Photodynamic therapy-mediated immune response against subcutaneous mouse tumors

The curative ability of photodynamic therapy (PDT) is severely compromised if treated tumors are growing in immunodeficient hosts. Reconstitution of severe combined immunodeficient (scid) mice with splenocytes from naive immunologically intact BALB/c mice did not improve the response to Photofrin-based PDT of EMT6 tumors growing in these animals. In contrast, adoptive transfer of BALB/c splenocytes containing EMT6 tumor-sensitized immune cells had a dramatic effect on tumor regrowth after PDT. For instance, full restoration of the curative effect of PDT was achieved with scid mice that received splenocytes from BALB/c donors that were cured of EMT6 tumors by PDT 5 weeks before adoptive transfer. Splenocytes obtained from donors cured of EMT6 tumors using X-rays were much less effective. Selective in vitro depletion of specific T-cell populations from engrafting splenocytes indicated that CTLs are the main immune effector cells responsible for conferring the curative outcome to PDT in this experimental model, whereas helper T lymphocytes play a supportive role. The immune specificity of these T-cell populations was demonstrated by the absence of cross-reactivity between the EMT6 and Meth-A tumor models (mismatch between tumors growing in splenocyte donors and recipients). The immunocompetent BALB/c mice that received adoptively transferred splenocytes containing PDT-generated, tumor-sensitized immune cells also benefited from the improved outcome of PDT of tumors they were bearing. This was demonstrated not only with the fairly immunogenic EMT6 tumor model but also with weakly immunogenic Line 1 carcinomas. The results of this study indicate that PDT is a highly effective means of generating tumor-sensitized immune cells that can be recovered from lymphoid sites distant to the treated tumor at protracted time intervals after PDT, which asserts their immune memory character. It is also shown that the treatment of tumors by PDT creates the conditions necessary for converting the inactive adoptively transferred pre-effector, tumor-sensitized immune cells into fully functional antitumor effector cells. An additional finding of this study is the evidence of NK cell activation in PDT-treated Meth-A sarcomas.     

重组人催乳素(rhPRL)在huPBL/SCID嵌合模型中对人直肠癌的治疗效应及免疫机理初探

通过体内实验进一步证实rhPRL对机体免疫功能的调节作用,以及用于过继细胞免疫治疗的可能性。我们选用CB17 SCID纯系小鼠,腹腔注入人结肠腺癌细胞(HT29),2小时后腹腔注入尼龙毛纯化的人淋巴细胞(T/NK细胞),同时开始进行rhPRL治疗,并设HBSS对照和rhIL-2治疗对照组。结果表明,rhPRL在体内外均无直接抗癌效应,反而可促进肿瘤生长。当SCID荷瘤鼠同时移植入人的T/NK细胞时出rhPRL可明显提高T/NK细胞的抗癌效果,生存期明显延长(P<0.05)。平均生存期由70.4天延至112.1天,在对照组全部死亡时(d105),rhPRL治疗组有60％存活,且在实验终止时(>160天)仍有40％存活。分析其抗癌机理发现,rhPRL体外可直接促进人T/NK细胞增殖,其分泌上清可抑制肿瘤生长,同时发现人T/NK细胞与HT29共育4小时中rhPRL亦可直接促进杀伤活性。     

Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells

Reprogramming of NK cells with a chimeric antigen receptor (CAR) proved an effective strategy to increase NK cell reactivity and recognition specificity toward tumor cells. To enhance the cytotoxicity of NK cells against CD138-positive multiple myeloma (MM) cells, we generated genetically modified NK-92MI cells carrying a CAR that consists of an anti-CD138 single-chain variable fragment (scFv) fused to the CD3ζ chain as a signaling moiety. The genetic modification through a lentiviral vector did not affect the intrinsic cytolytic activity of NK-92MI toward human erythroleukemic cell line K562 cells or CD138-negative targets. However, these retargeted NK-92MI (NK-92MI-scFv) displayed markedly enhanced cytotoxicity against CD138-positive human MM cell lines (RPMI8226, U266 and NCI-H929) and primary MM cells at various effector-to-target ratios (E:T) as compared to the empty vector-transfected NK-92MI (NK-92MI-mock). In line with the enhanced cytotoxicity of NK-92MI-scFv, significant elevations in the secretion of granzyme B, interferon-γ and proportion of CD107a expression were also found in NK-92MI-scFv in response to CD138-positive targets compared with NK-92MI-mock. Most importantly, the enhancement in the cytotoxicity of NK-92MI-scFv did not attenuate with 10Gy-irradiation that sufficiently blocked cell proliferation. Moreover, the irradiated NK-92MI-scFv exerted definitely intensified anti-tumor activity toward CD138-positive MM cells than NK-92MI-mock in the xenograft NOD-SCID mouse model. This study provides the rationale and feasibility for adoptive immunotherapy with CD138-specific CAR-modified NK cells in CD138-positive plasmacytic malignancies, which potentially further improves remission quality and prolongs the remission duration of patients with MM after upfront chemotherapy.     

NK cells are essential for effective BCG immunotherapy

Adjuvant intravesical bacillus Calmette-Guérin (BCG) therapy is a well-established and successful adjuvant immunotherapy in the treatment of superficial bladder cancer. Although the function of natural killer (NK) cells in other immunotherapeutic regimens (e.g., lymphokine-activated killer [LAK] cell or interleukin-2 [IL-2] therapy) has been established, the contribution of NK cells to effective BCG immunotherapy is not clear. We used a human in vitro system to analyze the role of NK cells in BCG-induced cellular cytotoxicity. After stimulation of mononuclear cells with BCG for 7 days, these BCG-activated killer (BAK) cells displayed substantial cytotoxicity against bladder tumor cells. Magnetic depletion experiments and fluorescence activated cell sorting revealed that NK cells were the major effector cell population. To address NK cell function in vivo, we studied a syngeneic orthotopic murine bladder cancer model and compared BCG immunotherapy in C57BL/6 wild-type mice, NK-deficient beige mice and mice treated with anti-NK1.1 monoclonal antibody. Four weekly instillations of viable BCG significantly prolonged survival in wild-type mice compared with control mice treated with solvent alone. In contrast, BCG therapy was completely ineffective in NK-deficient beige mice and in mice treated with anti-NK1.1 monoclonal antibody. These findings suggest a key role for NK cells during BCG immunotherapy.     

DNAX accessory molecule-1 mediated recognition of freshly isolated ovarian carcinoma by resting natural killer cells

Although natural killer (NK) cells are well known for their ability to kill tumors, few studies have addressed the interactions between resting (nonactivated) NK cells and freshly isolated human tumors. Here, we show that human leukocyte antigen class I(low) tumor cells isolated directly from patients with advanced ovarian carcinoma trigger degranulation by resting allogeneic NK cells. This was paralleled by induction of granzyme B and caspase-6 activities in the tumor cells and significant tumor cell lysis. Ovarian carcinoma cells displayed ubiquitous expression of the DNAX accessory molecule-1 (DNAM-1) ligand PVR and sparse/heterogeneous expression of the NKG2D ligands MICA/MICB and ULBP1, ULBP2, and ULBP3. In line with the NK receptor ligand expression profiles, antibody-mediated blockade of activating receptor pathways revealed a dominant role for DNAM-1 and a complementary contribution of NKG2D signaling in tumor cell recognition. These results show that resting NK cells are capable of directly recognizing freshly isolated human tumor cells and identify ovarian carcinoma as a potential target for adoptive NK cell-based immunotherapy.     

