A2A Adenosine Receptor Gene Deletion or Synthetic A2A Antagonist Liberate Tumor-Reactive CD8+ T Cells from Tumor-Induced Immunosuppression.

Tumor hypoxia-driven accumulation of extracellular adenosine was shown to facilitate tumor evasion by engaging the immunosuppressive, intracellular cAMP-elevating A2 adenosine receptors (A2R) on tumor-reactive effector T cells, but there remains a need for careful evaluation of the limiting factors and properties of A2R blockade-enabled antitumor immunity. In studies of A2AR and/or A2BR gene-deficient mice, we found that A2AR deletion-but not A2BR deletion-liberates endogenous CD8+ T cell antitumor immunity against weakly immunogenic MCA205 sarcomas. Studies of adoptively transferred A2AR-/-, A2BR-/-, or A2AR-/-/A2BR-/- tumor-reactive T cells confirmed that immunosuppression in the tumor microenvironment was mediated by A2AR on CD8+ T cells. Treatment with A2AR antagonist mimicked A2AR gene deletion in adoptive T cell immunotherapy. This therapeutic benefit of targeting A2AR was independent of the anatomical location of tumor growth. The enhanced antitumor reactivity also led to the eradication of established intracranial tumors, which was associated with mouse survival and the maintenance of long-lasting, tumor-specific immunological memory. The blockade of the A2AR on adoptively transferred T cells by synthetic A2AR antagonist led to higher levels of IFN-γ secretion by tumor-infiltrating CD8+ T cells. These data clarify the mechanism of hypoxia-driven immunosuppression in the tumor microenvironment by A2AR on tumor-reactive CD8+ T cells and show that selective A2AR antagonists can be effective in improving the outcomes of T cell-based immunotherapies. Demonstration of the T cell dose dependency of tumor rejection points to a major limitation of current cancer immunotherapies, in which the presence of sufficient numbers of tumor-reactive T cells in a patient is not known.

Hypoxia-adenosinergic immunosuppression: tumor protection by T regulatory cells and cancerous tissue hypoxia.

Cancerous tissue protection from tumor-recognizing CD8(+) and CD4(+) T cells (antitumor T cells) limits the therapeutic potential of immunotherapies. We propose that tumor protection is to a large extent due to (a) inhibition of antitumor T cells by hypoxia-driven accumulation of extracellular adenosine in local tumor microenvironment and due to (b) T regulatory cell-produced extracellular adenosine. The adenosine triggers the immunosuppressive signaling via intracellular cyclic AMP-elevating A2A adenosine receptors (A2AR) on antitumor T cells. In addition, the activated antitumor T cells in hypoxic tumor microenvironment could be inhibited by elevated levels of immunosuppressive hypoxia-inducible factor-1alpha. Complete rejection or tumor growth retardation was observed when A2AR has been genetically eliminated or antagonized with synthetic drug or with natural A2AR antagonist 1,3,7-trimethylxanthine (caffeine). The promising strategy may be in combining the anti-hypoxia-adenosinergic treatment that prevents inhibition of antitumor T cells by tumor-produced and T regulatory cell-produced adenosine with targeting of other negative regulators, such as CTL antigen-4 blockade. Observations of tumor rejection in mice and massive prospective epidemiologic studies support the feasibility of anti-hypoxia-adenosinergic combined immunotherapy.

Everolimus (RAD) inhibits in vivo growth of murine squamous cell carcinoma (SCC VII).

OBJECTIVE: Everolimus (RAD) is an mTOR inhibitor closely related to rapamycin. A potent immunosuppressive agent, it has also shown evidence of antineoplastic properties. SCC VII is a spontaneously arising murine squamous cell carcinoma line. This study examines the effect of everolimus on SCC VII proliferation. The data may provide support for the use of everolimus in transplant recipients with a history of malignancy. METHODS: A dose efficacy study was conducted that used a murine model of intradermal tumor growth and pulmonary metastases. The development of intradermal tumors and pulmonary metastases were studied. Of 80 total mice, 40 received intradermal injection of 1 x 10 SCC VII cells and 40 received intravenous injection of 1 x 10 cells to establish pulmonary metastases. Within each group, animals were subdivided into four subgroups that received 1) 1 mg/kg everolimus twice a day, 2) 0.5 mg/kg everolimus twice a day, 3) 7.5 mg/kg cyclosporine per day, and 4) no treatment. Intradermal tumors were measured three times per week. Animals receiving an intravenous tumor injection were killed after 17 days and pulmonary metastases were quantified. Medication trough levels were measured in all treated animals. RESULTS: Everolimus showed statistically significant tumor inhibition at 1.0 mg/kg twice a day and 0.5 mg/kg twice a day when compared with animals treated with cyclosporine and with untreated animals (P < .0001). Tumor inhibition was evident in both models studied (intradermal tumors and pulmonary metastasis generation). CONCLUSIONS: Everolimus provides potent tumor inhibition in animals inoculated with SCC VII cells. Inhibition of both local and distant spread of disease is evident. Although most immunosuppressives are known to potentiate neoplastic disease, this study supports the use of everolimus immunosuppression in the face of prior malignancy. This data has significant implication for laryngeal transplantation after laryngectomy.

