Blockade of TGF-ß signaling to enhance the antitumor response is accompanied by dysregulation of the functional activity of CD4+CD25+Foxp3+ and CD4+CD25-Foxp3+ T cells.

BACKGROUND: The pleiotropic cytokine, transforming growth factor (TGF)-ß, and CD4+CD25+Foxp3+ regulatory T cells (Tregs) play a critical role in actively suppressing antitumor immune responses. Evidence shows that TGF-ß produced by tumor cells promotes tolerance via expansion of Tregs. Our group previously demonstrated that blockade of TGF-ß signaling with a small molecule TGF-ß receptor I antagonist (SM16) inhibited primary and metastatic tumor growth in a T cell dependent fashion. In the current study, we evaluated the effect of SM16 on Treg generation and function. METHODS: Using BALB/c, FoxP3eGFP and Rag-/- mice, we performed FACS analysis to determine if SM16 blocked de novo TGF-ß-induced Treg generation in vitro and in vivo. CD4+ T cells from lymph node and spleen were isolated from control mice or mice maintained on SM16 diet, and flow cytometry analysis was used to detect the frequency of CD4+CD25-FoxP3+ and CD4+CD25+FoxP3+ T cells. In vitro suppression assays were used to determine the ability to suppress naive T cell proliferation in vitro of both CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ T cell sub-populations. We then examined whether SM16 diet exerted an inhibitory effect on primary tumor growth and correlated with changes in FoxP3+expression. ELISA analysis was used to measure IFN-γ levels after 72 h co-culture of CD4+CD25+ T cells from tumor-bearing mice on control or SM16 diet with CD4+CD25- T cells from naive donors. RESULTS: SM16 abrogates TGF-ß-induced Treg generation in vitro but does not prevent global homeostatic expansion of CD4+ T cell sub-populations in vivo. Instead, SM16 treatment causes expansion of a population of CD4+CD25-Foxp3+ Treg-like cells without significantly altering the overall frequency of Treg in lymphoreplete naive and tumor-bearing mice. Importantly, both the CD4+CD25-Foxp3+ T cells and the CD4+CD25+Foxp3+ Tregs in mice receiving SM16 diet exhibited diminished ability to suppress naive T cell proliferation in vitro compared to Treg from mice on control diet. CONCLUSIONS: These findings suggest that blockade of TGF-ß signaling is a potentially useful strategy for blunting Treg function to enhance the anti-tumor response. Our data further suggest that the overall dampening of Treg function may involve the expansion of a quiescent Treg precursor population, which is CD4+CD25-Foxp3+.

A pilot study of an autologous tumor-derived autophagosome vaccine with docetaxel in patients with stage IV non-small cell lung cancer.

BACKGROUND: Tumor-derived autophagosome vaccines (DRibbles) have the potential to broaden immune response to poorly immunogenic tumors. METHODS: Autologous vaccine generated from tumor cells harvested from pleural effusions was administered to patients with advanced NSCLC with the objectives of assessing safety and immune response. Four patients were vaccinated and evaluable for immune response; each received two to four doses of vaccine. Study therapy included two cycles of docetaxel 75 mg/m2 on days 1 and 29 to treat the tumor, release hidden antigens and produce lymphopenia. DRibbles were to be administered intradermally on days 14, 43, 57, 71, and 85, together with GM-CSF (50 µg/d x 6d, administered via SQ mini pump). Peripheral blood was tested for immune parameters at baseline and at each vaccination. RESULTS: Three of four patients had tumor cells available for testing. Autologous tumor-specific immune response was seen in two of the three, manifested by IL-5 (1 patient after 3 doses), and IFN-γ, TNF-α, IL-5, IL-10 (after 4 doses in one patient). All 4 patients had evidence of specific antibody responses against potential tumor antigens. All patients came off study after 4 or fewer vaccine treatments due to progression of disease. No significant immune toxicities were seen during the course of the study. CONCLUSIONS: DRibble vaccine given with GM-CSF appeared safe and capable of inducing an immune response against tumor cells in this small, pilot study. There was no evidence of efficacy in this small poor-prognosis patient population, with treatment not feasible. Trial registration NCT00850785, initial registration date February 23, 2009.

The Role of α1-Adrenoceptor Antagonists in the Treatment of Prostate and Other Cancers.

This review evaluates the role of α-adrenoceptor antagonists as a potential treatment of prostate cancer (PCa). Cochrane, Google Scholar and Pubmed were accessed to retrieve sixty-two articles for analysis. In vitro studies demonstrate that doxazosin, prazosin and terazosin (quinazoline α-antagonists) induce apoptosis, decrease cell growth, and proliferation in PC-3, LNCaP and DU-145 cell lines. Similarly, the piperazine based naftopidil induced cell cycle arrest and death in LNCaP-E9 cell lines. In contrast, sulphonamide based tamsulosin did not exhibit these effects. In vivo data was consistent with in vitro findings as the quinazoline based α-antagonists prevented angiogenesis and decreased tumour mass in mice models of PCa. Mechanistically the cytotoxic and antitumor effects of the α-antagonists appear largely independent of α 1-blockade. The proposed targets include: VEGF, EGFR, HER2/Neu, caspase 8/3, topoisomerase 1 and other mitochondrial apoptotic inducing factors. These cytotoxic effects could not be evaluated in human studies as prospective trial data is lacking. However, retrospective studies show a decreased incidence of PCa in males exposed to α-antagonists. As human data evaluating the use of α-antagonists as treatments are lacking; well designed, prospective clinical trials are needed to conclusively demonstrate the anticancer properties of quinazoline based α-antagonists in PCa and other cancers.

