T cell augments the antitumor activity of tumor-targeting <Emphasis Type="Italic">Salmonella</Emphasis>

Systemic administration of Salmonella to tumor-bearing mice leads to preferential accumulation within tumor sites and retardation of tumor growth. However, the detailed mechanism of Salmonella-induced antitumor immune response via host T cell remains uncertain. Herein, we used wild-type, CD4⁺ T-cell-deficient, and CD8⁺ T-cell-deficient mice to study the role of T cell in the antitumor immune responses induced by Salmonella enterica serovar Choleraesuis (Salmonella Choleraesuis). When systemically administered into mice bearing tumors, Salmonella Choleraesuis significantly inhibited tumor growth by 50%. In contrast, in T-cell-deficient mice, there was only 34–42% inhibition of tumor growth. We found that treatment with Salmonella Choleraesuis significantly upregulates interferon-γ in wild-type and CD8⁺ T-cell-deficient mice, but not in CD4⁺ T-cell-deficient mice. Furthermore, immunohistochemical staining of the tumors revealed more infiltration of macrophages and neutrophils in wild-type mice after Salmonella Choleraesuis treatment compared with those in T-cell-deficient mice. The antitumor therapeutic effect mediated by Salmonella Choleraesuis is associated with an inflammatory immune response at the tumor site and a tumor T helper 1-type immune response. In conclusion, these results suggest that tumor-targeted therapy using Salmonella Choleraesuis, which exerts tumoricidal effects and stimulates T cell activities, represents a potential strategy for the treatment of tumor.     

Induction of tumor regression by administration of B7-Ig fusion proteins: mediation by type 2 CD8+ T cells and dependence on IL-4 production

CD28 signals contribute to either type 1 or type 2 T cell differentiation. Here, we show that administration of B7.2-Ig fusion proteins to tumor-bearing mice induces tumor regression by promoting the differentiation of antitumor type 2 CD8(+) effector T cells along with IL-4 production. B7.2-Ig-mediated regression was not induced in IL-4(-/-) and STAT6(-/-) mice. However, it was elicited in IFN-gamma(-/-) and STAT4(-/-) mice. By contrast, IL-12-induced tumor regression occurred in IL-4(-/-) and STAT6(-/-) mice, but not in IFN-gamma(-/-) and STAT4(-/-) mice. Moreover, B7.2-Ig treatment was effective in a tumor model not responsive to IL-12. B7.2-Ig administration elicited elevated levels of IL-4 production. Tumor regression was predominantly mediated by CD8(+) T cells, although the induction of these effector cells required CD4(+) T cells. Tumor regression induced by CD8(+) T cells alone was inhibited by neutralizing the IL-4 produced during B7.2-Ig treatment. Thus, these results indicate that stimulation in vivo of CD28 with B7.2-Ig in tumor-bearing mice results in enhanced induction of antitumor type 2 CD8(+) T cells (Tc2) leading to Tc2-mediated tumor regression.     

Divergent roles for CD4+ T cells in the priming and effector/memory phases of adoptive immunotherapy

The requirement for CD4(+) Th cells in the cross-priming of antitumor CTL is well accepted in tumor immunology. Here we report that the requirement for T cell help can be replaced by local production of GM-CSF at the vaccine site. Experiments using mice in which CD4(+) T cells were eliminated, either by Ab depletion or by gene knockout of the MHC class II beta-chain (MHC II KO), revealed that priming of therapeutic CD8(+) effector T cells following vaccination with a GM-CSF-transduced B16BL6-D5 tumor cell line occurred independently of CD4(+) T cell help. The adoptive transfer of CD8(+) effector T cells, but not CD4(+) effector T cells, led to complete regression of pulmonary metastases. Regression of pulmonary metastases did not require either host T cells or NK cells. Transfer of CD8(+) effector T cells alone could cure wild-type animals of systemic tumor; the majority of tumor-bearing mice survived long term after treatment (>100 days). In contrast, adoptive transfer of CD8(+) T cells to tumor-bearing MHC II KO mice improved survival, but eventually all MHC II KO mice succumbed to metastatic disease. WT mice cured by adoptive transfer of CD8(+) T cells were resistant to tumor challenge. Resistance was mediated by CD8(+) T cells in mice at 50 days, while both CD4(+) and CD8(+) T cells were important for protection in mice challenged 150 days following adoptive transfer. Thus, in this tumor model CD4(+) Th cells are not required for the priming phase of CD8(+) effector T cells; however, they are critical for both the complete elimination of tumor and the maintenance of a long term protective antitumor memory response in vivo.     