Active immunotherapy for advanced intracranial murine tumors by using dendritic cell-tumor cell fusion vaccines.

OBJECT: Immunotherapy for malignant brain tumors by active immunization or adoptive transfer of tumor antigen-specific T lymphocytes has the potential to make up for some of the limitations of current clinical therapy. In this study, the authors tested whether active immunotherapy is curative in mice bearing advanced, rapidly progressive intracranial tumors. METHODS: Tumor vaccines were created through electrofusion of dendritic cells (DCs) and irradiated tumor cells to form multinucleated heterokaryons that retained the potent antigen processing and costimulatory function of DCs as well as the entire complement of tumor antigens. Murine hosts bearing intracranial GL261 glioma or MCA 205 fibrosarcoma were treated with a combination of local cranial radiotherapy, intrasplenic vaccination with DC/tumor fusion cells, and anti-OX40R (CD134) monoclonal antibody (mAb) 7 days after tumor inoculation. Whereas control mice had a median survival of approximately 20 days, the treated mice underwent complete tumor regression that was immunologically specific. Seven days after vaccination treated mice demonstrated robust infiltration of CD4+ and CD8+ T cells, which was exclusively confined to the tumor without apparent neurological toxicity. Cured mice survived longer than 120 days with no evidence of tumor recurrence and resisted intracranial tumor challenge. CONCLUSIONS: These data indicate a strategy to achieve an antitumor response against tumors in the central nervous system that is highly focused from both immunological and anatomical perspectives.

Tumor-dendritic cell fusion as a basis for cancer immunotherapy.

OBJECTIVE: To establish the basis for use of allogeneic dendritic-tumor fusion cells. STUDY DESIGN: Fusion cells were created by electrofusion. We used 2 allogeneic murine tumor lines (D5 and 4T1) that were virally transduced to express the antigen (beta-galactosidase) as a surrogate tumor marker. RESULTS: Cross-immunization was achieved with irradiated allogenic tumor cells. Successful electrofusion of dendritic cells and tumor cells was confirmed by using fluorescence-activated cell sorting and cytospin. Significant responses were shown in immunized mice against tumor challenge and established 3-day pulmonary metastasis with fusion cells. CONCLUSIONS: Allogeneic tumor sharing a common tumor antigen can immunize against syngeneic tumor challenge. Fusion cells showed successful immunization against tumor challenge and showed regression of 3-day established pulmonary metastasis. SIGNIFICANCE: These preclinical studies provide evidence that an allogenic tumor-dendritic cell fusion vaccine is a valid approach for head and neck cancer immunotherapy.

Immunological mechanisms of the antitumor effects of supplemental oxygenation.

Antitumor T cells either avoid or are inhibited in hypoxic and extracellular adenosine-rich tumor microenvironments (TMEs) by A2A adenosine receptors. This may limit further advances in cancer immunotherapy. There is a need for readily available and safe treatments that weaken the hypoxia-A2-adenosinergic immunosuppression in the TME. Recently, we reported that respiratory hyperoxia decreases intratumoral hypoxia and concentrations of extracellular adenosine. We show that it also reverses the hypoxia-adenosinergic immunosuppression in the TME. This, in turn, stimulates (i) enhanced intratumoral infiltration and reduced inhibition of endogenously developed or adoptively transfered tumor-reactive CD8 T cells, (ii) increased proinflammatory cytokines and decreased immunosuppressive molecules, such as transforming growth factor-ß (TGF-ß), (iii) weakened immunosuppression by regulatory T cells, and (iv) improved lung tumor regression and long-term survival in mice. Respiratory hyperoxia also promoted the regression of spontaneous metastasis from orthotopically grown breast tumors. These effects are entirely T cell- and natural killer cell-dependent, thereby justifying the testing of supplemental oxygen as an immunological coadjuvant to combine with existing immunotherapies for cancer.