Analysis of the relationship between stage of differentiation and nk/lak susceptibility of colon carcinoma cells

NK and LAK cells which are able to lyse tumor target cells in an MHC-unrestricted manner are not equally effective against targets of the same nature. In the case of colorectal tumors, some cells are highly sensitive, whereas others are resistant to NK and can even be quite resistant to LAK-mediated lysis. In the present paper, we tried to correlate the stage of differentiation of 17 human colorectal tumor cell lines with their NK- or LAK-cell susceptibility. It was observed that NK cells killed colorectal target cells independently from their stage of differentiation defined according to histopathological criteria from xenografting in nude mice. NK susceptibility was not correlated either with in vitro-defined criteria of differentiation, such as cell polarity and morphology, brush-border enzyme expression and CEA production. A LAK-resistant HT-29 sub-line (HT-29 LAK) was selected which could not be distinguished from HT-29 in terms of features of differentiation. It was further observed that HT-29 Glc^?/+ cell line, a highly differentiated enterocytic-like variant of HT-29, obtained after glucose starvation, was killed by LAK cells as efficiently as the moderately differentiated parental HT-29, and that Caco-2 cells, which differentiate spontaneously after confluence in standard culture conditions, were equally sensitive to NK-mediated lysis whatever their stage of differentiation. In contrast, HT29 MTX10^?5, a highly differentiated mucus-secreting variant of HT29 obtained by methotrexate selection, was much more resistant to LAK cells than parental HT29 cells.     

异体NK细胞过继免疫在治疗血液肿瘤中的机制及应用

 自然杀伤（NK）细胞是人体天然免疫系统的主要组成部分,其对血液肿瘤细胞的杀伤主要是由其表面抑制性受体和活化性受体所传递的信号共同决定。近年来,异体半相合NK细胞在造血干细胞移植和过继性细胞免疫治疗方面均显示出对人类血液系统肿瘤的积极治疗作用,为以NK细胞为基础的过继免疫治疗的临床应用提供了可能。     

Macrophage-directed immunotherapy as adjuvant to photodynamic therapy of cancer.

The effect of Photofrin-based photodynamic therapy (PDT) and adjuvant treatment with serum vitamin D3-binding protein-derived macrophage-activating factor (DBPMAF) was examined using a mouse SCCVII tumour model (squamous cell carcinoma). The results show that DBPMAF can markedly enhance the curative effect of PDT. The most effective DBPMAF therapy consisted of a combination of intraperitoneal and peritumoral injections (50 and 0.5 ng kg-1 respectively) administered on days 0, 4, 8 and 12 after PDT. Used with a PDT treatment curative to 25% of the treated tumours, this DBPMAF regimen boosted the cures to 100%. The DBPMAF therapy alone showed no notable effect on the growth of SCCVII tumour. The PDT-induced immunosuppression, assessed by the evaluation of delayed-type contact hypersensitivity response in treated mice, was greatly reduced with the combined DBPMAF treatment. These observations suggest that the activation of macrophages in PDT-treated mice by adjuvant immunotherapy has a synergistic effect on tumour cures. As PDT not only reduces tumour burden but also induces inflammation, it is proposed that recruitment of the activated macrophages to the inflamed tumour lesions is the major factor for the complete eradication of tumours.     

Retargeting NK92 cells using an HLA-A2-restricted, EBNA3C-specific chimeric antigen receptor

Advances in adoptive cell immunotherapy have led to several promising options for cancer patients. Single-chain variable fragments (scFvs) were isolated from a human phage display library by panning on recombinant human leukocyte antigen (HLA)-A2-peptide complexes. A scFv (EBNA Clone 315) specific for HLA-A2 carrying a 10 amino acid peptide (LLDFVRFMGV) derived from the Epstein-Barr virus latent protein EBNA3C was fully characterized. EBNA Clone 315 displayed exquisite specificity toward its targeted T-cell epitope (TCE) and did not cross-react with the free peptide, HLA-A2 complexes, which carried irrelevant peptides, or HLA-A2(-) cells. Furthermore, after engineering into a scFv-Fc fusion protein, we were able to determine its affinity, detection sensitivity, and ability to induce antibody-dependent cellular cytotoxicity (ADCC). As a proof-of-principle, a chimeric antigen receptor (CAR) version of EBNA Clone 315 was used to reprogram NK92MI cells. CAR-expressing NK92MI cells showed highly specific and potent cytotoxicity toward the targeted TCE, with detection sensitivity of approximately 25 molecules and cytolytic capacity threefold greater than scFv-Fc-mediated ADCC. For the first time, we show the successful reprogramming of non-T cells toward a specific TCE using a CAR.     