Potent tumor-specific protection ignited by adoptively transferred CD4+ T cells

Administration of anti-CD25 mAb before an aggressive murine breast tumor inoculation provoked effective antitumor immunity. Compared with CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that did not reject tumor, CD4(+) T cells purified from anti-CD25 mAb-pretreated mice that rejected tumor stimulated by dendritic cells (DCs) produced more IFN-gamma and IL-2, and less IL-17 in vitro, and ignited protective antitumor immunity in vivo in an adoptive transfer model. Tumor Ag-loaded DCs activated naive CD8(+) T cells in the presence of these CD4(+) T cells in vitro. Tumor Ag and adoptively transferred CD4(+) T cells were both required for inducing a long-term tumor-specific IFN-gamma-producing cellular response and potent protective antitumor activity. Although adoptively transferred CD4(+) T cells ignited effective tumor-specific antitumor immunity in wild-type mice, they failed to do so in endogenous NK cell-depleted, Gr-1(+) cell-depleted, CD40(-/-), CD11c(+) DC-depleted, B cell(-/-), CD8(+) T cell-depleted, or IFN-gamma(-/-) mice. Collectively, the data suggest that adoptively transferred CD4(+) T cells orchestrate both endogenous innate and adaptive immunity to generate effective tumor-specific long-term protective antitumor immunity. The data also demonstrate the pivotal role of endogenous DCs in the tumor-specific protection ignited by adoptively transferred CD4(+) T cells. Thus, these findings highlight the importance of adoptively transferred CD4(+) T cells, as well as host immune components, in generating effective tumor-specific long-term antitumor activity.     

Role of effector cell-derived IL-4, IL-5, and perforin in early and late stages of type 2 CD8 effector cell-mediated tumor rejection.

Type 2 CD8 T cells (Tc2) secrete IL-4 and IL-5 and display perforin-dependent cytolysis in vitro. Using an OVA-transfected B16-melanoma model, we show that tumor-reactive Tc2 effector cells accumulated at the tumor site and induced tumor regression that enhanced survival in mice with pulmonary tumors. Transfer of perforin-deficient Tc2 cells generated from perforin gene knockout mice showed no differences in therapeutic efficiency when compared with wild-type Tc2 cells. In contrast, Tc2 cells derived from select cytokine gene-deficient mice showed that therapeutic effects were dependent on effector cell-derived IL-4 and IL-5 that led to a local elevation in lung-derived chemoattractants and accumulation of activated host-derived CD8/CD44(high), CD4/CD44(high), and OVA-specific tetramer-positive CD8 cells in vivo. Host-derived T and non-T immune cells increased in the lung over time and correlated with an elevated production of type 1-related chemokines. Conversely, donor Tc2 cell numbers markedly diminished at later times, suggesting that prolonged therapeutic responses were due to host-derived mechanisms. Moreover, type 1 host responses were detectable with increased levels of IFN-gamma production by lung-derived CD4 and CD8 T cells from surviving Tc2-treated mice. Transfer of Tc2 cells into IFN-gamma-deficient tumor-bearing mice was markedly less effective then into wild-type mice, suggesting that host-derived IFN-gamma-dependent mechanisms play a role in Tc2-mediated antitumor responses.     