Systemic oxygenation weakens the hypoxia and hypoxia inducible factor 1α-dependent and extracellular adenosine-mediated tumor protection.

UNLABELLED: Intratumoral hypoxia and hypoxia inducible factor-1α (HIF-1-α)-dependent CD39/CD73 ectoenzymes may govern the accumulation of tumor-protecting extracellular adenosine and signaling through A2A adenosine receptors (A2AR) in tumor microenvironments (TME). Here, we explored the conceptually novel motivation to use supplemental oxygen as a treatment to inhibit the hypoxia/HIF-1α-CD39/CD73-driven accumulation of extracellular adenosine in the TME in order to weaken the tumor protection. We report that hyperoxic breathing (60 % O2) decreased the TME hypoxia, as well as levels of HIF-1α and downstream target proteins of HIF-1α in the TME according to proteomic studies in mice. Importantly, oxygenation also downregulated the expression of adenosine-generating ectoenzymes and significantly lowered levels of tumor-protecting extracellular adenosine in the TME. Using supplemental oxygen as a tool in studies of the TME, we also identified FHL-1 as a potentially useful marker for the conversion of hypoxic into normoxic TME. Hyperoxic breathing resulted in the upregulation of antigen-presenting MHC class I molecules on tumor cells and in the better recognition and increased susceptibility to killing by tumor-reactive cytotoxic T cells. Therapeutic breathing of 60 % oxygen resulted in the significant inhibition of growth of established B16.F10 melanoma tumors and prolonged survival of mice. Taken together, the data presented here provide proof-of principle for the therapeutic potential of systemic oxygenation to convert the hypoxic, adenosine-rich and tumor-protecting TME into a normoxic and extracellular adenosine-poor TME that, in turn, may facilitate tumor regression. We propose to explore the combination of supplemental oxygen with existing immunotherapies of cancer. KEY MESSAGES: Oxygenation decreases levels of tumor protecting hypoxia. Oxygenation decreases levels of tumor protecting extracellular adenosine. Oxygenation decreases expression of HIF-1alpha dependent tumor-protecting proteins. Oxygenation increases MHC class I expression and enables tumor regression.

Mechanism of third signals provided by IL-12 and OX-40R ligation in eliciting therapeutic immunity following dendritic-tumor fusion vaccination.

Dendritic-tumor heterokaryons generated by electrofusion are highly immunogenic. In animal studies, a single vaccination was therapeutic for tumors established in the lung, skin, and brain. However, effective therapy required a third signal which could be provided by exogenous IL-12 or the agonistic anti-OX-40R monoclonal antibody (mAb). In this study, we investigated the mechanism and mode of actions of these two seemingly distinct adjuvants. In immunotherapy of the MCA205 sarcoma, administration of the neutralizing anti-IL-12 mAb nearly completely blocked the adjuvant effect of IL-12, but had minimal inhibitory effects on anti-OX-40R mAb. By contrast, in vivo administration of the antagonistic anti-OX-40L mAb inhibited the adjuvant effects of both IL-12 and anti-OX-40R mAb. Thus, a common pathway of endogenous OX-40 interaction is critical for the development of a therapeutic immune response. Analysis of the third signal mechanism revealed that in the absence of an adjuvant, vaccination with fusion hybrids led to IL-10 production without eliciting IFN-gamma secreting cells. The addition of IL-12 to vaccination suppressed IL-10 production and initiated sensitization of specific IFN-gamma secreting cells, resulting in a type 1-like antitumor immunity. These findings underscore the significance of the third signal in the design of dendritic cell-based cancer vaccines.

Electrofusion of syngeneic dendritic cells and tumor generates potent therapeutic vaccine.

