Fractalkine mediates natural killer-dependent antitumor responses in vivo

CX3CR1 has been described previously as a marker of human cytotoxic effector cells. We evaluated the possibility of using its ligand, CX3CL1, to redirect immune response against tumors. When murine lymphoma cell lines (EL4 and its derivative EG7) stably transfected with human-CX3CL1 were injected s.c. into C57BL/6 mice, the tumor growth was severely impaired when compared with the growth of control cell lines. This antitumor effect of CX3CL1 was also found in T- and B-cell-deficient Rag1-/- mice but vanished in natural killer (NK) cell-deficient beige mice and in CX3CR1-/- mice, suggesting the involvement of CX3CR1-expressing NK cells. In addition, increased NK cell infiltration was observed in CX3CL1-producing tumors compared with controls. The effect of CX3CR1 on tumor growth required host cytotoxic effector cell functions because both IFNgamma-/- and perforin-/- mice were resistant to CX3CL1 antitumor effect. Finally, intratumoral injection of DNA plasmid coding for a chimeric immunoglobulin presenting the CX3CL1 chemokine domain provided strong antitumor activity. Together, these data demonstrate that the CX3CL1 can reduce incidence and size of lymphoma in vivo through increased recruitment of activated NK cytotoxic cells. These findings offer the first evidence of the potential of chimeric immunoglobulin-chemokines in anticancer therapy.     

Differential Hsp70 plasma-membrane expression on primary human tumors and metastases in mice with severe combined immunodeficiency

To study the role of cell-surface expression of a tumor-selective heat-shock protein 70 (Hsp70) in vivo, the colon-carcinoma cell line CX2, and the clonal sub-lines CX⁺ and CX⁻, which differ in Hsp70 cell-surface expression, but not in MHC and adhesion-molecule expression, were implanted into immunodeficient SCID/beige mice by s.c., i.p., i.v. and orthotopic (o.t.) inoculation. On day 18 after s.c. injection, all animals developed s.c. tumors, ranging in size from 2.5 to 3 cm². Phenotypic characterization of single-cell suspensions generated from freshly isolated tumor material revealed that the pattern of cell-surface expression is identical to that of the injected tumor cells from cell culture. Comparable results were obtained following i.p. inoculation of CX⁺ and CX⁻ cells. Macroscopic and microscopic evaluation of lymph nodes, lung, liver and spleen at autopsy of tumor-bearing mice showed no tumor burden except the primary tumor, following s.c. or i.p. injection. After i.v. inoculation of CX⁺ and of CX⁻ cells, weak tumor growth was observed in lung and liver, the Hsp70 cell-surface-expression pattern on these tumors being identical to that of the injected cells. However, o.t. injection of colon-carcinoma cell lines CX⁺ and CX⁻ into the cecum resulted in tumor growth at the injection site and in spread of distant metastases in lung, liver and spleen. Most interestingly, and in contrast to the primary colon carcinomas, metastases of CX⁺ and of CX⁻ tumor cells both revealed strong Hsp70 plasma-membrane expression, although the total amount of cytoplasmic Hsp70 was comparable. Int. J. Cancer 77:942–948, 1998.© 1998 Wiley-Liss, Inc.     

Coexpression of CD40L and CD70 by semiallogenic tumor cells induces anti-tumor immunity

The immune system is potentially qualified to detect and eliminate tumor cells, but various mechanisms developed by tumor cells allow tumor escape. Strategies selected to promote antitumor responses have included genetic modifications of tumor cells to induce expression of costimulatory molecules. Moreover, alloantigens can also act as strong enhancers of the immune response. In this work, we have associated the expression of two costimulatory members of the TNF superfamily, CD40L and CD70 along with an allogenic MHC Class I (H-2K(d)) molecule expression on melanoma cells (B16F10, H-2(b)) to favor the antitumor immune response. B16F10 tumor growth slows significantly when CD40L and CD70 are coexpressed by tumor cells and the association with the allogenic molecule (H-2K(d)) enhances this effect. Growth kinetics of mock and CD40L-CD70-H-2K(d)-expressing B16F10 tumors in immunocompetent versus nu/nu and beige mice suggested that CD8(+) T lymphocytes and NK cells were involved in this antitumor immunity. A delay in mock tumor growth was observed when CD40L-CD70-H-2K(d)-expressing B16F10 cells and mock tumor cells were injected simultaneously and contralaterally. It was also shown that in vivo immunization of immunocompetent mice with CD40L-CD70-H-2K(d) B16F10 tumor cells improved the generation of cytotoxic lymphocytes against the wild-type melanoma cells expressing the syngenic MHC Class I molecule H-2K(b) (B16K1). These observations lay a path for new immunotherapeutic trials using semiallogenic fibroblasts expressing costimulatory molecules and tumor-associated antigens.     

