Christian H. Poehlein

Signaling Through OX40 Enhances Antitumor Immunity

The existence of tumor-specific T cells, as well as their ability to be primed in cancer patients, confirms that the immune response can be deployed to combat cancer. However, there are obstacles that must be overcome to convert the ineffective immune response commonly found in the tumor environment to one that leads to sustained destruction of tumor. Members of the tumor necrosis factor (TNF) superfamily direct diverse immune functions. OX40 and its ligand, OX40L, are key TNF members that augment T-cell expansion, cytokine production, and survival. OX40 signaling also controls regulatory T-cell differentiation and suppressive function. Studies over the past decade have demonstrated that OX40 agonists enhance antitumor immunity in preclinical models using immunogenic tumors; however, treatment of poorly immunogenic tumors has been less successful. Combining strategies that prime tumor-specific T cells together with OX40 signaling could generate and maintain a therapeutic antitumor immune response.

TNF Plays an Essential Role in Tumor Regression after Adoptive Transfer of Perforin/IFN-γ Double Knockout Effector T Cells

We have recently shown that effector T cells (TE) lacking either perforin or IFN-γ are highly effective mediators of tumor regression. To rule out compensation by either mechanism, TE deficient in both perforin and IFN-γ (perforin knockout (PKO)/IFN-γ knockout (GKO)) were generated. The adoptive transfer of PKO/GKO TE mediated complete tumor regression and cured wild-type animals with established pulmonary metastases of the B16BL6-D5 (D5) melanoma cell line. PKO/GKO TE also mediated tumor regression in D5 tumor-bearing PKO, GKO, or PKO/GKO recipients, although in PKO/GKO recipients efficacy was reduced. PKO/GKO TE exhibited tumor-specific TNF-α production and cytotoxicity in a 24-h assay, which was blocked by the soluble TNFRII-human IgG fusion protein (TNFRII:Fc). Blocking TNF in vivo by administering soluble TNFR II fusion protein (TNFRII:Fc) significantly reduced the therapeutic efficacy of PKO/GKO, but not wild-type TE. This study identifies perforin, IFN-γ, and TNF as a critical triad of effector molecules that characterize therapeutic antitumor T cells. These insights could be used to monitor and potentially tune the immune response to cancer vaccines.

Tumour-induced polarization of tumour vaccine-draining lymph node T cells to a type 1 cytokine profile predicts inherent strong immunogenicity of the tumour and correlates with therapeutic efficacy in adoptive transfer studies.

Previously we have shown that vaccination with the poorly immunogenic B16BL6‐D5 melanoma (D5) elicits a dominant type 2 (T2) cytokine response that fails to protect the host from a subsequent tumour challenge. Here we investigated whether the inherent immunogenicity of a tumour can be correlated with its ability to bias the anti‐tumour cytokine response towards either a type 1 (T1) or a T2 profile. The immune response to six tumours of different inherent immunogenicity was assayed. By isolating l‐selectinlow T cells from tumour vaccine draining lymph nodes (TVDLN), it was possible to detect tumour‐specific cytokine responses from both immunogenic, poorly immunogenic and non‐immunogenic tumours. Immunogenic tumours (MCA‐304, MCA‐309, MPR‐4) induced a predominant tumour‐specific T1 cytokine response. In contrast, weakly (MCA‐310, MPR‐3) and poorly/non‐immunogenic tumours (MPR‐5, D5) sensitized T cells with a predominant tumour‐specific T2 cytokine response. A significant correlation (P < 0·025) between immunogenicity and the ratio of tumour‐specific interferon‐γ : interleukin‐4 (IL‐4) secretion by TVDLN T cells was identified. We then documented that non‐therapeutic T cells primed by the poorly immunogenic D5, recognized ‘tumour‐rejection’ antigens and that reprogramming their cytokine response, by in vitro culture with IL‐12 and anti‐IL‐4, to a T1 profile uncovered therapeutic efficacy. In contrast, TVDLN T cells primed by a therapeutic vaccine lose therapeutic efficacy when cultured with IL‐4. These results provide insights into the development of a protective anti‐tumour immune response and strengthen the hypothesis that a T1 cytokine response is critical for T‐cell‐mediated tumour regression.