Antigen presentation by dendritic cells (DCs) has the potential to elicit therapeutic immune responses against malignant tumors. One strategy utilizing DC-tumor fusion hybrids as cancer vaccine is particularly attractive because of polyclonal presentation of a diverse array of unaltered tumor antigens. We have recently developed a large-scale electrofusion technique for generating DC-tumor heterokaryons and demonstrated their superb immunogenicity. Here, employing the weakly immunogenic MCA205 sarcoma, a single vaccination with electrofusion hybrids eradicated tumors established in the lung, skin, and brain. Immunotherapy required intra-lymphoid vaccine delivery and co-administration of adjuvants such as OX-40R antibody. Tumor eradication was immunologically specific and involved the participation of both CD4 and CD8 T cells. Consistent with DC's functionality of MHC-restriction, the use of syngeneic DCs for fusion was an obligatory requirement. Fusion with allogeneic DCs completely lacked therapeutic effects. These findings provide a strong impetus for treating cancer patients with similarly generated DC-tumor hybrids.

Therapeutic efficacy of adoptive immunotherapy is predicated on in vivo antigen-specific proliferation of donor T cells.

Activated T cells with down-regulated L-selectin expression (L-sel(-)) from tumor-draining lymph nodes represent a potent source of specific immune effectors in adoptive immunotherapy. Using congenic pairs of mice and carboxyfluorescein diacetate succinimidyl ester-labeled L-sel(-) T cells, the current study analyzed in vivo proliferation of transferred cells. In the lung of MCA205 tumor-bearing mice, 6% or 0.3 x 10(6) of the 5 x 10(6) donor cells were identified 24 h after transfer. Vigorous proliferation of donor cells was evident on day 2, reaching a maximum on day 6. The proliferation was tumor-specific and CD4 T cells divided with greater magnitude than CD8 cells. Successful adoptive immunotherapy also required sublethal whole-body irradiation (WBI) of the recipient. WBI exerted its effects on facilitating specific T cell proliferation at the tumor site. Taken together, our results demonstrate that adoptively transferred T cells undergo extensive proliferation in response to the tumor and this response is associated with therapeutic efficacy.

Depletion of CD4+ CD25+ regulatory cells augments the generation of specific immune T cells in tumor-draining lymph nodes.

Recent studies have identified a unique population of CD4+CD25+ regulatory T cells that is crucial for the prevention of spontaneous autoimmune diseases. Further studies demonstrated that depletion of CD4+CD25+ T cells enhances immune responses to nonself antigens. Because immune responses to malignant tumors are weak and ineffective, depletion of regulatory T cells has been reported to result in tumor regression. In the current study, using the weakly immunogenic MCA205 sarcoma and the poorly immunogenic B16/BL6/D5 (D5) melanoma, depletion of CD4+CD25+ T cells by the administration of anti-CD25 monoclonal antibodies (mAb), PC61 induced some tumor growth retardation, but all mice eventually succumbed to tumors. In our laboratory, immunotherapy by the transfer of tumor-immune T cells has demonstrated potent antitumor effects. A reliable source of tumor-reactive T cells has been lymph nodes (LN) draining progressive tumors. Therapeutic effector T cells can be generated by in vitro activation of draining LN cells with anti-CD3 mAb followed by culture in interleukin-2. In this system, PC61 mAb depletion of CD4+CD25+ T cells before or on day 8 of tumor growth resulted in increased sensitization in the draining LN. The therapeutic efficacy of activated tumor-draining LN cells from mAb depleted mice increased approximately three fold while maintaining specificity when tested in adoptive immunotherapy of established pulmonary metastases. Specific interferon-gamma secretion by LN T cells from mice treated with PC61 mAb 1 day before tumor inoculation increased significantly. However, this increase was not demonstrated with LN T cells from mice treated on day 8 despite their enhanced therapeutic reactivities. Our results indicate that although the antitumor immunity enhanced by the depletion of CD4+CD25+ T cells is insufficient to eradicate tumors, it augments the sensitization of immune T cells in the draining LN, thus, facilitating adoptive immunotherapy.

Memory T cells originate from adoptively transferred effectors and reconstituting host cells after sequential lymphodepletion and adoptive immunotherapy.