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.     

An Hsp70 peptide initiates NK cell killing of leukemic blasts after stem cell transplantation

In contrast to solid tumors, leukemic blasts frequently present both Hsp70 and HLA-E on their cell surface and thereby present activating and inhibitory signals to CD94(+) NK cells. In the first 12 months after stem cell transplantation (SCT) CD94(+) NK cells clearly dominate over CD3(+)/CD16(-)/56(-) T and CD3(+)/CD16(+)/56(+) NK-like T cells. An incubation of post-SCT-derived peripheral blood lymphocytes with the Hsp70 peptide TKD and IL-15 enhances the cell surface density of CD56/CD94 and initiates the cytolytic activity of NK cells against Hsp70/HLA-E double-positive autologous and allogeneic leukemic blasts. Hsp70 was identified as the target structure for TKD-activated NK cells.     

Heat shock protein 70 (Hsp70) membrane expression on head-and-neck cancer biopsy-a target for natural killer (NK) cells

PURPOSE: Heat shock protein 70 (Hsp70) was detected on the cell membrane of human tumor cell lines, but not on normal cells. Here we studied Hsp70 membrane expression as a target for natural killer (NK) cells on tumor material and control tissues of head-and-neck cancer patients. METHODS AND MATERIALS: Membrane-bound Hsp70 was determined by flow cytometry on single-cell suspensions of tumors and the corresponding normal tissues of head-and-neck cancer patients. The cytolytic activity of NK cells against Hsp70-positive tumor cells was measured in a standard cytotoxicity assay. RESULTS: In total, 54 of 74 primary tumors were found to be Hsp70 membrane-positive (73%); tongue/mouth, 21 of 24 (88%); oropharynx, 13 of 20 (65%); hypopharynx, 3 of 6 (50%); larynx, 8 of 11 (73%); trachea 1 of 2 (50%); esophagus, 4 of 5 (80%); lymph node metastases, 4 of 6 (67%). The corresponding control tissue was negative for membrane-bound Hsp70. Biopsies (6 of 6) of patients after in vivo gamma-irradiation (fractionated 5 x 2 Gy) were strongly Hsp70 membrane-positive. Irradiated, Hsp70-positive tumor cells are targets for Hsp70-peptide stimulated NK cells. CONCLUSION: An irradiation-inducible, tumor-selective Hsp70 membrane localization provides a target structure for Hsp70-peptide stimulated human NK cells.     

Natural killer cell-mediated ablation of metastatic liver tumors by hydrodynamic injection of IFNalpha gene to mice

Interferon (IFN) alpha is a pleiotropic cytokine acting as an antiviral substance, cell growth inhibitor and immunomodulator. To evaluate the therapeutic efficacy and mechanisms of IFNalpha on hepatic metastasis of tumor cells, we hydrodynamically injected naked plasmid DNA encoding IFNalpha1 (pCMV-IFNa1) into Balb/cA mice having 2 days hepatic metastasis of CT-26 cells. Single injection of pCMV-IFNa1 efficiently enhanced the natural killer (NK) activity of hepatic mononuclear cells, induced production of IFNgamma in serum and led to complete rejection of tumors in the liver. Mice protected from hepatic metastasis by IFNalpha therapy displayed a tumor-specific cytotoxic T cell response and were resistant to subcutaneous challenge of CT-26 cells. NK cells were critically required for IFNalpha-mediated rejection of hepatic metastasis, because their depletion by injecting anti-asialo GM1 antibody completely abolished the antimetastatic effect. To find whether NK cells are directly activated by IFNalpha and are sufficient for the antimetastatic effect, the responses to IFNalpha were examined in SCID mice lacking T cells, B cells and NKT cells. IFNalpha completely rejected hepatic metastasis in SCID mice and efficiently activated SCID mononuclear cells, as evidenced by activation of STAT1 and a variety of genes, such as MHC class I, granzyme B, tumor necrosis factor-related apoptosis-inducing ligand and IFNgamma, and also enhanced Yac1 lytic ability. Study of IFNgamma knockout mice revealed that IFNgamma was not necessary for IFNalpha-mediated NK cell activation and metastasis protection. In conclusion, IFNalpha efficiently activates both innate and adaptive immune responses, but NK cells are critically required and sufficient for IFNalpha-mediated initial rejection of hepatic metastasis of microdisseminated tumors.     

