and epidermis, as well as in most of the lymphoid cells within the infiltrates for both diseases. Macrophages (Leu-M3) in EDP dermal infiltrates were generally found adjacent to extracellular melanin pigment. Lymphocytes expressing TAC (CD25) surface antigens were also present in the dermal infiltrates. These morphological observations suggest a possible immune cell participation in the dyschromia of such cutaneous disorders.     

Combined blockade of TIM-3 and TIM-4 augments cancer vaccine efficacy against established melanomas

Cancer vaccines have been developed to instruct the endogenous immune responses to autologous tumors and to generate durable clinical responses. However, the therapeutic benefits of cancer vaccines remain insufficient due to the multiple immunosuppressive signals delivered by tumors. Thus, to improve the clinical efficacy of cancer immunotherapy, it is important to develop new modalities to overcome immunosuppressive tumor microenvironments and elicit effective antitumor immune responses. In this study, we show that novel monoclonal antibodies (mAbs) specifically targeting either T cell immunoglobulin mucin protein-3 (TIM-3) or T cell immunoglobulin mucin protein-4 (TIM-4) enhance the therapeutic effects of vaccination against established B16 murine melanomas. This is true for vaccination with irradiated B16 melanoma cells engineered to express the flt3 ligand gene (FVAX). More importantly, combining anti-TIM-3 and anti-TIM-4 mAbs markedly increased vaccine-induced antitumor responses against established B16 melanoma. TIM-3 blockade mainly stimulated antitumor effector activities via natural killer cell-dependent mechanisms, while CD8⁺ T cells served as the main effectors induced by anti-TIM-4 mAb. Our findings reveal that therapeutic manipulation of TIM-3 and TIM-4 may provide a novel strategy for improving the clinical efficacy of cancer immunotherapy.     

In vitro propagated dendritic cells from patients with human-papilloma virus-associated preneoplastic lesions of the uterine cervix: use of Flt3 ligand

Dendritic cells (DC) are the most efficient antigen presenting cells. The clinical use of DC as vectors for antitumor and anti-infectious disease immunotherapy has been limited by their low level and accessibility in normal tissue. Substantial numbers of DC can be generated from peripheral blood cultured in the presence of interleukin-4 (IL-4) and granulocyte/macrophage-colony-stimulating factor (GM-CSF). We showed in this study that substantial numbers of DC can be obtained from the peripheral blood of patients with (pre)neoplastic lesions of the uterine cervix. The procedure required relatively small blood samples (10 ml) and the presence of 100 U/ml IL-4 and 800 U/ml GM-CSF in the culture medium. There was no significant difference in the morphology, yield, phenotype and function of generated DC between patients with cervical (pre)neoplastic lesions and healthy individuals. When the hematopoietic factor Flt3 ligand (Flt3L, 40 ng/ml) was added, there was an average increase in the DC population of 26% compared to cultures with GM-CSF and IL-4 alone. Approximately 1.2 × 10⁶ cells with the characteristics of dendritic cells could be obtained when Flt3L was included in the medium. The addition of Flt3L did not modify the phenotypic profile of DC (HLA-DR⁺, CD1a⁺, CD4⁺, CD54⁺, CD80⁺, CD86⁺, CD40⁺, CD3⁻ and CD14⁻). In addition, Flt3L generated functional DC capable of stimulating the proliferation of alloreactive T cells. These results suggest that Flt3L, in association with GM-CSF and IL-4, provides an advantageous tool for the large-scale generation of DC and that an immunotherapy based on the use of DC generated in vitro is possible in patients with (pre)neoplastic lesions of the uterine cervix. Received: 8 January 1998 / Accepted: 30 April 1998     

Immune restoration in head and neck cancer patients after in vivo COX-2 inhibition

Purpose To determine the immunomodulatory effects of in vivo COX-2 inhibition on leukocyte infiltration and function in patients with head and neck cancer. Experimental design Patients with squamous cell carcinoma of the head and neck preoperatively received a specific COX-2 inhibitor (rofecoxib, 25 mg daily) orally for 3 weeks. Serum and tumor specimens were collected at the start of COX-2 inhibition (day 0) and again on the day of surgery (day 21). Adhesion to peripheral blood monocytes to ICAM-1 was examined. Percentages of tumor-infiltrating monocytes (CD68, CCR5) and lymphocytes (CCR5, CD4, CD8 and CD25) were determined by immunohistochemistry. Results Monocytes obtained from untreated cancer patients showed lower binding to ICAM-1 compared to monocytes of healthy donors but significantly regained adhesion affinity following incubation in sera of healthy donors. Conversely, sera of cancer patients inhibited adhesion of healthy donors’ monocytes. Tumor monocyte adhesion to ICAM-1 was increased (P < 0.001) after 21 days of COX-2 inhibition, and concomitant increases in tumor infiltrating monocytes (CD68+), lymphocytes (CD68− CCR5+, CD4+ and CD8+) and activated (CD25+) T cells were observed. Conclusions Short-term administration of a COX2 inhibitor restored monocyte binding to ICAM-1 and increased infiltration into the tumor of monocytes and Th1 and CD25+ activated lymphocytes. Thus, in vivo inhibition of the COX-2 pathway may be useful in potentiating specific active immunotherapy of cancer.     