Synergistic antitumor activity of the novel SN-38-incorporating polymeric micelles, NK012, combined with 5-fluorouracil in a mouse model of colorectal cancer, as compared with that of irinotecan plus 5-fluorouracil

The authors reported in a previous study that NK012, a 7-ethyl-10-hydroxy-camptothecin (SN-38)-releasing nano-system, exhibited high antitumor activity against human colorectal cancer xenografts. This study was conducted to investigate the advantages of NK012 over irinotecan hydrochloride (CPT-11) administered in combination with 5-fluorouracil (5FU). The cytotoxic effects of NK012 or SN-38 (an active metabolite of CPT-11) administered in combination with 5FU was evaluated in vitro in the human colorectal cancer cell line HT-29 by the combination index method. The effects of the same drug combinations was also evaluated in vivo using mice bearing HT-29 and HCT-116 cells. All the drugs were administered i.v. 3 times a week; NK012 (10 mg/kg) or CPT11 (50 mg/kg) was given 24 hr before 5FU (50 mg/kg). Cell cycle analysis in the HT-29 tumors administered NK012 or CPT-11 in vivo was performed by flow cytometry. NK012 exerted more synergistic activity with 5FU compared to SN-38. The therapeutic effect of NK012/5FU was significantly superior to that of CPT-11/5FU against HT-29 tumors (p = 0.0004), whereas no significant difference in the antitumor effect against HCT-116 tumors was observed between the 2-drug combinations (p = 0.2230). Cell-cycle analysis showed that both NK012 and CPT-11 tend to cause accumulation of cells in the S phase, although this effect was more pronounced and maintained for a more prolonged period with NK012 than with CPT-11. Optimal therapeutic synergy was observed between NK012 and 5FU, therefore, this regimen is considered to hold promise of clinical benefit, especially for patients with colorectal cancer.     

Natural killer cells: of-the-shelf cytotherapy for cancer immunosurveillance

Natural killer (NK) cells are advantaged innate cytotoxic lymphocytes with characteristics of tumor immunosurveillance and microorganism elimination. Distinguish from the adaptive T and B lymphocytes, the autologous or allogeneic NK cells efficaciously fulfil the function of combating transformed hematological malignancies and metastatic solid tumors via the proverbial mechanisms including direct cytolytic effect and antibody-dependent cell-mediated cytotoxicity (ADCC) as well as paracrine effects dispense with antigen presentation. Herein, we review the candidate sources (e.g., peripheral blood, umbilical cord blood, placental blood, cell lines and stem cells) for large-scale and clinical-grade NK cell manufacturing, ex vivo cultivation (feeder-, cytokine cocktail- or physicochemical irritation-dependent strategies) for NK cell persistence and activation. Furthermore, we also figure out the promising prospects as well as the accompanied challenges of NK cell- or chimeric antigen receptor-transduced NK (CAR-NK) cell-based adoptive immunotherapy in standardizations for industrialized preparation and clinical practices.     

Hypericin-mediated photodynamic therapy induces lipid peroxidation and necrosis in nasopharyngeal cancer

Photoactivation of hypericin is known to generate singlet oxygen and superoxide anion radicals. Reactive oxygen species (ROS) produced by photodynamic therapy (PDT) has the capacity to induce oxidative damage and tumor destruction. We have previously shown that hypericin-PDT induces tumor shrinkage and regression in the human nasopharyngeal cancer (NPC)/HK1 murine tumor model. In this extended study, we show by electron microscopy that subcutaneously implanted HK1 NPC cells from Balb/c nude mice perished by cell necrosis with hypericin-PDT treatment. There was evidence of cytoplasmic swelling accompanied by loss of cell membrane integrity and autophagic vacuolization of cytoplasm but no nuclear changes. There was also no significant difference in the apoptotic index of control and PDT-treated tumors, when analyzed by in situ end labeling of DNA strand breakage to detect apoptosis. This further supports the observation that cell death in PDT-treated NPC/HK1 tumors was by necrosis. Lipid peroxidative stress analyzed by the malonaldehyde assay was significantly elevated in PDT-treated cells. However, PDT had no effect on the activity of superoxide dismutase, an intracellular antioxidant enzyme. The findings show that hypericin-PDT of nasopharyngeal tumors in vivo induces tumor necrosis with accompanying lipid peroxidation.     

