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Tumor Regulatory T Cells Potently Abrogate Antitumor Immunity

Regulatory T cell (Treg) from mice bearing a breast tumor were elevated (tumor Treg). In vitro, whereas tumor Treg ability to inhibit tumor-primed CD4+ T cell activity is comparable to Treg from naive mice (naive Treg), only tumor Treg suppress naive CD8+ T cell activation and DC function. Neither tumor Treg nor naive Treg can suppress antitumor immunity at the effector phase of the immune response induced by adoptively transferred tumor-primed CD4+ T cells. This is consistent with the observation that, in this model, neither tumor Treg nor naive Treg can inhibit effectors in vitro or in vivo. However, tumor Treg abrogate tumor-specific CD8+ T cell responses in tumor-draining lymph nodes and antitumor immunity at the early stage of the immune response induced by adoptively transferred tumor-primed CD4+ T cells. These data indicate that, in this model, tumor Treg potently abrogate tumor-specific CD8+ T cell responses in tumor-draining lymph nodes, thereby suppressing antitumor immunity at the early stage of the immune response induced by adoptively transferred tumor-primed CD4+ T cells.

Targeting the Primary Tumor to Generate CTL for the Effective Eradication of Spontaneous Metastases

Metastatic disease is the major cause of morbidity and mortality in cancer. Although surgery, chemotherapy, or radiation can often control primary tumor growth, successful eradication of disseminated metastases remains rare. We have now tested whether direct targeting tumor tissues to generate antitumor immune response before surgical excision produces sufficient CTL against micrometastases. One unsolved problem is whether such response allows coming CTL to be educated and then exit the tumor site. Another unsolved problem is whether these CTL can then patrol and effectively eliminate spontaneously metastasized tumor cells in the periphery. In this study, we have shown that adenovirus-expressing TNFSF14 [LIGHT (name derived from homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes); Ad-LIGHT] inoculated directly into primary 4T1 tumor, a highly aggressive, spontaneously metastasizing mammary carcinoma, followed by surgical removal of the primary tumor can eradicate established and disseminated metastatic tumor cells in the peripheral tissues. Furthermore, we clearly show with a fibrosarcoma model Ag104Ld that local treatment can generate plenty of tumor-specific CTL that exit the primary tumor and infiltrate distal tumors to completely eradicate distal tumors. Therefore, targeting the primary tumor with Ad-LIGHT before surgical excision is a new strategy to elicit better immune response for the eradication of spontaneous metastases.

Knockdown of HMGB1 in Tumor Cells Attenuates Their Ability To Induce Regulatory T Cells and Uncovers Naturally Acquired CD8 T Cell-Dependent Antitumor Immunity

Although high mobility group box 1 (HMGB1) in tumor cells is involved in many aspects of tumor progression, its role in tumor immune suppression remains elusive. Host cell-derived IL-10 suppressed a naturally acquired CD8 T cell-dependent antitumor response. The suppressive activity of tumor-associated Foxp3+CD4+CD25+ regulatory T cells (Treg) was IL-10 dependent. Neutralizing HMGB1 impaired tumor cell-promoted IL-10 production by Treg. Short hairpin RNA-mediated knockdown of HMGB1 (HMGB1 KD) in tumor cells did not affect tumor cell growth but uncovered naturally acquired long-lasting tumor-specific IFN-γ– or TNF-α–producing CD8 T cell responses and attenuated their ability to induce Treg, leading to naturally acquired CD8 T cell- or IFN-γ–dependent tumor rejection. The data suggest that tumor cell-derived HMGB1 may suppress naturally acquired CD8 T cell-dependent antitumor immunity via enhancing Treg to produce IL-10, which is necessary for Treg-mediated immune suppression.

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-γ 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-γ-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-γ−/− 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.