Hsp70-H22肿瘤抗原肽复合物激活树突状细胞诱导抗肿瘤免疫的研究

目的　探讨通过树突状细胞 (DC)提呈途径降低肿瘤抗原肽用量的可行性 ,了解热休克蛋白 70 (Hsp70 )和抗原肽修饰DC作用的特点。方法　以Hsp70于体外结合抗原肽 ,并于体外修饰DC ,检测修饰后DC的代谢活性及分泌的细胞因子 ;比较修饰后DC和Hsp70 H2 2肽对淋巴细胞的激活作用 ;检测激活的淋巴细胞对H2 2瘤细胞的细胞毒作用以及注射DC和Hsp70 H2 2肽对小鼠肿瘤的抑制作用。结果　Hsp70结合 0 .15 μgH2 2肽可使 2× 10 5DC成熟 ,4× 10 3 成熟DC可激活 2× 10 6淋巴细胞 ;Hsp70结合 0 .0 0 3μg肽修饰的DC或Hsp70结合 0 .15 μg肽直接刺激 ,可激活相同数量的淋巴细胞 ,产生同样的杀瘤效果。激活DC后再回输的治疗方式与直接注射Hsp70 肽复合物的治疗方式相比 ,抗原肽的用量可以降低 5 0倍。正常肝组织来源的混合肽结合Hsp70后 ,不能使DC成熟 ,亦不能通过DC途径活化脾淋巴细胞。结论　DC提呈抗原肽激活淋巴细胞的途径能够有效降低Hsp70 肿瘤抗原肽复合物使用剂量。正常细胞的混合肽不能通过Hsp70和DC提呈激活淋巴细胞 ,其诱发自身免疫应答的可能性极低     

Treatment of colon and lung cancer patients with ex vivo heat shock protein 70-peptide-activated, autologous natural killer cells: a clinical phase i trial.

PURPOSE: The 14 amino acid sequence (aa(450-463)) TKDNNLLGRFELSG (TKD) of heat shock protein 70 (Hsp70) was identified as a tumor-selective recognition structure for natural killer (NK) cells. Incubation of peripheral blood lymphocyte cells with TKD plus low-dose interleukin 2 (IL-2) enhances the cytolytic activity of NK cells against Hsp70 membrane-positive tumors, in vitro and in vivo. These data encouraged us to test tolerability, feasibility, and safety of TKD-activated NK cells in a clinical Phase I trial. EXPERIMENTAL DESIGN: Patients with metastatic colorectal cancer (n = 11) and non-small cell lung cancer (n = 1) who had failed standard therapies were enrolled. After ex vivo stimulation of autologous peripheral blood lymphocytes with Hsp70-peptide TKD (2 microg/ml) plus low-dose IL-2 (100 units/ml), TKD was removed by extensive washing, and activated cells were reinfused i.v. The procedure was repeated for up to six cycles, applying a dose escalation schedule in 4 patients. RESULTS: The percentage of activated NK cells in the reinfused leukapheresis products ranged between 8 and 20% of total lymphocytes, corresponding to total NK cell counts of 0.1 up to 1.5 x 10(9). Apart from restless feeling in 1 patient and itching in 2 patients, no negative side effects were observed. Concomitant with an enhanced CD94 cell surface density, the cytolytic activity of NK cells against Hsp70 membrane-positive colon carcinoma cells was enhanced after TKD/IL-2 stimulation in 10 of 12 patients. Concerning tumor response, 1 patient was in stable disease during therapy by formal staging criteria and another patient showed stable disease in one metastases and progression in another. CONCLUSIONS: Reinfusion of Hsp70-activated autologous NK cells is safe. Immunological results warrant additional studies in patients with lower tumor burden.     

