Peritumoral CpG oligodeoxynucleotide treatment inhibits tumor growth and metastasis of B16F10 melanoma cells

Although melanoma mostly affects the skin, it is notorious for its propensity to easily develop metastasis. Metastatic melanoma is highly resistant to a variety of therapies. We examined the anti-metastatic potential of peritumoral monotherapy against murine cutaneous B16F10 melanoma with synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. We demonstrated that repeated peritumoral injections of CpG ODN significantly reduced skin tumor size. Peritumoral CpG ODN-treatment of skin tumors prevented the development of pulmonary B16F10 colonies. Adoptive transfer of splenocytes obtained from CpG ODN-treated mice markedly reduced the number of previously established pulmonary colonies in recipient na?ve mice. T-lymphocyte depletion studies indicated that the anti-metastatic effect was dependent on both CD4+ and CD8+ T cells. These results suggest that CpG ODN are promising as a preventive and therapeutic anti-metastatic measure against melanoma.     

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.     

Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model

Mesenchymal stem cells (MSCs) represent a new tool for delivery of therapeutic agents to tumor cells. In this study, we have evaluated the anti-tumor activity of human MSCs stably transduced with a retroviral vector expressing the cytokine interleukin-12 (IL-12) in a mouse melanoma model. Application of MSC(IL-12) but not control MSCs strongly reduced the formation of lung metastases of B16F10 melanoma cells. The activity of the MSC(IL-12) cells was dependent on the presence of natural killer (NK) cells in this experimental setting. Further, MSC(IL-12) cells elicited a pronounced retardation of tumor growth and led to prolonged survival when injected into established subcutaneous melanoma in a therapeutic regimen. The therapeutic effect of the MSC(IL-12) was in part mediated by CD8(+) T cells, while NK cells and CD4(+) T cells appeared to play a minor role. The anti-tumor effect of MSC(IL-12) cells was of similar efficiency as observed for application of naked plasmid DNA encoding IL-12. The presented data demonstrate that these two different strategies can induce a similar therapeutic anti-tumor efficacy in the mouse melanoma tumor model.     

Production and characterization of tumor infiltrating lymphocyte clones derived from B16-F10 murine melanoma

The adoptive transfer of tumor infiltrating lymphocytes (TIL) in conjunction with recombinant interleukin-2 (rIL-2) for the treatment of advanced cancer has recently been under intense investigation. Despite extensive research, the precise surface phenotype of TIL remains to be fully defined. To elucidate this unsolved problem, we established 11 TIL clones derived from rIL-2 expanded TIL obtained from B16-F10 murine melanoma tumors. These clones could be divided phenotypically into four groups: CD8 (+) T-cell clones, natural killer (NK)-cell clones, NK-like CD8 (+) T-cell clones, and double negative T-cell clones. Functionally, CD8 (+) T-cell clones demonstrated specific cytotoxic activity against B16-F10 melanoma cells, whereas NK-cell clones and double negative T-cell clones demonstrated only non-specific cytotoxic activity against NK-sensitive YAC-1 cells. NK-like CD8 (+) T-cell clones showed dual cytotoxic activity. Clones T1 [a CD8 (+) T-cell clone] and T2 [an NK-like CD8 (+) T-cell clone] which had cytotoxic activity against B16-F10 melanoma cells, demonstrated a proliferative response against immunoblotted B16-F10 melanoma antigens, whereas clones T7 (an NK-cell clone) and T10 (a double negative T-cell clone), which had no cytotoxic activity against B16-F10 cells, demonstrated no proliferative response against them. Winn assays revealed that only the CD8 (+) T-cell clone (T1) had an antitumor effect in vivo, whereas the double negative T-cell clone (T10) and NK-like CD8 (+) T-cell clone (T2) stimulated tumor growth in vivo. Adoptive immunotherapy using tumor-specific, highly cytotoxic TIL clones may represent a useful future immunotherapeutic option for the treatment of human tumors.     