The generation of anti-tumoral cells using dentritic cells from the peripheral bloood of patients with malignant brain tumors

 Dendritic cells (DCs) can be the principal initiators of antigen-specific immune responses. We analyzed the in vitro-responses against brain tumor cells using DCs from the peripheral blood of patients with brain tumors. Peripheral blood mononuclear cells (PBMC) were obtained from 19 patients with malignant brain tumors: 12 metastatic brain tumors of lung adenocarcinoma, 7 high-grade astrocytomas. PBMC were cultured with 100 ng/ml of GM-CSF and 10 ng/ml of IL-4 for 5–7 days in order to produce mature DCs. The autologous tumor lysate (5 mg/ml, containing 1 × 10⁶ cells) was then added to the cultured DCs. Using the DCs generated by these treatments, we assessed the changes that occurred in their immune responses against brain tumor via ⁵¹Cr-release and lymphocyte proliferation assays. We found that the matured DCs displayed the typical surface phenotype of CD3⁺, CD45⁺, CD80⁺ and CD86⁺. After the pulsation treatment with tumor lysate, DCs were found to have strong cytotoxic T lymphocyte activity, showing 42.5 ± 12.7% killing of autologous tumor cells. We also found an enhancement of allogeneic T cell proliferation after pulsing the DC with tumor lysate. These data support the efficacy of DC-based immunotherapy for patients with malignant brain tumors. Received: 2 October 2000 / Accepted: 26 April 2001     

Targeting melanoma inhibitor of apoptosis protein with cancer immunotherapy

Aberrantly expressed or mutated proteins in cancer cells evoke immune recognition, but host reactions are usually insufficient to prevent disease progression. Vaccination with irradiated tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) augments host immunity through improved tumor antigen presentation by recruited dendritic cells and macrophages. By analyzing the immune response of a metastatic melanoma patient who achieved a long-term response to vaccination, we identified melanoma inhibitor of apoptosis protein (ML-IAP) as a target for immune-mediated tumor destruction. Vaccination stimulated a coordinated cellular and humoral reaction to ML-IAP that was associated with extensive tumor necrosis, whereas lethal disease progression was linked with the loss of ML-IAP expression and the absence of intra-tumoral lymphocyte infiltrates. These findings demonstrate that ML-IAP can serve as a tumor rejection antigen, although additional vaccine targets will be required to circumvent immune escape and tumor heterogeneity.     

DCs-CIK细胞免疫治疗联合化疗治疗晚期非小细胞肺癌的疗效观察

目的:探讨树突状细胞(DCs)和细胞因子诱导的杀伤(CIK)细胞免疫治疗联合化疗对晚期非小细胞肺癌患者的治疗效果。方法:将我院2012年2月到2014年2月就诊的72例晚期非小细胞肺癌患者随机分为对照组(n=36,单纯化疗组)和实验组(n=36,DCs-CIK细胞免疫联合化疗组)。比较两组患者治疗后的疗效、治疗前后免疫功能,并运用Kamofsky(KPS)评分来评估两组患者治疗后生活质量的改善情况。结果:实验组的疾病控制率(DCR)77.78%显著高于对照组的52.78%(P0.05)。治疗后实验组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前均上升显著(P0.05);治疗后对照组患者外周血CD3+、CD8+及NK细胞所占的比值较治疗前下降显著(P0.05)。治疗后实验组KPS评分提高率明显高于对照组(P0.05)。结论:DCs-CIK细胞免疫联合化疗能够提高晚期非小细胞肺癌患者的DCR,且显著改善患者的免疫功能和生活质量。     

CD25+ regulatory T cell depletion augments immunotherapy of micrometastases by an IL-21-secreting cellular vaccine

IL-21 is an IL-2-like cytokine, signaling through a specific IL-21R and the IL-2R gamma-chain. Because the TS/A mammary adenocarcinoma cells genetically modified to secrete IL-21 (TS/A-IL-21) are strongly immunogenic in syngeneic mice, we analyzed their application as vaccine. In mice bearing TS/A-parental cell (pc) micrometastases, vaccination with irradiated TS/A-IL-21 cells significantly increased the animal life span, but cured only 17% of mice. Spleen cells from cured mice developed CTL activity and produced IFN-gamma in response to stimulation by the AH1 epitope of the gp70env Ag of TS/A-pc. We tested whether the low therapeutic outcome might be due to CD4+CD25+ regulatory T cells (Treg) present in TS/A-pc tumors and draining lymph nodes and whether IL-21 had any effect on these cells. Indeed, CD4+CD25+ cells suppressed IFN-gamma production by splenocytes from immune mice in response to stimulation by the AH1 peptide. Low concentrations of IL-21 (10 ng/ml) failed to reverse the inhibitory activity of CD4+CD25+ cells in an allogeneic MLR, whereas 60 ng/ml rIL-21 partially restored responder T cell proliferation. IL-21R expression on CD25- lymphocytes suggested that IL-21 could be more effective in mice depleted of CD25+ cells. Depletion of Treg cells by a single dose of anti-CD25 mAb combined with TS/A-IL-21 cell vaccine cured >70% of mice bearing micrometastases, whereas anti-CD25 mAb treatment alone had no effect. Successful combined immunotherapy required NK cells, CD8+ T cells, and IFN-gamma. In conclusion, immunotherapy of micrometastases by an IL-21-based cellular vaccine is strongly potentiated by CD25+ cell depletion.     