In vivo induction of antitumor immunity and protection against tumor growth by injection of CD154-expressing tumor cells

As they should enhance tumor-specific antigen presentation by dendritic cells, tumor cell lines genetically modified to express CD154 molecules have been used in an attempt to induce protective antitumor immunity. Two murine models were used: the major histocompatibility complex (MHC) class I negative melanoma B16F10 and the MHC class I positive mammary adenocarcinoma TS/A. CD154 or mock-transfected B16F10 or TS/A cells were injected subcutaneously into H-2-compatible B6D2 mice. CD154 expression by tumor cells induced a complete rejection (in the TS/A model) or a striking reduction (in the B16F10 model) of modified tumors growth, but also a significant protection against the growth of mock tumor cells injected simultaneously, either mixed with the CD154-expressing tumor cells, or in the other flank of mice. Thirty days after CD154-expressing tumor rejection, splenic lymphocytes from surviving tumor-free mice were able to inhibit tumor proliferation in vitro and significant amounts of IFN-gamma were detected in the sera of these mice. Growth kinetics of mock and CD154-expressing tumors in immunocompetent versus nude mice suggest that T lymphocytes and natural killer cells responses are implicated in this antitumor immunity. The injection of CD154-expressing tumor cell induced an antitumor protective response, both locally and distant from the injection site. The effect was most pronounced in MHC class I expressing TS/A tumor model.     

Orthotopic reconstitution of human small-cell lung carcinoma after intravenous transplantation in SCID mice.

We have constructed an orthotopically reconstituted model of human small-cell lung carcinoma (SCLC) by intravenous transplantation in severe combined immunodeficient (SCID) mice. Two human SCLC xenografts, H-69 and Lu-130, were disaggregated and injected through the tail vein of SCID mice. Human SCLCs were orthotopically reconstituted with multi-focal lung tumor growth in all SCID mice after intravenous injection of 5 x 10(6) tumor cells per mouse. The heart and liver were also seeded with actively growing SCLC. This orthotopic reconstitution model of human SCLC in SCID mice should be useful for further studies on the biological behavior and treatment of human SCLC.     

Spontaneous and induced primary oncogenesis in natural killer (NK)-cell-deficient beige mutant mice

Spontaneous tumor development and primary oncogenesis were compared in a large number of NK4-deficient, homozygous C57Bl/6-bg/bg mice and their NK normal, heterozygous +/bg littermate controls. In a group of 167 retired breeders followed for spontaneous tumors, the probability of survival for mice eventually dying with a tumor was greater for the NK-competent, +/bg than for the NK-deficient, homozygous C57BL/6-bg/bg mice (p = 0.0019), although the higher overall incidence of tumors in the bg/bg group (48%) was not significantly different from that in the +/bg group (37%). In the bg/bg group the incidence of tumor death appeared to increase relatively sharply in the 25- to 29-month age bracket compared to the fairly regular increase in incidence observed in the +/bg group. All the spontaneous tumors except 2 (discovered in +/bg mice) were classified histologically as widely disseminated malignant lymphomas. The other two were one squamous-cell carcinoma and one sarcoma. A total of 73 bg/bg mice injected s.c. with benzo[alpha]pyrene (BP) had a higher overall incidence of tumors (81%) (rhabdomyosarcomas) than 138 +/bg mice (64%) and in the largest group (0.3 mg, n = 85) the bg/bg group developed tumors, at a higher incidence (p = 0.01) and with a shorter latency (p = 0.025) than the +/bg group. On the other hand, mice injected with dimethylbenzanthracene or given 4 weekly doses of 160 rads of gamma irradiation showed no difference in overall tumor incidence. In addition, mice injected with various doses of DMBA-induced murine leukemia virus (DMBA-LV) also showed no significant difference in tumor incidence. Others have reported that some of these treatments (DMBA, split-dose irradiation) cause profound NK suppression, thereby reducing NK differences between the two groups of mice. These results suggest that a partial NK impairment in beige mutant mice early in life may lead to significantly greater rates of death with spontaneous malignant tumors late in life. Some primary oncogenesis treatments (BP) but not others (DMBA, split-dose irradiation, leukemia viruses) cause tumors with a greater incidence and shorter latency in beige mice. The results suggest, but do not prove, that NK cells play a role in surveillance against spontaneously arising, and possibly some types of carcinogen-induced, tumors.     