T-钙黏蛋白对小鼠皮下氮烯咪胺耐药黑素瘤B16F10细胞株的影响

目的观察T-钙黏蛋白(cadherin)对小鼠皮下氮烯咪胺(dacarbazine,DTIC)耐药黑素瘤B16F10细胞株的影响。方法通过体外分步诱导法诱导B16F10氮烯咪胺耐药细胞株(DTIC-R B16F10)。采用CCK-8法检测DTIC-R B16F10细胞的增殖情况。将T-cadherin基因转染至DTIC-R B16F10细胞。免疫组织化学检测转染后T-cadherin的表达情况。培养DTIC-R B16F10细胞,分为6组:对照组、pEGFP-N1组、T-cadherin组、DTIC组、pEGFP-N1联合DTIC组和T-cadherin联合DTIC组。通过CCK-8法和Transwell小室实验检测T-cadherin与氮烯咪胺联合对DTIC-R B16F10细胞活性和迁移的影响。建立荷瘤小鼠模型,观察T-cadherin对小鼠皮下注射DTIC-R B16F10细胞后肿瘤生长的影响。采用析因分析法评价T-cadherin联合氮烯咪胺对细胞活性、迁移和小鼠皮下瘤生长的影响。结果DTIC-R B16F10组、B16F10组与DTIC-R B16F10+DTIC组A值差异无统计学意义(P>0.05)。免疫组织化学检测表明细胞表达T-cadherin。T-cadherin联合DTIC组细胞活性、迁移细胞数及肿瘤重量均显著低于其他组(P<0.05)。析因分析显示,T-cadherin联合氮烯咪胺对DTIC-R B16F10的细胞活性、迁移和小鼠皮下瘤生长的抑制有协同作用。结论T-cadherin可抑制黑素瘤小鼠皮下瘤的生长,增加黑素瘤对氮烯咪胺的敏感性。     

CpG immunostimulatory sequences enhance contact hypersensitivity responses in mice

Bacterial DNA and synthetic cytidine-phosphate-guanosine-oligodeoxynucleotides (CpG ODN) potently activate dendritic cells (DC) and therefore have been proposed as adjuvants for vaccination strategies. Although CpG ODN are considered as safe adjuvants this study shows that CpG ODN are responsible for enhanced antigen-specific skin inflammatory reactions. We used the murine model of contact hypersensitivity (CHS) to 2,4-dinitrofluorobenzene (DNFB) in which hapten-specific CD8+T cytotoxic 1 cells are effector cells. Subcutaneous injection of CpG ODN, 1 d before sensitization enhanced the CHS response to DNFB and resulted in increased recruitment of CD8+ T cells at the challenge sites, whereas control ODN injection did not have any effect. This effect was local and not systemic as it was only observed when DNFB was applied at the same site as the CpG motifs. CpG ODN-induced enhancement of CHS was due to increased antigen-presenting cell functions of DC since: (i) CpG ODN-injected skin revealed upregulated expression of major histocompatibility complex class II, CD80, and CD86 molecules and (ii) CpG ODN treatment of DNFB-derivatized DC enhanced the intensity of CHS responses after in vivo transfer. Taken together, the results show that CpG ODN may be responsible for immune side-effects such as worsening of T cell-mediated skin diseases.     

CDK2基因沉默联合达卡巴嗪对B16-F1黑素瘤的生长抑制作用

目的 探讨CDK2基因沉默后对达卡巴嗪(DTIC)治疗B16-F1黑素瘤抑瘤效应的影响.方法 实验设对照组、CDK2-shRNA组、DTIC组、CDK2-shRNA+ DTIC组,MTT法检测各组细胞生长抑制情况,计算药物相互作用指数(CDI值),AnnexinV-FITC/PI双染法检测细胞凋亡情况.建立C57 BL/6小鼠B16-F1细胞移植瘤模型,分组实验,持续观察18d,绘制肿瘤生长曲线,计算肿瘤生长抑制率,TUNEL检测肿瘤组织细胞凋亡情况.结果 与对照组相比,CDK2-shRNA组、DTIC组、CDK2-shRNA+ DTIC组在72h的细胞相对存活率分别为(40.6±2.8)％、(45.2±3.7)％、(28.7±2.1)％,细胞凋亡率分别为(25.1±3.3)％、(15.6±2.2)％和(45.6±3.5)％,细胞相对存活率显著降低(F=458.04,P＜ 0.05)而细胞凋亡率显著升高(F=115.46,P＜0.05),其中CDK2-shRNA+ DTIC组比DTIC组的细胞相对存活率显著降低(P＜0.01),而细胞凋亡率显著升高(P＜0.01);两药相互作用指数(CDI值)＜0.7.治疗第6天,对照组、CDK2-shRNA组、DTIC组及CDK2-shRNA+ DTIC组的肿瘤体积分别为(185.44±68.97) mm3、(83.91±14.33)mm3、(123.70±20.85) mm3、(34.54±10.72) mm3.从治疗的第6天开始,与对照组相比,CDK2-shRNA组、DTIC组及CDK2-shRNA+ DTIC组的肿瘤生长速度明显降低(F=11.819,P＜0.05),而CDK2-shRNA+ DTIC组的肿瘤生长速度明显低于DTIC组(P=0.04);与对照组相比,CDK2-shRNA组、DTIC组、CDK2-shRNA+ DTIC组的肿瘤生长抑制率分别为52.2％、41.2％、86.4％,组织细胞凋亡指数分别为(32.93±3.72)％、(21.62±3.54)％、(63.29±4.74)％,组织细胞凋亡指数显著升高(F=222.25,P＜0.05),其中CDK2-shRNA+ DTIC组的组织细胞凋亡指数显著高于DTIC组(P＜0.01).结论 沉默CDK2基因的表达可以增加DTIC对黑素瘤的生长抑制作用,二者体外有协同效应,通过增加肿瘤细胞的凋亡是其机制之一.     