Disruption of TGF-β Signaling Prevents the Generation of Tumor-Sensitized Regulatory T Cells and Facilitates Therapeutic Antitumor Immunity

Regulatory T (Treg) cells represent a major roadblock to the induction of antitumor immunity through vaccine approaches. TGF-β is a cytokine implicated in the generation and maintenance of Treg cells, as well as in their suppressive function. These experiments examined whether the generation of tumor-sensitized Treg cells was TGF-β dependent and evaluated whether TGF-β produced by Treg cells blocked the priming of tumor-specific T cells in vaccinated reconstituted lymphopenic mice. We show that tumor-sensitized Treg cells (CD25+/FoxP3+) obtained from tumor-bearing mice block the generation of tumor-specific T cells in reconstituted lymphopenic mice. Strikingly, this suppression is absent if tumor-sensitized Treg cells are acquired from tumor-bearing mice expressing the dominant-negative TGFβRII in T cells. This loss of suppression was a result of the crucial role of TGF-β in generating tumor-sensitized Treg cells, and not due to the insensitivity of naive or tumor-primed effector T cells to the direct suppressive influence of TGF-β. We conclude that blocking TGF-β in a tumor-bearing host can inhibit the induction of highly suppressive tumor-sensitized Treg cells. These data suggest that an integrative strategy combining “up-front” Treg cell ablation followed by vaccination and TGF-β blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.

Depletion of tumor-induced Treg prior to reconstitution rescues enhanced priming of tumor-specific, therapeutic effector T cells in lymphopenic hosts.

We reported previously that vaccination of reconstituted, lymphopenic mice resulted in a higher frequency of tumor‐specific effector T cells with therapeutic activity than vaccination of normal mice. Here, we show that lymphopenic mice reconstituted with spleen cells from tumor‐bearing mice (TBM), a situation that resembles the clinical condition, failed to generate tumor‐specific T cells with therapeutic efficacy. However, depletion of CD25+ Treg from the spleen cells of TBM restored tumor‐specific priming and therapeutic efficacy. Adding back TBM CD25+ Treg to CD25− naïve and TBM donor T cells prior to reconstitution confirmed their suppressive role. CD25+ Treg from TBM prevented priming of tumor‐specific T cells since subsequent depletion of CD4+ T cells did not restore therapeutic efficacy. This effect may not be antigen‐specific as three histologically distinct tumors generated CD25+ Treg that could suppress the T‐cell immune response to a melanoma vaccine. Importantly, since ex vivo depletion of CD25+ Treg from TBM spleen cells prior to reconstitution and vaccination fully restored the generation of therapeutic effector T cells, even in animals with established tumor burden, we have initiated a translational clinical trial of this strategy in patients with metastatic melanoma.

Cancer Immunotherapy: The Role Regulatory T Cells Play and What Can be Done to Overcome their Inhibitory Effects

Since multiple lines of experimental and clinical data clearly identified regulatory T cells as an integral part of the immune response, these cells have become a major focus of investigation in tumor immunology. Regulatory T cells are in place to dampen ongoing immune responses and to prevent autoimmunity, but they also have profound effects in blocking therapeutic anti-tumor activity. Therefore regulatory T cells are seen as a major hurdle that must be overcome in order for cancer immunotherapy to reach its therapeutic potential. Regulatory T cells are heterogeneous with sub-populations that exhibit distinct functional features. Here we will review the individual sub-populations in regards to their mode of action and their potential impact on blocking anti-tumor immunity. Approaches to measure function and frequency of regulatory T cells in model systems and clinical trails will be discussed. Finally, we will describe possible ways to interfere with regulatory T cell-mediated immune suppression with the focus on recent pre-clinical and clinical findings.