Effect of isocaloric low-fat diet on human LAPC-4 prostate cancer xenografts in severe combined immunodeficient mice and the insulin-like growth factor axis.

Over-consumption of dietary fat has been suggested to promote the development and progression of prostate cancer in men. The present study was conducted to answer the following questions: (a) Can dietary fat reduction decrease tumor growth rates of Los Angeles prostate cancer (LAPC)-4 xenografts in severe combined immunodeficient (SCID) mice independent of total caloric intake? and (b) Is the insulin-like growth factor (IGF) axis involved in the effects of dietary fat on LAPC-4 tumor growth in SCID mice? Twenty-eight male CB17 beige SCID mice (8 weeks old) were individually caged, randomized, and fed an isocaloric high-fat (HF, 42% kcal) or low-fat (LF, 12% kcal) diet. Each mouse was s.c. injected with 1 x 10(5) LAPC-4 cells, and tumor volumes were measured weekly. At week 16, all animals were sacrificed, and serum and tumors were obtained for analysis. Although caloric intakes and mouse weights were equal between groups, the LF mice had significantly slower tumor growth rates and lower serum prostate-specific antigen levels compared with the HF mice. LF mice had significantly lower levels of serum insulin, tumor IGF-1 mRNA expression, and tumor IGFBP-2 immunostaining and higher levels of serum IGFBP-1 (by Western ligand blot) relative to the HF mice. There were no differences in the serum levels of IGFBP-3 and IGFBP-4 between the groups. LAPC-4 cells cultured in vitro with media containing serum from LF mice demonstrated slower growth than LAPC-4 cells cultured in media containing HF mice serum. These results demonstrate that intake of an LF diet was associated with slower LAPC-4 prostate tumor growth relative to mice fed an HF diet, independent of total caloric intake, and this effect may be mediated through modulation of the insulin/IGF axis.     

A mouse model for the Sézary syndrome

Background

The Sézary syndrome is an aggressive leukemic form of cutaneous T cell lymphoma and there is no cure of this disease. Until now there is no true animal model for Sézary syndrome, by which new drugs against the disease could be tested.

Methods

Immune deficient CB-17 SCID beige mice were injected subcutaneously with HUT78 cells, a cell line, derived from a Sézary syndrome patient. Developing tumors were analyzed by immunohistochemistry.

Results

Injected HUT78 cells formed tumors at the site of injection. In contrast to the Sézary syndrome in man, no malignant cells were observed in the blood of tumor bearing CB-17 SCID beige mice. The tumors appeared 44-62 days after injection and tumor bearing mice survived further 25 - 62 days until they had to be euthanized according to the guidelines of the Swiss animal protection law, since the tumors had reached the maximal allowed size.

Conclusion

Although the mouse model does not exactly match the human disease, it will be suited for tests of new substances for the treatment of the Sézary syndrome. The formation of an isolated tumor on the skin has the advantage that the effect of a potential drug can be directly monitored without the use of invasive methods.

     

Growth and metastasis of fresh human melanoma tissue in mice with severe combined immunodeficiency

Cryopreserved cell suspensions of freshly excised melanoma metastases from nine patients were injected s.c. into C.B-17 severe combined immunodeficiency (SCID) mice. All 9 tumors grew as s.c. masses and six of nine were successfully transplanted into other SCID mice. Transplant inocula as low as 5 x 10(5) cells resulted in 100% tumor incidence. Moreover, seven of nine tumors metastasized, five from the original s.c. implants and two from transplanted s.c. tumors. Metastases were detected mainly in the lungs but also were found in abdominal viscera (liver, spleen, and pancreas) and thoracic lymph nodes. Flow cytometric analysis showed that expression of a panel of melanoma antigens, melanoma-associated proteoglycan, ganglioside GD3, and ganglioside GD2, was maintained with SCID passage. The original tumor inocula contained a variable percentage of tumor-associated lymphocytes (1-76%). Flow cytometry analysis indicated that these were mainly CD3+ T-cells. However, there was no correlation between the percentage of tumor-associated lymphocytes and the time required for development of a palpable tumor after s.c. injection or the ability to metastasize. These results demonstrate the growth and spontaneous metastasis of fresh human melanoma in SCID mice and suggest that this model could be important for therapeutic and basic biological studies.     