Comparison of a treatment strategy combining CCI-779 plus DTIC versus DTIC monotreatment in human melanoma in SCID mice

This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.     

Tumor regression of human and murine melanoma after intratumoral injection of IL-12-encoding plasmid DNA in mice

DNA coding for murine interleukin 12 (IL-12) prevents the formation of B16-melanoma metastasis when administered intramuscularly. Here, the antitumor effect of IL-12-encoding DNA on established mouse B16 melanoma and human melanoma tumors was investigated in vivo using two animal models: B16 melanoma in C57B/6 mice and human melanoma in nude mice. In B16 melanoma, intratumoral injections of IL-12-encoding DNA resulted in highly significant growth retardation when compared with mice injected with control vector. In the case of the human melanoma model, treatment with DNA coding for IL-12 induced regression of tumors in all cases, with complete disappearance of the tumor in two out of five animals. DNA treatment did not induce systemic side-effects. In the animals injected with control vector the human melanoma tumors grew expansively. The therapeutic effect of the DNA injection was mediated in part by natural killer (NK) cells as shown by NK-depletion experiments. An antivascular effect of IL-12 treatment was evident in histological examination with endothelial thickening and abrupt changes in vessel diameters. These results suggest that intratumoral plasmid DNA coding for IL-12 holds some promise as a new therapeutic tool for accessible melanoma lesions and should be tested in clinical trial.     

Regulatory T cells stimulate B7-H1 expression in myeloid-derived suppressor cells in ret melanomas

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells, and they promote an immunosuppressive environment in tumor-bearing hosts. To characterize MDSCs in melanoma, we examined the expression of inhibitory B7 molecules by CD11b(+)Gr1(+) cells isolated from mice with transplantable ret tumors. B7 molecules were expressed on CD11b(+)Gr1(+) cells, which also expressed CD124 and inducible nitric oxide synthase, thus verifying their relation to MDSCs. In developing melanomas, CD11b(+)Gr1(+) cells express only low levels of B7-H1. In contrast, B7-H1 is upregulated in large tumors, and functional analysis demonstrates that CD11b(+)Gr-1(+) cells suppress the proliferation of CD4(+) T cells through B7-H1. Depletion of regulatory T cells (Tregs) significantly downregulated the expression of B7-H1, B7-H3, and B7-H4 on MDSCs and reduced tumor growth, indicating a concerted immunosuppressive activity of Tregs and MDSCs. No differences in the suppressive function of MDSCs between CD25-depleted and non-depleted mice were recorded. Instead, tumor-derived MDSCs from Treg-depleted hosts produced less IL-10 and more IFN-γ as compared with Treg-harboring mice. These studies indicate that Tregs in tumors not only suppress effector T cells directly, but also modify the phenotype of tumor-infiltrating CD11b(+) cells to express inhibitory B7-H molecules and to produce IL-10.     

NKG2D ligands expression and NKG2D-mediated NK activity in Sezary patients

Sezary syndrome (SS) is a rare lymphoma characterized by the clonal expansion in the skin and in blood of CD4(+)CD158k(+) T cells. Natural killer (NK) activation against tumors in leukemia models is partly based on the recognition of the target through the NKG2D/NKG2D ligands interactions. We analyzed ex vivo SS malignant lymphocytes for the expression of the NKG2D ligands such as the major histocompatibility complex class I-related molecules (MIC) A and B and the UL16 binding proteins (ULBP). The expressions of NKG2D, the natural cytotoxicity receptors (NKp30, NKp44, and NKp46) and the activating receptor DNAM-1 were simultaneously investigated on circulating patients NK and CD8(+) nonmalignant lymphocytes. Interestingly, although at least one of the NKG2D ligands was expressed on the circulating malignant lymphocytes of 9 out of 10 patients, NKG2D was expressed on effector lymphocytes. We found that soluble MICA in patient's sera was increased, which may constitute a mechanism to escape the immune response. In vitro, SS tumor lymphocytes induced the degranulation of perforin by the NKL cell line. More importantly, NKG2D expressed on SS patients NK cells is functional and capable to induce degranulation. Altogether, these data could suggest that stimulating NK function in SS patients may be a promising strategy to reduce tumor invasion.     