Accumulation of immunosuppressive CD11b+ myeloid cells correlates with the failure to prevent tumor growth in the anterior chamber of the eye

The purpose of these studies is to determine why an immunogenic tumor grows unchecked in the anterior chamber (a.c.) of the eye. The OVA-expressing EL4 tumor, E.G7-OVA, was injected into the a.c. or skin of immunocompetent and immunodeficient mice. Tumor growth and tumor-specific immune responses were monitored. Ocular tumor-infiltrating leukocytes were characterized phenotypically and functionally. Growth of E.G7-OVA was inhibited when limiting numbers of cells were injected in the skin but not in the a.c. of C57BL/6 mice, although both routes primed OVA-specific immune responses, which prevented the growth of a subsequent injection with E.G7-OVA in the skin or opposite eye. Tumor regression was OVA-specific because growth of the parental EL-4 tumor was not inhibited in primed mice. E.G7-OVA growth in the skin was not inhibited in immunodeficient Rag(-/-) or CD8 T cell-deficient mice, suggesting that CD8(+) CTLs mediate tumor elimination. CD8(+) T cell numbers were significantly increased in eyes of mice primed with E.G7-OVA, but few were detected in primary ocular tumors. Nevertheless, growth of E.G7-OVA was retarded in the a.c. of TCR-transgenic OT-I mice, and CD8(+) T cell numbers were increased within eyes, suggesting that tumor-specific CD8(+) CTLs migrated into and controlled primary ocular tumor growth. E.G7-OVA did not lose antigenicity or become immunosuppressive after 13 days of growth in the eye. However, CD11b(+) cells accumulated in primary ocular tumors and contained potent immunosuppressive activity when assayed in vitro. Thus, CD11b(+) cells that accumulate within the eye as tumors develop in the a.c. may contribute to immune evasion by primary ocular tumors by inhibiting CTLs within the eye.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Tumor-associated myeloid cells can be activated in vitro and in vivo to mediate antitumor effects

Tumor growth is often accompanied by the accumulation of myeloid cells in the tumors and lymphoid organs. These cells can suppress T cell immunity, thereby posing an obstacle to T cell-targeted cancer immunotherapy. In this study, we tested the possibility of activating tumor-associated myeloid cells to mediate antitumor effects. Using the peritoneal model of B16 melanoma, we show that peritoneal cells (PEC) in tumor-bearing mice (TBM) had reduced ability to secrete nitric oxide (NO) following in vitro stimulation with interferon gamma and lipopolysaccharide, as compared to PEC from control mice. This reduced function of PEC was accompanied by the influx of CD11b(+) Gr-1(+) myeloid cells to the peritoneal cavity. Nonadherent PEC were responsible for most of the NO production in TBM, whereas in na?ve mice NO was mainly secreted by adherent CD11b(+) F4/80(+) macrophages. Sorted CD11b(+) Gr-1(-) monocytic and CD11b(+) Gr-1(+) granulocytic PEC from TBM had a reduced ability to secrete NO following in vitro stimulation (compared to na?ve PEC), but effectively suppressed proliferation of tumor cells in vitro. In vivo, treatment of mice bearing established peritoneal B16 tumors with anti-CD40 and CpG resulted in activation of tumor-associated PEC, reduction in local tumor burden and prolongation of mouse survival. Inhibition of NO did not abrogate the antitumor effects of stimulated myeloid cells. Taken together, the results indicate that in tumor-bearing hosts, tumor-associated myeloid cells can be activated to mediate antitumor effects.     

Immune mechanism of the antitumor effects generated by bortezomib

Bortezomib, a proteasome inhibitor, is a chemotherapeutic drug that is commonly used to treat a variety of human cancers. The antitumor effects of bortezomib-induced tumor cell immunogenicity have not been fully delineated. In this study, we examined the generation of immune-mediated antitumor effects in response to treatment by bortezomib in a murine ovarian tumor model. We observed that tumor-bearing mice that were treated with bortezomib had CD8(+) T cell-mediated inhibition of tumor growth. Furthermore, the comparison of tumor cell-based vaccines that were produced from tumor cells treated or untreated with bortezomib showed vaccination with drug-treated tumor cell-based vaccines elicited potent tumor-specific CD8(+) T cell immune response with improved therapeutic antitumor effect in tumor-bearing mice. Conversely, the untreated tumor cell-based vaccines led to no appreciable antitumor response. Treatment of tumor cells with bortezomib led to the upregulation of Hsp60 and Hsp90 on the cell surface and promoted their phagocytosis by dendritic cells (DCs). However, cell surface expression of Hsp60, instead of Hsp90, is the more important determinant of whether bortezomib-treated tumor cells can generate tumor-specific CD8(+) T cells. CD11c(+) DCs that were treated with bortezomib in vitro had enhanced phagocytic activities. In addition, CD11c(+) DCs from bortezomib-treated tumor-bearing mice had increased maturation. At lower concentrations, bortezomib had no inhibitory effects on T cell proliferation. Taken together, our data indicate that bortezomib can render tumor cells immunogenic by upregulating the cell surface expression of heat shock protein 60 and heat shock protein 90, as well as improve DC function, which results in potent immune-mediated antitumor effects.     