Potential role of natural killer cells in controlling growth and infiltration of AIDS-associated primary effusion lymphoma cells

Natural killer (NK) cells are an important component of the innate immune response against microbial infections and tumors. Direct involvement of NK cells in tumor growth and infiltration has not yet been demonstrated clearly. Primary effusion lymphoma (PEL) cells were able to produce tumors and ascites very efficiently with infiltration of cells in various organs of T-, B- and NK-cell knock-out NOD/SCID/gammac(null) (NOG) mice within 3 weeks. In contrast, PEL cells formed small tumors at inoculated sites in T- and B-cell knock-out NOD/SCID mice with NK-cells while completely failing to infiltrate into various organs. Immunosupression of NOD/SCID by treatment with an antimurine TM-beta1 antibody, which transiently abrogates NK cell activity in vivo, resulted in enhanced tumorigenicity and organ infiltration in comparison with non-treated NOD/SCID mice. Activated human NK cells inhibited tumor growth and infiltration in NOG mice. Our results suggest that NK cells play an important role in growth and infiltration of PEL cells, and activated NK cells could be a promising immunotherapeutic tool against tumor or virus-infected cells either alone or in combination with conventional therapy. The rapid and efficient engraftment of PEL cells in NOG mice also suggests that this new animal model could provide a unique opportunity to understand and investigate the mechanism of pathogenesis and malignant cell growth.     

Tumor cells expressing anti-CD137 scFv induce a tumor-destructive environment

For immunotherapy to become more effective, there is a need to maximize the antitumor response at the tumor site as well as to eliminate tumor cell variants that lack a given tumor antigen or the ability to present it. We have previously shown that wild-type (WT) cells from the K1735 melanoma (K1735-WT) are rejected following vaccination with cells (K1735-1D8) transfected to express scFv from the anti-CD137 monoclonal antibody 1D8, and that CD4(+) T cells and natural killer (NK) cells are needed for this rejection. We now show that tumors harvested 4 to 10 days after mice had been transplanted with K1735-1D8 cells or a mixture of K1735-1D8 and K1735-WT cells contained more NK cells and that they had an increased percentage of CD4(+) T lymphocytes producing IFNgamma or tumor necrosis factor-alpha. We further show that the percentage of NK cells was higher in B16-1D8 melanomas expressing anti-CD137 scFv than in the WT tumors and that the percentage of FoxP3(+) cells was lower. Admixture of 10% K1735-1D8 cells prevented the progressive growth of transplanted K1735-WT cells in syngeneic mice and also of cells from the antigenically different sarcoma Ag104. Inhibition of WT tumor cells by tumor cells transfected to express anti-CD137 scFv was shown also with the TC1 carcinoma and B16 melanoma. Furthermore, injection of an adenovirus vector, Ad-1D8, which encodes anti-CD137 scFv into established B16 melanomas, significantly prolonged the survival of tumor-bearing mice and could induce regression. Our data suggest that targeting of anti-CD137 scFv to tumors should be explored for therapy for some human cancers.     

Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism

Regulation of innate immunity may be an effective means of cancer control. Delaying cancer onset is regarded as an important mode of action in cancer prevention. We have been investigating the chemopreventive mechanisms of Lactobacillus casei Shirota (LcS), a probiotic strain. In this study, we evaluated the effect of LcS on tumor onset and the involvement of natural killer (NK) cells using a 3-methylcholanthrene-induced carcinogenesis model. C3H/HeN mice were divided into three groups, according to treatment: vehicle-treated, treated with vehicle only; control, 3-methylcholanthrene treated; LcS, 3-methylcholanthrene and LcS treated. 3-Methylcholanthrene was injected intradermally at 7 weeks of age. LcS was mixed into the diet (0.05%, w/w), which the mice were fed from the day of 3-methylcholanthrene injection onward. Tumor incidence was observed weekly. Profiles of splenic NK cells, in vitro cytotoxicity and the proportion, in the early stage of carcinogenesis were analyzed at 5 weeks after the injection. The tumor suppressive effect of LcS was also evaluated in a beige mouse model that is genetically deficient in NK cells. LcS delayed tumor onset and reduced tumor incidence in the results with C3H/HeN mice (P< 0.05). More specifically, tumor incidence in the control group was 33% at 6 weeks after the injection and 83% at 11 weeks as opposed to 0 and 42%, respectively, in the LcS group. NK cell cytotoxicity was significantly higher than in the control group, and the number of NK cells also increased in the LcS group of C3H/HeN mice. However, LcS failed to suppress tumorigenesis in the beige mouse. These findings suggest that enhancement of the cytotoxicity of NK cells by LcS delays tumor onset.     