Attenuation of the glucocorticoid response during Ad5IL-12 adenovirus vector treatment enhances natural killer cell-mediated killing of MHC class I-negative LNCaP prostate tumors

Tumor cells can evolve to evade immune responses by down-modulating surface MHC class I expression and become refractory to T cell-directed immunotherapy. We employed a strategy to bypass this escape mechanism using a recombinant adenovirus vector expressing interleukin-12 (Ad5IL-12) to target natural killer (NK) cell-mediated killing of human prostate tumors in NOD.scid mice. Fluorescence-activated cell sorting analysis revealed that LNCaP tumor cells bear negligible levels of MHC class I molecules; yet, they express MICA/B molecules, ligands for the NKG2D receptors found on NK cells. Transduction of LNCaP cells with the Ad5IL-12 vector prevented tumor formation in NOD.scid mice, indicating that NK cells alone can conduct tumor immunosurveillance and mediate protection. Intratumor injection of the Ad5IL-12 vector to established LNCaP tumors in NOD.scid mice resulted in a significant delay of tumor growth mediated by NK cell killing activity. The dependency of NK cells in this protective response was shown by the complete loss of Ad5IL-12 therapeutic efficacy on LNCaP tumors established in NOD.Cg-Rag1(tm1Mom)Prf1(tm1Sdz) congenic mice, which are devoid of NK cell activity. More pronounced attenuation of tumor growth and enhanced NK killing activity was observed when pharmacologic adrenalectomy with mitotane was done in combination with Ad5IL-12 vector treatment. The Ad5IL-12 vector treatment also induced killing of MICA/B-negative MHC class I-positive PC3 tumors formed in NOD.scid mice. Together, these results indicate that a targeted NK cell response could provide a generic approach for cancer immunotherapy, and that enhancing the NK cell response via control of cortisol levels may provide an additional therapeutic avenue in cancer.     

Natural killer cell‐mediated lysis of freshly isolated human tumor cells

Studies on interactions between natural killer (NK) cells and freshly isolated human tumor cells are still relatively scarce. With respect to the understanding of NK cell interactions with human tumors in vivo, and attempts to better predict the outcome of NK cell‐mediated adoptive immunotherapy, such studies merit further attention. They may provide information that is not readily evident through studies of human tumor cell lines established through long‐term culturing in vitro. The latter undergo several changes and adaptations to their environment, many of which may affect interactions with NK cells. Studies of NK cell interactions with freshly explanted human tumors, however, are cumbersome for several reasons including; often poor accessibility of human tumor material per se, difficulties in sample processing and contamination of tumors with nontumor cells, spontaneous apoptosis of tumor cells, as well as methodological challenges in assessing tumor cell lysis following interactions with NK cells. Here, we review the current knowledge on NK cell interaction with freshly isolated human tumors. © 2008 Wiley‐Liss, Inc.     