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood. We administered non-Ag-loaded, immature dendritic cells (DC) to CD8alpha knockout (KO) mice and studied their anti-CNS tumor immune responses. DC administration induced dramatic antitumor immune protection in CD8alpha KO mice that were challenged with B16 melanoma both s.c. and in the brain. The CNS antitumor immunity was dependent on both CD4+ T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4+ T cell and NK cell expansion in the draining lymph nodes at 6 days postvaccination, which persisted for 2 wk. Finally, DC administration in CD8alpha KO mice was associated with robust infiltration of CD4+ T cells and NK cells into the brain tumor parenchyma. These results represent the first demonstration of a potent innate antitumor immune response against CNS tumors in the absence of toxicity. Thus, non-Ag-loaded, immature DC administration, in the setting of CD8 genetically deficient mice, can induce dramatic antitumor immune responses within the CNS that surpass the effects observed in wild-type mice. Our results suggest that a better understanding of the cross-talk between DC and innate immune cells may provide improved methods to vaccinate patients with tumors located both systemically and within the CNS.     

Pivotal roles of CD4+ effector T cells in mediating agonistic anti-GITR mAb-induced-immune activation and tumor immunity in CT26 tumors

Glucocorticoid-induced TNF receptor family related protein (GITR) is a member of the TNFR superfamily. Previous studies have shown that in vivo administration of a GITR agonistic Ab (DTA-1) is able to overcome tolerance and induce tumor rejection in several murine syngeneic tumor models. However, little is known about the in vivo targets and the mechanisms of how this tolerance is overcome in a tumor-bearing host, nor is much known about how the immune network is regulated to achieve this antitumor response. In this study, we demonstrate that the in vivo ligation of GITR on CD4(+) effector T cells renders them refractory to suppression by regulatory T (T(reg)) cells in the CT26 tumor-bearing mouse. GITR engagement on T(reg) cells does not appear to directly abrogate their suppressive function; rather, it increases the expansion of T(reg) cells and promotes IL-10 production, a cytokine important for their suppressive function. Moreover, CD4(+) effector T cells play a crucial role in mediating DTA-1-induced immune activation and expansion of CD8(+), NK, and B cells in the tumor-draining lymph nodes. This includes increased CD69 expression on all of these subsets. In addition, NK and tumor-specific CD8(+) T cells are generated that are cytolytic, which show increased intracellular IFN-gamma production and CD107a mobilization, the latter a hallmark of cytolytic activities that lead to tumor killing.     

Synergistic anti-tumor responses after administration of agonistic antibodies to CD40 and IL-2: coordination of dendritic and CD8+ cell responses

In cancer, the coordinate engagement of professional APC and Ag-specific cell-mediated effector cells may be vital for the induction of effective antitumor responses. We speculated that the enhanced differentiation and function of dendritic cells through CD40 engagement combined with IL-2 administration to stimulate T cell expansion would act coordinately to enhance the adaptive immune response against cancer. In mice bearing orthotopic metastatic renal cell carcinoma, only the combination of an agonist Ab to CD40 and IL-2, but neither agent administered alone, induced complete regression of metastatic tumor and specific immunity to subsequent rechallenge in the majority of treated mice. The combination of anti-CD40 and IL-2 resulted in significant increases in dendritic cell and CD8(+) T cell number in advanced tumor-bearing mice compared with either agent administered singly. The antitumor effects of anti-CD40 and IL-2 were found to be dependent on CD8(+) T cells, IFN-gamma, IL-12 p40, and Fas ligand. CD40 stimulation and IL-2 may therefore be of use to promote antitumor responses in advanced metastatic cancer.     