Cytotoxic antimelanoma drugs suppress the activation of M2 macrophages

Together with regulatory T cells (Tregs), tumor‐associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Although cytotoxic antimelanoma drugs such as dacarbazine (DTIC), nimustine hydrochloride (ACNU) and vincristine (VCR) have been used for the treatment of malignant melanoma as adjuvant therapy in Japan, the detailed mechanisms of their immunomodulatory effects are not fully understood. As the majority of TAMs are alternatively activated M2 macrophages that favour tumor development, the aim of this study was to elucidate the immunomodulatory effects of these reagents on human monocyte‐derived M2 macrophages. First, mRNA expressions and protein production of immune checkpoint molecules, PD‐L1 and chemokines by CD163⁺ CD206⁺ M2 macrophages derived from peripheral blood mononuclear cells were investigated to determine the immunomodulatory effects of DTIC, ACNU, and VCR. DTIC and VCR significantly decreased PD‐L1 mRNA expression, which was confirmed by flow cytometry. Moreover, the mRNA expression and production of CCL22 were significantly decreased by DTIC, which suggested that DTIC might suppress the recruitment of Tregs in the tumor site. Furthermore, the decreased expression of PD‐L1 and production of CCL22 were validated in vivo, using the B16F10 mouse melanoma model, leading to abrogation of the suppressive function of T‐cell proliferation. The present report suggests one of the possible antimelanoma mechanisms of DAV combination chemotherapy for melanoma patients.     

Interleukin-18 and the costimulatory molecule B7-1 have a synergistic anti-tumor effect on murine melanoma; implication of combined immunotherapy for poorly immunogenic malignancy

Interleukin-18 has been described recently as a cytokine secreted primarily by Kupffer cells. Furthermore, it has been shown that it has significant anti-tumor effects, which are mediated by T cells and natural killer cells, in a manner similar to interleukin-12. Here, we report the evaluation of the effects of the systemic administration of interleukin-18 in combination with B7-1 (CD80) expressed on tumor cells [interleukin-18 + B7-1] on the growth of murine B16 melanoma in vivo. After the subcutaneous inoculation of B16 melanoma, B16 tumors grew progressively in immunocompetent syngeneic C57BL/6 mice. Mice treated with either interleukin-18 or immunized with B7-1-transduced B16 did not demonstrate significant anti-tumor effect. The combination of the two treatments, however, resulted in dramatic suppression of melanoma formation, tumor growth, and a significant improvement in survival. Inhibitory effects of [interleukin-18 + B7-1] on lung metastasis in mice were also detected. Additionally, mice treated with [interleukin-18 + B7-1] showed an increase of natural killer cytotoxicity and interferon-gamma production in vivo. Unlike [interleukin-18 + B7-1], [interleukin-12 + B7-1] did not have a strong anti-tumor effect against B16 melanoma. Histologic characterization after the [interleukin-18 + B7-1] treatment confirmed the infiltration of natural killer cells into the tumor, suggesting that natural killer cells may be involved in the [interleukin-18 + B7-1]-induced anti-tumor effect. This finding was confirmed by showing that depletion of NK1.1+ cells before immunization inhibits the [interleukin-18 + B7-1]-induced anti-tumor effect. Depletion of CD3+ cells in vivo also decreased the anti-tumor effect of [interleukin-18 + B7-1], suggesting the importance of CD3+ T cells. Collectively, combination with interleukin-18 and B7-1 expression has synergistic anti-tumor effects against B16 murine melanoma.     