'Survival gene' Bcl-xl potentiates DNA-raised antitumor immunity

T-cell priming is strongly affected by the longevity of antigen-bearing dendritic cells (DCs), which are typically short-lived in lymphoid tissues. ‘Survival gene’ Bcl-xl is critical for the lifespan of DCs in vivo. Here, we showed that in vivo coadministration of Bcl-xl under control of the DC-specific promoter (CD11c-Bcl-xl) and TRP2hsp70 DNA prolonged T-cell stimulation by DCs and augmented TRP2-specific-IFN-γ-producing CD8+ T-cell responses. Consistent with these findings, enhanced protection and significant therapeutic immunity to B16 melanoma was generated by this coimmunization strategy, which also augmented therapeutic immunity to GL-26 tumor. In this B16 melanoma model, results from animal experiments with depletion of immune cells indicate that CD8+ T cells and NK cells are important in the antitumor immunity induced by this coimmunization strategy. These observations suggest that ‘survival gene’ Bcl-xl potentiates the magnitude of antigen-specific-CD8+ T-cell responses and the efficacy of antitumor immunity induced by DNA vaccine, and is relevant for the design of in vivo targeted DC-based vaccine strategies to improve immunity against cancer.

Therapeutic immunity by adoptive tumor-primed CD4(+) T-cell transfer in combination with in vivo GITR ligation.

Tumor-primed CD4+ T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8+ T cell-dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4+ T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4+ T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-γ-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Tregs ability to suppress IL-12 production by DCs and IFN-γ production by effectors ex vivo. Importantly, tumor-primed CD4+ CD25- T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.Tumor-primed CD4(+) T cells from splenocytes of tumor-rejection mice in combination with in vivo glucocorticoid-induced tumor necrosis factor receptor (GITR) ligation (the combination therapy) elicited effective host CD8(+) T cell-dependent therapeutic immunity against a murine breast tumor. GITR ligation in vitro enhanced tumor-primed CD4(+) T-cell activity and partially abrogated regulatory T cells (Treg) suppressor function. Dendritic cells (DCs) from tumor-draining lymph nodes (TDLNs) of tumor-bearing mice treated by the combination therapy stimulated Ag-specific T cells and produced interleukin (IL)-12 ex vivo. Whereas tumor-primed CD4(+) T cells or in vivo GITR ligation alone induced a tumor-specific interferon (IFN)-gamma-producing cellular response, the combination therapy enhanced and sustained it. Furthermore, the combination therapy in vivo attenuated Tregs ability to suppress IL-12 production by DCs and IFN-gamma production by effectors ex vivo. Importantly, tumor-primed CD4(+) CD25(-) T cells from splenocytes of untreated tumor-bearing mice in combination with in vivo GITR ligation also elicited an effective therapeutic effect in this model. These data suggest that the combination therapy may improve DC function, accentuate tumor-specific T-cell responses, and attenuate Treg suppressor function, thereby eliciting effective therapeutic immunity.

Genetic Vaccines To Potentiate the Effective CD103+ Dendritic Cell–Mediated Cross-Priming of Antitumor Immunity

The development of effective cancer vaccines remains an urgent, but as yet unmet, clinical need. This deficiency is in part due to an incomplete understanding of how to best invoke dendritic cells (DC) that are crucial for the induction of tumor-specific CD8+ T cells capable of mediating durable protective immunity. In this regard, elevated expression of the transcription factor X box–binding protein 1 (XBP1) in DC appears to play a decisive role in promoting the ability of DC to cross-present Ags to CD8+ T cells in the therapeutic setting. Delivery of DNA vaccines encoding XBP1 and tumor Ag to skin DC resulted in increased IFN-α production by plasmacytoid DC (pDC) from skin/tumor draining lymph nodes and the cross-priming of Ag-specific CD8+ T cell responses associated with therapeutic benefit. Antitumor protection was dependent on cross-presenting Batf3+ DC, pDC, and CD8+ T cells. CD103+ DC from the skin/tumor draining lymph nodes of the immunized mice appeared responsible for activation of Ag-specific naive CD8+ T cells, but were dependent on pDC for optimal effectiveness. Similarly, human XBP1 improved the capacity of human blood- and skin-derived DC to activate human T cells. These data support an important intrinsic role for XBP1 in DC for effective cross-priming and orchestration of Batf3+ DC–pDC interactions, thereby enabling effective vaccine induction of protective antitumor immunity.

Dendritic cell-derived interleukin-15 is crucial for therapeutic cancer vaccine potency.