Characterization of stem cell factor gene-modified human natural killer cell line, NK-92 cells: implication in NK cell-based adoptive cellular immunotherapy

NK-92 cells are effective against a broad range of malignant targets both in vitro and in vivo. Stem cell factor (SCF) is an important early acting cytokine for NK cell development. The characterization and implication in clinic of SCF gene-modified NK-92 cells need to be investigated. SCF cDNA was inserted into pcDNA3 eukaryotic expression vector and the recombinant vector (pcDNA3-SCF) was transfected into NK-92 cells. The SCF gene-modified NK-92 cells (NK92-SCF) were cloned and characterized by cytokine gene expression, proliferation potential, cytotoxic function and surface phenotype. NK92-SCF cells continuously produced a high level of SCF in culture supernatant, which made the cells proliferate significantly more rapidly in response to IL-2 or IL-15 stimulation, the cumulative amount of cells in long-term culture was significantly higher. NK92-SCF cells exerted stronger cytotoxicity against a broad range of target tumor cells than their parent NK-92 cells, which was correlated to the increased expression of cytotoxic effector molecules such as perforin and Fas ligand. NK92-SCF cells became more heterogeneous; the CD56(High) and CD56(Low) subsets appeared, which may, at least partly, explain the increased proliferating and cytotoxic potential of NK92-SCF cells. SCF gene-modified NK-92 cells (NK92-SCF cells) are more promising than their parent cells for adoptive cellular immunotherapy.     

Comparison of the growth and metastasis of four human intestinal tumor cell line xenografts

The growth and metastasis of four human intestinal tumor cell lines: one duodenal adenocarcinoma (HTB-40) and three adenocarcinomas of the colon (CCL-218, CCL-222 and HT-29) have been compared in vitro and in vivo in nude mice. HTB-40 was the fastest growing cell line in vitro with a doubling time (DT) of 14.8 h. CCL-218 and CCL-222 grew more slowly in vitro with doubling times of 21.6 and 22.8 hours, respectively. All three of these tumors grew more slowly in vivo with doubling times ranging from 39.1 h (CCL-218 in male nude mice) to 65.3 h (CCL-222). The growth of CCL-218 cells was significantly slower in female nude mice DT 51.0 h). HT-29 was the slowest growing in vitro (DT 23.8 h) and in vivo (DT about 100 h). HT-29 also showed the greatest discrepancy between its DT measured in vivo as compared to in vitro, suggesting a greater clonogenic cell loss from HT-29 tumors in vivo. Histologic evaluation of these tumors grown subcutaneously in nude mice showed all to be anaplastic and to produce liver micrometastases. However, more extensive abdominal and liver metastases were observed in the nude mice injected with HT-29 cells, and some of these metastases had morphologic features of moderately well-differentiated epithelium. These results indicate the usefulness of the HT-29 tumor cell line as an experimental model of metastasis from a human colonic adenocarcinoma.     

Systemic antitumor effect of intratumoral injection of dendritic cells in combination with local photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT), which is used clinically for the palliative treatment of cancer, induces local tumor cell death but has no effect on tumors in untreated sites. The purpose of this study was to determine if local PDT followed by intratumoral injection of na?ve dendritic cells (IT-DC) induces systemic antitumor immunity that can inhibit the growth of untreated as well as PDT + IT-DC-treated tumors. EXPERIMENTAL DESIGN: BALB/c or C57Bl/6 mice were injected s.c. with CT26 colorectal carcinoma cells and B16 melanoma cells, respectively, and following 10 to 12 days of tumor growth, the tumors were treated with PDT alone or PDT followed by IT-DC or IT-PBS. In other studies, tumors were established simultaneously in both lower flanks or in one flank and in the lungs, but only one flank was treated. RESULTS: Whereas neither PDT nor IT-DC alone was effective, PDT + IT-DC eradicated both CT26 and B16 tumors in a significant proportion of animals and prolonged the survival of mice of which the tumors were not cured. The spleens of mice treated with PDT + IT-DC contained tumor-specific cytotoxic and IFN-gamma-secreting T cells whereas the spleens of control groups did not. Moreover, adoptive transfer of splenocytes from successfully treated CT26 tumor-free mice protected na?ve animals from a subsequent challenge with CT26, and this was mediated mainly by CD8 T cells. Most importantly, PDT plus IT-DC administered to one tumor site led to tumor regression at distant sites, including multiple lung metastases. CONCLUSIONS: PDT + IT-DC induces potent systemic antitumor immunity in mice and should be evaluated in the treatment of human cancer.