Adoptive transfer of tumor-specific effector T cells induces regression of advanced tumors and induces a long term memory response; however, the origin of this response has not been clearly defined. In this study Thy1.2+ mice bearing advanced MCA-205 tumors were treated with sublethal total body irradiation, followed by adoptive transfer of congenic Thy1.1+ T cells that had been sensitized to tumor in vivo and then activated ex vivo with anti-CD3, IL-2, and IL-7. Splenocytes were recovered >140 days after the initial therapy, and the L-selectinlow memory cell subset was separated into host Thy1.2+ and transferred Thy1.1+ cells and restimulated ex vivo. Both adoptively transferred Thy1.1+ cells as well as reconstituted host Thy1.2+ cells could specifically eliminate MCA-205 pulmonary metastases. Interestingly, hosts with partial responses followed by tumor recurrence nevertheless harbored memory cells that could be isolated and numerically amplified ex vivo to regenerate potent effector function. Memory cells were recovered after adoptive transfer into lymphodepleted nontumor-bearing hosts, indicating that they were not dependent on continued Ag exposure. These experiments establish that rapid ex vivo expansion of tumor Ag-primed T cells does not abrogate their capacity to become long-lived memory cells. Moreover, immune-mediated tumor regression coincident with lymphoid reconstitution produces another wave of host memory cells. These data suggest an approach to rescuing antitumor immune function even in hosts with long-standing progressive tumor through restorative ex vivo activation.

Comparative analysis of antigen loading strategies of dendritic cells for tumor immunotherapy.

Dendritic cells (DCs) loaded with antigens can effectively stimulate host immune responses to syngeneic tumors, but there is considerable controversy as to which forms of antigen-loading are most immunogenic. Here, the authors compared immunotherapeutic reactivities of DCs loaded with a variety of antigen preparations. Because DC maturation stages affect their capacities of antigen processing and presentation, two DC populations were used for the current analysis: in vivo Flt-3 ligand-induced mature DCs and in vitro bone marrow-derived DCs, which were less mature. To facilitate a direct comparison, the LacZ gene-transduced B16 melanoma model system was used, where beta-galactosidase served as the surrogate tumor-rejection antigen. DC loading strategies included pulsing with the beta-galactosidase protein, H-2K restricted peptide, tumor cell lysate, and irradiated tumor cells and fusion of DCs with tumor cells. Our results demonstrated that electrofusion of DCs and tumor cells generated a therapeutic vaccine far superior to other methods of DC loading. For the treatment of 3-day established pulmonary tumor nodules, a single intranodal vaccination plus IL-12 resulted in a significant reduction of metastatic nodules, while other DC preparations were only marginally effective. Immunotherapy mediated by the fusion cells was tumor antigen-specific. Consistent with their therapeutic activity, fusion hybrids were the most potent stimulators to induce specific IFN-gamma secretion from immune T cells. Furthermore, fusion cells also stimulated a small amount of IL-10 production from immune T cells. However, this IL-10 secretion was also induced by other DC preparations and did not correlate with in vivo therapeutic reactivity.

Tumor-induced L-selectinhigh suppressor T cells mediate potent effector T cell blockade and cause failure of otherwise curative adoptive immunotherapy.

Tumor-specific effector T cells (T(E)) are naturally sensitized within the L-selectin(low) (CD62L(low)) fraction of tumor-draining lymph nodes (TDLN). Whether isolated from day 9 (D9) or day 12 (D12) TDLN, 5 million L-selectin(low) T(E) could be culture activated and adoptively transferred to achieve complete rejection of established intradermal, pulmonary, and brain tumors. Surprisingly, although 25 million unfractionated T cells from D9 TDLN were equally effective, even 100 million unfractionated T cells from D12 TDLN seldom prevented lethal intradermal tumor progression, despite a pronounced therapeutic excess of T(E). This highly reproducible treatment failure was due to cotransfer of tumor-induced, L-selectin(high) suppressor T cells (T(S)) which were also present in D12 TDLN. In contrast, D9 TDLN and normal spleens lacked L-selectin(high) T(S). Only those L-selectin(high) D12 TDLN T cells that down-regulated L-selectin during culture activation were suppressive in vivo and in vitro, and, like L-selectin(low) T(E), trafficked promptly into tumors following i.v. administration. This is the first demonstration that adoptive immunotherapy can fail as a direct result of passenger T(S) that share certain phenotypic and trafficking features of T(E), even when otherwise curative doses of T(E) have been administered. Furthermore, in contrast to recently described CD4(+)CD25(+) T(S) and plasmacytoid dendritic cell-activated T(S), tumor-induced L-selectin(high) T(S) prevent tumor rejection via blockade of sensitized, activated T(E) rather than via afferent blockade.