IL-15 supports improved antitumor immunity. How to best incorporate IL-15 into vaccine formulations for superior cancer immunotherapy remains a challenge. DC-derived IL-15 (DCIL-15) notably has the capacity to activate DC, to substitute for CD4+ Th and to potentiate vaccine efficacy making IL-15-based therapies attractive treatment options. We observed in transplantable melanoma, glioma and metastatic breast carcinoma models that DCIL-15-based DNA vaccines in which DC specifically express IL-15 and simultaneously produce tumor Aghsp70 were able to mediate potent therapeutic efficacy that required both host Batf3+ DC and CD8+ T cells. In an inducible BrafV600E/Pten-driven murine melanoma model, DCIL-15 (not rIL-15)-based DNA vaccines elicited durable therapeutic CD8+ T cell-dependent antitumor immunity. DCIL-15 was found to be superior to rIL-15 in “licensing” both mouse and human DC, and for activating CD8+ T cells. Such activation occurred even in the presence of Treg, without a need for CD4+ Th, but was IL-15/IL-15Rα-dependent. A single low-dose of DCIL-15 (not rIL-15)-based DC vaccines induced therapeutic antitumor immunity. CD14+ DC emigrating from human skin explants genetically-immunized by IL-15 and Aghsp70 were more effective than similar DC emigrating from the explants genetically-immunized by Aghsp70 in the presence of rIL-15 in expressing membrane-bound IL-15/IL-15Rα and activating CD8+ T cells. These results support future clinical use of DCIL-15 as a therapeutic agent in battling cancer.

Transcriptional IL-15-directed in vivo DC targeting DNA vaccine

Dendritic cells (DC) engineered in vitro by DNA encoding OVAhsp70 and IL-15 up-regulated their expressions of CD80, CD86, CCR7 and IL-15Rα and promoted their productions of IL-6, IL-12 and TNF-α. Transcriptional IL-15-directed in vivo DC targeting DNA vaccine encoding OVAhsp70 elicited long-lasting Th1 and CTL responses and anti-B16OVA activity. CD8T cell-mediated primary tumor protection was abrogated by DC or CD4T cell depletion during the induction phase of immune responses. However, CD4T cell depletion during immunization did not impair CD8T cell-dependent long-lasting tumor protection. Furthermore, in vivo DC-derived IL-15 exerted the enhancements of cellular and humoral immune responses and antitumor immunity elicited by OVAhsp70 DNA vaccine. Importantly, the potency of this novel DNA vaccine strategy was proven using a self/tumor Ag (TRP2) in a clinically relevant B16 melanoma model. These findings have implications for developing next generation DNA vaccines against cancers and infectious diseases in both healthy and CD4 deficient individuals.

Extranodal induction of therapeutic immunity in the tumor microenvironment after intratumoral delivery of Tbet gene-modified dendritic cells

Murine dendritic cells (DC) transduced to express the Type-1 transactivator T-bet (i.e. mDC.Tbet) and delivered intratumorally as a therapy are superior to control wild-type DC in slowing the growth of established subcutaneous MCA205 sarcomas in vivo. Optimal antitumor efficacy of mDC.Tbet-based gene therapy was dependent on host natural killer (NK) cells and CD8+ T cells, and required mDC.Tbet expression of major histocompatibility complex class I molecules, but was independent of the capacity of the injected mDC.Tbet to produce proinflammatory cytokines (interleukin-12 family members or interferon-γ) or to migrate to tumor-draining lymph nodes based on CCR7 ligand chemokine recruitment. Conditional (CD11c-DTR) or genetic (BATF3−/−) deficiency in host antigen-crosspresenting DC did not diminish the therapeutic action of intratumorally delivered wild-type mDC.Tbet. Interestingly, we observed that intratumoral delivery of mDC.Tbet (versus control mDC.Null) promoted the acute infiltration of NK cells and naive CD45RB+ T cells into the tumor microenvironment (TME) in association with elevated expression of NK- and T-cell-recruiting chemokines by mDC.Tbet. When taken together, our data support a paradigm for extranodal (cross)priming of therapeutic Type-1 immunity in the TME after intratumoral delivery of mDC.Tbet-based gene therapy.