Rescue of tumor-infiltrating lymphocytes from activation-induced cell death enhances the antitumor CTL response in CD5-deficient mice

The CD5 coreceptor is expressed on all T cells and on the B1a B cell subset. It is associated with TCR and BCR, and modulates intracellular signals initiated by both Ag receptor complexes. Human CD5 contributes to regulation of the antitumor immune response and susceptibility of specific CTL to activation-induced cell death (AICD) triggered by the tumor. In this study, we compared the T cell response to the B16F10 melanoma engrafted into CD5-deficient and wild-type C57BL/6 mice. Compared with wild-type mice, CD5 knockout animals displayed delayed tumor growth, associated with tumor infiltration by T cell populations exhibiting a more activated phenotype and enhanced antitumor effector functions. However, control of tumor progression in CD5(-/-) mice was transient due to increased AICD of CD8(+) tumor-infiltrating T lymphocytes. Remarkably, in vivo protection of T cells from TCR-mediated apoptosis by an adenovirus engineered to produce soluble Fas resulted in a dramatic reduction in tumor growth. Our data suggest that recruitment of tumor-specific T cells in the tumor microenvironment occurs at early stages of cancer development and that tumor-mediated AICD of tumor-infiltrating T lymphocytes is most likely involved in tumor escape from the immune system.     

Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models

BACKGROUND: Some anaerobic and facultatively anaerobic bacteria have been used experimentally as anticancer agents because of their selective growth in tumors. In this study, we exploited attenuated Salmonella choleraesuis as a tumoricidal agent and a vector to deliver the endostatin gene for tumor-targeted gene therapy. METHODS: Attenuated S. choleraesuis carrying a eukaryotic expression plasmid encoding reporter gene was used to evaluate its abilities of tumor targeting and gene delivery in three syngeneic murine tumor models. Furthermore, S. choleraesuis carrying the endostatin expression vector was administered intraperitoneally into tumor-bearing mice, and its antitumor effect was evaluated. RESULTS: Systemically administered S. choleraesuis preferentially accumulated within tumors for at least 10 days, forming tumor-to-normal tissue ratios exceeding 1000-10,000 : 1. Transgene expression via S. choleraesuis-mediated gene transfer also persisted for at least 10 days. Host immune responses and tumor hypoxia may influence tumor-targeting potential of S. choleraesuis. When systemically administered into mice bearing melanomas or bladder tumors, S. choleraesuis carrying the endostatin expression vector significantly inhibited tumor growth by 40-70% and prolonged survival of the mice. Furthermore, immunohistochemical studies in the tumors revealed decreased intratumoral microvessel density, reduced expression of vascular endothelial growth factor (VEGF), and increased infiltration of CD8(+) T cells. CONCLUSIONS: These results suggest that tumor-targeted gene therapy using S. choleraesuis carrying the endostatin expression vector, which exerts tumoricidal and antiangiogenic activities, represents a promising strategy for the treatment of solid tumors.     

CD11b+/Gr-1+ immature myeloid cells mediate suppression of T cells in mice bearing tumors of IL-1beta-secreting cells

Tumor cells secreting IL-1beta are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1beta-secreting cells restored immune reactivity and hematological alterations within 7-10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1beta-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1beta, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.     