Adenoviral gene transfer of stromal cell-derived factor-1 to murine tumors induces the accumulation of dendritic cells and suppresses tumor growth

The human CXC chemokine, stromal cell-derived factor 1 (SDF-1alpha), is known to function in vitro as a chemotactic factor for lymphocytes, monocytes, and dendritic cells. In the context that dendritic cells are powerful antigen-presenting cells, we hypothesized that adenoviral gene transfer of SDF-1alpha to tumors might inhibit growth of preexisting tumors through attracting dendritic cells to the tumor. AdSDF-1alpha mediated the expression of SDF-1alpha mRNA and protein in A549 cells in vitro, and the supernatant of the AdSDF-1alpha-infected A549 cells showed chemotactic activity for dendritic cells. When syngeneic murine CT26 colon carcinoma tumors (BALB/c) and B16 melanoma and Lewis lung cell carcinoma (C57Bl/6) were injected with AdSDF-1alpha (5 x 10(8) plaque-forming units), there was an accumulation of dendritic cells and CD8(+) cells within the tumor and significant inhibition of tumor growth compared with tumors injected with PBS or AdNull (control vector). The injection of AdSDF-1alpha into tumors induced the inflammatory enlargement and the accumulation of dendritic cells in the draining lymph node. Intratumoral AdSDF-1alpha administration elicited tumor-specific CTLs and adoptive transfer of splenocytes from AdSDF-1alpha-treated mice resulted in the elongation of survival after tumor challenge. Interestingly, in wild-type and CD4(-/-) mice but not in CD8(-/-) mice, AdSDF-1alpha inhibited the growth of the tumor. These observations suggest that adenoviral gene transfer of SDF-1alpha may be a useful strategy to accumulate dendritic cells in tumors and evoke antitumor immune responses to inhibit tumor growth.     

Immune stimulatory effects of CD70 override CD70-mediated immune cell apoptosis in rodent glioma models and confer long-lasting antiglioma immunity in vivo

CD70 (CD27 ligand) promotes the expansion of primed lymphocytes by enhancing cell survival. Surprisingly, we previously observed that CD70 aberrantly expressed on human glioma cells promoted immune cell apoptosis and inhibited alloreactive lysis. Here we report that ectopic expression of CD70 in mouse glioma cells enhances apoptosis of T, B and NK cells in coculture, but nevertheless promotes glioma cell lysis by NK cells in vitro. In nude mice, CD70 expression in SMA-560 gliomas delays the glioma growth upon subcutaneous (s.c.) or intracerebral (i.c.) inoculation, suggesting a role for CD70/CD27-dependent NK cell activity in tumor surveillance. In syngeneic immunocompetent VM/Dk mice, CD70 allows the rejection of s.c. and i.c. implanted SMA-560 tumors. The tumorigenicity of CD70-expressing glioma cells is abrogated when TGF-beta signaling is blocked. Moreover, mice surviving the s.c. CD70 glioma challenge subsequently also reject wild-type glioma cells administered i.c. Similarly, CD70-expressing GL-261 gliomas are rejected in syngeneic C57BL/6 mice, while glioma growth is restored in C57BL/6 CD27(-/-) mice, suggesting that the CD70/CD27 interaction recruits a tumor-specific T-cell repertoire and induces tumor-specific memory. Altogether, these observations indicate that the net effect of aberrant CD70 expression in gliomas is immune stimulatory rather than immune paralytic and encourage its application in tumor immunotherapy.