Active-specific immunotherapy for non-small cell lung cancer

Non-small cell lung cancer constitutes about 85% of all newly diagnosed cases of lung cancer and continues to be the leading cause of cancer-related deaths worldwide. Standard treatment for this devastating disease, such as systemic chemotherapy, has reached a plateau in effectiveness and comes with considerable toxicities. For all stages of disease fewer than 20% of patients are alive 5 years after diagnosis; for metastatic disease the median survival is less than one year. Until now, the success of active-specific immunotherapy for all tumor types has been sporadic and unpredictable. However, the active-specific stimulation of the hosts own immune system still holds great promise for achieving non-toxic and durable antitumor responses. Recently, sipuleucel-T (Provenge(®); Dendreon Corp., Seattle, WA) was the first therapeutic cancer vaccine to receive market approval, in this case for advanced prostate cancer. Other phase III clinical trials using time-dependent endpoints, e.g. in melanoma and follicular lymphoma, have recently turned out positive. More sophisticated specific vaccines have now also been developed for lung cancer, which, for long, was not considered an immune-sensitive malignancy. This may explain why advances in active-specific immunotherapy for lung cancer lag behind similar efforts in renal cell cancer, melanoma or prostate cancer. However, various vaccines are now being evaluated in controlled phase III clinical trials, raising hopes that active-specific immunotherapy may become an additional effective therapy for patients with lung cancer. This article reviews the most prominent active-specific immunotherapeutic approaches using protein/peptide, whole tumor cells, and dendritic cells as vaccines for lung cancer.

Therapeutic T cells induce tumor-directed chemotaxis of innate immune cells through tumor-specific secretion of chemokines and stimulation of B16BL6 melanoma to secrete chemokines

BackgroundThe mechanisms by which tumor-specific T cells induce regression of established metastases are not fully characterized. In using the poorly immunogenic B16BL6-D5 (D5) melanoma model we reported that T cell-mediated tumor regression can occur independently of perforin, IFN-γ or the combination of both. Characterization of regressing pulmonary metastases identified macrophages as a major component of the cells infiltrating the tumor after adoptive transfer of effector T cells. This led us to hypothesize that macrophages played a central role in tumor regression following T-cell transfer. Here, we sought to determine the factors responsible for the infiltration of macrophages at the tumor site.MethodsThese studies used the poorly immunogenic D5 melanoma model. Tumor-specific effector T cells, generated from tumor vaccine-draining lymph nodes (TVDLN), were used for adoptive immunotherapy and in vitro analysis of chemokine expression. Cellular infiltrates into pulmonary metastases were determined by immunohistochemistry. Chemokine expression by the D5 melanoma following co-culture with T cells, IFN-γ or TNF-α was determined by RT-PCR and ELISA. Functional activity of chemokines was confirmed using a macrophage migration assay. T cell activation of macrophages to release nitric oxide (NO) was determined using GRIES reagent.ResultsWe observed that tumor-specific T cells with a type 1 cytokine profile also expressed message for and secreted RANTES, MIP-1α and MIP-1β following stimulation with specific tumor. Unexpectedly, D5 melanoma cells cultured with IFN-γ or TNF-α, two type 1 cytokines expressed by therapeutic T cells, secreted Keratinocyte Chemoattractant (KC), MCP-1, IP-10 and RANTES and expressed mRNA for MIG. The chemokines released by T cells and cytokine-stimulated tumor cells were functional and induced migration of the DJ2PM macrophage cell line. Additionally, tumor-specific stimulation of wt or perforin-deficient (PKO) effector T cells induced macrophages to secrete nitric oxide (NO), providing an additional effector mechanism for T cell-mediated tumor regression.ConclusionThese data suggest two possible sources for chemokine secretion that stimulates macrophage recruitment to the site of tumor metastases. Both appear to be initiated by T cell recognition of specific antigen, but one is dependent on the tumor cells to produce the chemokines that recruit macrophages.