Nonviral transfection of human umbilical cord blood dendritic cells is feasible, but the yield of dendritic cells with transgene expression limits the application of this method in cancer immunotherapy

Dendritic cells (DC) generated from human umbilical cord blood might replace patients' DC in attempts to elicit tumor-specific immune response in cancer patients. We studied the efficiency of transfection of human cord blood DC with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene, to test if nonviral gene transfer would be a method to load DC with protein antigens for immunotherapy purposes. Cord blood mononuclear cells were cultured in serum-free medium in the presence of granulocyte-monocyte colony stimulating factor (GM-CSF), stem cell factor (SCF) and Flt-3 ligand (FL), to generate DC from their precursors, and thereafter transfected by electroporation. Maturation of DC was induced by stimulation with GM-CSF, SCF, FL and phorbol myristate acetate (PMA). Transfected DC strongly expressed EGFP, but transfection efficiency of DC, defined as HLA-DR(+) cells lacking lineage-specific markers, did not exceed 2.5%. Expression of the reporter gene was also demonstrated in the DC generated from transfected, purified CD34(+) cord blood cells, by stimulation with GM-CSF, SCF, FL, and tumor necrosis factor alpha (TNF-alpha). Transfection of CD34(+) cells was very efficient, but proliferation of the transfected cells was much reduced as compared to the untransfected cells. Therefore, the yield of transgene-expressing DC was relatively low. In conclusion, nonviral transfection of cord blood DC proved feasible, but considering the requirements for immunotherapy in cancer patients, transfection of differentiated DC or generation of DC from transfected hematopoietic stem cells provide only a limited number of DC expressing the transgene.     

Granulocyte-macrophage colony-stimulating factor increases the proportion of circulating dendritic cells after autologous but not after allogeneic hematopoietic stem cell transplantation

Background aimsGranulocyte–macrophage (GM) colony-stimulating factor (CSF) has been used as an adjuvant in cancer immunotherapy. We tested the hypothesis that GM-CSF (Leukine^®; sargramostim) improves immune reconstitution after hematopoietic stem cell transplantation (HSCT) based on our prior in vitro work that demonstrated the pro-inflammatory effects of GM-CSF on dendritic cells (DC).MethodsGM-CSF was administered to donors, along with standard granulocyte (G) CSF, during stem cell mobilization, and to recipients from the day prior to transplant until engraftment. Eighteen patients consented to the GM-CSF⁺ protocol and were compared with 17 matched controls undergoing HSCT during the same time period (GM-CSF^?).ResultsNumbers of white blood cells (WBC) and CD34⁺ stem cells in the graft were comparable to controls. Surprisingly, contrary to our hypothesis, the allogeneic donor graft had significantly decreased numbers of CD3⁺ T cells and their subsets (CD4⁺, CD4⁺ CD45RA⁺, CD4⁺ CD45RO⁺, CD8⁺ and CD8⁺ CD45RO⁺), DC (both myeloid and plasmacytoid) and natural killer (NK) cells (CD16⁺ CD56⁺). In the GM-CSF arm, following allogeneic transplantation, the levels of DC, T cells and NK cells did not increase with treatment. Conversely, autologous transplant patients receiving GM-CSF had a higher proportion of DC at the time of engraftment.ConclusionsThese findings demonstrate that administration of GM-CSF improves DC reconstitution after autologous rather than allogeneic HSCT.     

Phenotype of peripheral blood leukocytes and survival of patients with metastatic colorectal cancer

Immune dysfunction is prevalent in metastatic cancer. Few patients with colorectal cancer metastases are cured, and among the strategies aimed at improving the therapeutic results in patients with metastatic colorectal cancer, immunotherapy is being increasingly investigated. We evaluated retrospectively the prognostic significance of peripheral blood leukocytes in 59 patients with metastatic colorectal cancer. The relative numbers of CD3+, CD3+CD4+, CD3+CD8+, NK (CD3-CD16+CD56+), CD3+DR+, CD3+CD25+, CD3+CD69+, CD19+, CD19+CD23+, CD8+CD28+, CD8-CD28+, CD8+CD57+, CD14+DR+ and CD14+CD16+ leukocytes were analyzed by two-color flow cytometry. A three-step approach was adopted to identify predictors of prognosis using regression analysis. Based on the results of univariate survival analysis, the absolute number of white blood cells, NK/CD3+CD69+ and NK/white cell count ratios were significant indicators of prognosis. In the multivariate regression analysis a model was obtained using a single parameter, the NK/CD3+CD69+ ratio, predicting the survival with 10-15% power of regression. The present results indicate that the NK/CD3+CD69+ ratio in peripheral blood may be an independent variable in a regression model predicting the overall survival of patients with colorectal cancer metastases to be tested in prospective studies.