The vast majority of lymphocytes infiltrating primary cutaneous melanoma express the CD27 costimulatory receptor: implications for melanoma progression

Melanoma progression is favoured by prevalence, within the micro-environment of primary cutaneous melanoma, of suppressive forces, e.g. exerted by CD4(+) CD25(+) FOXP3(+) regulatory T lymphocytes, over anti-melanoma immunity, e.g. exerted by CD8(+) cytolytic T lymphocytes. The CD27 glycoprotein is crucial because it is able to identify regulatory T cells endowed with strong suppressive ability, whilst CD8(+) T cells endowed with actual cytolytic ability become CD27(-). The present in situ quantitative immunohistochemical study, including a series of double labelling experiments and morphometrical cell analyses, shows that the vast majority of lymphocytes infiltrating primary cutaneous melanoma express CD27. Specifically, virtually the entire CD4(+) CD25(+) FOXP3(+) T subset infiltrating primary cutaneous melanoma also co-expressed CD27; CD27 was, moreover, co-expressed even by the vast majority of the CD8(+) T cells, and, conversely, effector/cytotoxic CD8(+)CD27(-) cells were very scarcely represented. The overwhelming CD27 co-expression may confer on the CD4(+)CD25(+)FOXP3(+) T subset a consistent capacity to suppress anti-melanoma immunity, whereas the too low CD8(+) CD27(-) cell proportion may presumably be insufficient to confer on the CD8(+) T subset a satisfactory anti-melanoma cytotoxic activity. We therefore propose that these CD27-discriminated pathways may trigger a functional imbalance within the microenvironment of primary cutaneous melanoma, thus favouring melanoma progression.     

Activation of human vascular endothelium in melanoma metastases induces ICAM‐1 and E‐selectin expression and results in increased infiltration with effector lymphocytes

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real‐time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM‐1 (CD54) and E‐selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3⁺ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF‐α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8⁺ cells were detected in TNF‐α–treated melanoma metastases compared with PBS‐treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI‐67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.     

HLA-A(*)0201(+) Plasmacytoid Dendritic Cells Provide a Cell-Based Immunotherapy for Melanoma Patients

Several sources of evidence suggest that tumor-specific T cells have the potential to control melanoma tumors. Current active and adoptive therapeutic approaches to elicit such T cells are either not sufficiently clinically efficient or require fastidious processes that impede their extensive clinical use. As plasmacytoid dendritic cells (pDCs) have a crucial role in triggering antitumor immunity especially in melanoma, we explored their potential as a cell-based approach for melanoma immunotherapy. An irradiated human HLA-A(*)0201(+) pDC line loaded with peptides derived from the major melanoma tumor antigens, MelA/MART-1, gp100/pmel17, tyrosinase, and MAGE-A3, was used to trigger functional multi-specific T cells ex vivo from peripheral blood mononuclear cells and tumor-infiltrating lymphocytes from stage I-IV HLA-A(*)0201(+) melanoma patients. pDCs loaded with melanoma-derived peptides promptly induced high levels of melanoma tumor-specific T cells from both sources. pDC-primed central/effector memory antitumor T cells were highly functional as indicated by the specific IFNγ secretion and membrane CD107 expression upon stimulation. Cells also exhibited strong cytotoxicity toward semi-allogeneic melanoma cells and patient-derived tumor cells. The simple design and potent efficacy of this promising approach provides a preclinical basis for the development of a pDC-based vaccine and an alternative means to produce tumor-specific T cells for adoptive cellular immunotherapy in melanoma patients.     

Immunomodulation by imiquimod in patients with high-risk primary melanoma

Imiquimod is a synthetic Toll-like receptor 7 (TLR7) agonist approved for the topical treatment of actinic keratoses, superficial basal cell carcinoma, and genital warts. Imiquimod leads to an 80-100% cure rate of lentigo maligna; however, studies of invasive melanoma are lacking. We conducted a pilot study to characterize the local, regional, and systemic immune responses induced by imiquimod in patients with high-risk melanoma. After treatment of the primary melanoma biopsy site with placebo or imiquimod cream, we measured immune responses in the treated skin, sentinel lymph nodes (SLNs), and peripheral blood. Treatment of primary melanomas with 5% imiquimod cream was associated with an increase in both CD4+ and CD8+ T cells in the skin, and CD4+ T cells in the SLN. Most of the CD8+ T cells in the skin were CD25 negative. We could not detect any increases in CD8+ T cells specifically recognizing HLA-A(*)0201-restricted melanoma epitopes in the peripheral blood. The findings from this small pilot study demonstrate that topical imiquimod treatment results in enhanced local and regional T-cell numbers in both the skin and SLN. Further research into TLR7 immunomodulating pathways as a basis for effective immunotherapy against melanoma in conjunction with surgery is warranted.