Tumor-specific T cells signal tumor destruction via the lymphotoxin β receptor

BackgroundPreviously, we reported that adoptively transferred perforin k/o (PKO), and IFN-γ k/o (GKO), or perforin/IFN-γ double k/o (PKO/GKO) effector T cells mediated regression of B16BL6-D5 (D5) pulmonary metastases and showed that TNF receptor signaling played a critical role in mediating tumor regression. In this report we investigated the role of lymphotoxin-α (LT-α) as a potential effector molecules of tumor-specific effector T cells.MethodsEffector T cells were generated from tumor vaccine-draining lymph node (TVDLN) of wt, GKO, LT-α deficient (LKO), or PKO/GKO mice and tested for their ability to mediate regression of D5 pulmonary metastases in the presence or absence of LT-βR-Fc fusion protein or anti-IFN-γ antibody. Chemokine production by D5 tumor cells was determined by ELISA, RT-PCR and Chemotaxis assays.ResultsStimulated effector T cells from wt, GKO, or PKO/GKO mice expressed ligands for LT-β receptor (LT-βR). D5 tumor cells were found to constitutively express the LT-βR. Administration of LT-βR-Fc fusion protein completely abrogated the therapeutic efficacy of GKO or PKO/GKO but not wt effector T cells (p < 0.05). Consistent with this observation, therapeutic efficacy of effector T cells deficient in LT-α, was greatly reduced when IFN-γ production was neutralized. While recombinant LT-α1β2 did not induce apoptosis of D5 tumor cells in vitro, it induced secretion of chemokines by D5 that promoted migration of macrophages.ConclusionThe contribution of LT-α expression by effector T cells to anti-tumor activity in vivo was not discernable when wt effector T cells were studied. However, the contribution of LT-β R signaling was identified for GKO or PKO/GKO effector T cells. Since LT-α does not directly induce killing of D5 tumor cells in vitro, but does stimulate D5 tumor cells to secrete chemokines, these data suggest a model where LT-α expression by tumor-specific effector T cells interacts via cross-linking of the LT-βR on tumor cells to induce secretion of chemokines that are chemotactic for macrophages. While the contribution of macrophages to tumor elimination in our system requires additional study, this model provides a possible explanation for the infiltration of inate effector cells that is seen coincident with tumor regression.

Increased Susceptibility to Immune Destruction of B16BL6 Tumor Cells Engineered to Express a Novel Pro-Smac Fusion Protein

Recent developments in immunology have provided new strategies to induce and augment the immune response to cancer. Nonetheless, objective clinical responses after vaccination are rare and even when high frequencies of tumor-specific T cells are achieved after adoptive immunotherapy, tumor cells continue to evade the immune response. We hypothesize that 1 mechanism of resistance of tumor cells to destruction by T cells is an elevated threshold for the induction of apoptosis. Inhibitor of apoptosis proteins (IAPs) are overexpressed in various tumors and have been associated with treatment failure and poor prognosis. As the mitochondrial peptide second mitochondria-derived activator of caspase (Smac) can antagonize IAPs, we designed a GFP-Smac fusion protein with a granzyme B (GrB) cleavage site. This fusion protein should be cleaved when tumor-specific cytolytic T cells recognize the tumor and, using the pore-forming protein perforin, insert GrB into the target. Here we report that transfer of a construct encoding a novel eGFP-Smac fusion protein (pro-Smac) containing a specific cleavage site for GrB, into the poorly immunogenic mouse melanoma cell line, B16BL6-D5 (D5), sensitizes tumor cells for killing by tumor-specific wild type, but not perforin-deficient (perforin-knockout), effector T cells in vitro and in vivo. These results describe the first example of a tumor-specific, T-cell–mediated approach to amplify the GrB-mediated cytotoxicity pathway with a pro-Smac fusion protein and provide an innovative approach to overcome IAPs and improve the efficacy of immunotherapy.