Tumor-induced CD11b+Gr-1+ myeloid cells suppress T cell sensitization in tumor-draining lymph nodes

Suppression of tumor-specific T cell sensitization is a predominant mechanism of tumor escape. To identify tumor-induced suppressor cells, we transferred spleen cells from mice bearing progressive MCA205 sarcoma into sublethally irradiated mice. These mice were then inoculated subdermally with tumor cells to stimulate T cell response in the tumor-draining lymph-node (TDLN). Tumor progression induced splenomegaly with a dramatic increase (22.1%) in CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSC) compared with 2.6% of that in normal mice. Analyses of therapeutic effects by the adoptive immunotherapy revealed that the transfer of spleen cells from tumor-bearing mice severely inhibited the generation of tumor-immune T cells in the TDLN. We further identified MDSC to be the dominant suppressor cells. However, cells of identical phenotype from normal spleens lacked the suppressive effects. The suppression was independent of CD4(+)CD25(+) regulatory T cells. Intracellular IFN-gamma staining revealed that the transfer of MDSC resulted in a decrease in numbers of tumor-specific CD4(+) and CD8(+) T cells. Transfer of MDSC from MCA207 tumor-bearing mice also suppressed the MCA205 immune response indicating a lack of immunologic specificity. Further analyses demonstrated that MDSC inhibited T cell activation that was triggered either by anti-CD3 mAb or by tumor cells. However, MDSC did not suppress the function of immune T cells in vivo at the effector phase. Our data provide the first evidence that the systemic transfer of MDSC inhibited and interfered with the sensitization of tumor-specific T cell responses in the TDLN.     

Single administration of low dose cyclophosphamide augments the antitumor effect of dendritic cell vaccine

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-γ secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (T_reg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ T_reg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine. J.-Y. Liu and Y. Wu contributed equally to this work.     

VEGF-C promotes immune tolerance in B16 melanomas and cross-presentation of tumor antigen by lymph node lymphatics

Tumor expression of the lymphangiogenic factor VEGF-C is correlated with metastasis and poor prognosis, and although VEGF-C enhances transport to the draining lymph node (dLN) and antigen exposure to the adaptive immune system, its role in tumor immunity remains unexplored. Here, we demonstrate that VEGF-C promotes immune tolerance in murine melanoma. In B16 F10 melanomas expressing a foreign antigen (OVA), VEGF-C protected tumors against preexisting antitumor immunity and promoted local deletion of OVA-specific CD8(+) T cells. Naive OVA-specific CD8(+) T cells, transferred into tumor-bearing mice, were dysfunctionally activated and apoptotic. Lymphatic endothelial cells (LECs) in dLNs cross-presented OVA, and naive LECs scavenge and cross-present OVA in vitro. Cross-presenting LECs drove the proliferation and apoptosis of OVA-specific CD8(+) T cells ex vivo. Our findings introduce a tumor-promoting role for lymphatics in the tumor and dLN and suggest that lymphatic endothelium in the local microenvironment may be a target for immunomodulation.     

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.) injection of IL-12 (0.25 microg/mouse) from day 18 to day 24 for a total of 7 doses. Their splenocytes were obtained on Day 31 for natural killer cells (NK), lymphokine activated killer cells (LAK) and cytotoxic T lymphocyte (CTL) activity assay and lymphocyte subsets phenotypic analysis. The tumor suppression effect of systemic IL-12 administration was evaluated based on the tumor outgrowth of the higher number of tumor cells rechallenged 24 hours after resectioning of the primary tumor. After evaluation on Day 31, the result of in vitro cytotoxicity assay revealed that systemic administration of IL-12 mainly enhanced the splenic LAK and CTL activities in non-tumor-primed naive mice, and the NK activity in tumor-primed D17TBM, respectively. However, in vivo administration of IL-12 with or without IL-2 failed to upgrade the proportions of either CD4+ CD44+ or CD8+ CD44+ T cells subsets in the spleens and regional inguinal lymph nodes (LNs) of both the D17TBM and naive mice. However, the splenic CD8+ CD44+ T-cell subset in the IL-12-treated D17TBM increased prominently after further culturing in the presence of IL-2 400 units/ml plus IL-12 25 ng/ml for 4 days. The fact that systemic administration of IL-12 significantly suppressed the outgrowth of Day-18 challenged tumor cells, especially in D17TBM, clearly indicates that the established specific antitumor immunity in tumor-primed D17TBM was efficiently augmented. From the results of this study, we conclude that, after surgical resection of a primary tumor, systemic administration of IL-12 can be an effective adjuvant therapy because it demonstrates a significant augmentation effect on the tumor-specific immune response in the tumor-primed host.