OX40 is a potent immune-stimulating target in late-stage cancer patients.

OX40 is a potent costimulatory receptor that can potentiate T-cell receptor signaling on the surface of T lymphocytes, leading to their activation by a specifically recognized antigen. In particular, OX40 engagement by ligands present on dendritic cells dramatically increases the proliferation, effector function, and survival of T cells. Preclinical studies have shown that OX40 agonists increase antitumor immunity and improve tumor-free survival. In this study, we performed a phase I clinical trial using a mouse monoclonal antibody (mAb) that agonizes human OX40 signaling in patients with advanced cancer. Patients treated with one course of the anti-OX40 mAb showed an acceptable toxicity profile and regression of at least one metastatic lesion in 12 of 30 patients. Mechanistically, this treatment increased T and B cell responses to reporter antigen immunizations, led to preferential upregulation of OX40 on CD4(+) FoxP3(+) regulatory T cells in tumor-infiltrating lymphocytes, and increased the antitumor reactivity of T and B cells in patients with melanoma. Our findings clinically validate OX40 as a potent immune-stimulating target for treatment in patients with cancer, providing a generalizable tool to favorably influence the antitumor properties of circulating T cells, B cells, and intratumoral regulatory T cells.

(Z)-1-(2-Hy-droxy-eth-yl)-4-(2-meth-oxy-benzyl-idene)-2-methyl-1H-imidazol-5(4H)-one.

In the title compound, C14H16N2O3, an analog of the chromophore in green fluorescent protein, the meth-oxy-phenyl substituent and the imidazole N adopt a Z conformation with respect to the C=C bond. Aside from the hy-droxy-ethyl group, the mol-ecule is approximately planar, with the five- and six-membered ring planes forming a dihedral angle of 9.3 (1)°. An intra-molecular C-H⋯N contact occurs. In the crystal, O-H⋯N hydrogen bonds link the mol-ecules, forming chains along the b-axis direction. C-H⋯O hydrogen bonds are also observed.

Revisited: Decomposition or melting? Formation mechanism investigation of LiCoO2 via in-situ time-resolved X-ray diffraction.

We report the first in-situ time-resolved X-ray diffraction investigation in conjunction with a non-isothermal kinetic study using the model-free isoconversional kinetic method to determine the formation mechanism for the solid-state synthesis of electrochemically active LiCoO(2) from Li(2)CO(3) and Co(3)O(4). Detailed information on the phase evolution as well as thermal events during the heating process was clearly observed, explained, and supported. This investigation provides structural as well as kinetic evidence for a multistep reaction and proposes the first plausible formation mechanism for the solid-state synthesis of LiCoO(2).

Phase 1 study of stereotactic body radiotherapy and interleukin-2--tumor and immunological responses.

Preclinical models suggest that focal high-dose radiation can make tumors more immunogenic. We performed a pilot study of stereotactic body radiation therapy (SBRT) followed by high-dose interleukin-2 (IL-2) to assess safety and tumor response rate and perform exploratory immune monitoring studies. Patients with metastatic melanoma or renal cell carcinoma (RCC) who had received no previous medical therapy for metastatic disease were eligible. Patients received one, two, or three doses of SBRT (20 Gy per fraction) with the last dose administered 3 days before starting IL-2. IL-2 (600,000 IU per kilogram by means of intravenous bolus infusion) was given every 8 hours for a maximum of 14 doses with a second cycle after a 2-week rest. Patients with regressing disease received up to six IL-2 cycles. Twelve patients were included in the intent-to-treat analysis, and 11 completed treatment per the study design. Response Evaluation Criteria in Solid Tumors criteria were used to assess overall response in nonirradiated target lesions. Eight of 12 patients (66.6%) achieved a complete (CR) or partial response (PR) (1 CR and 7 PR). Six of the patients with PR on computed tomography had a CR by positron emission tomography imaging. Five of seven (71.4%) patients with melanoma had a PR or CR, and three of five (60%) with RCC had a PR. Immune monitoring showed a statistically significantly greater frequency of proliferating CD4(+) T cells with an early activated effector memory phenotype (CD3(+)CD4(+)Ki67(+)CD25(+)FoxP3(-)CCR7(-)CD45RA(-)CD27(+)CD28(+/-)) in the peripheral blood of responding patients. SBRT and IL-2 can be administered safely. Because the response rate in patients with melanoma was significantly higher than expected on the basis of historical data, we believe that the combination and investigation of CD4(+) effector memory T cells as a predictor of response warrant further study.

Defining a functionally distinct subset of human memory CD4+ T cells that are CD25POS and FOXP3NEG.

Surface expression of the IL-2 receptor α-chain (CD25) has been used to discriminate between CD4(+) CD25(HI) FOXP3(+) regulatory T (Treg) cells and CD4(+) CD25(NEG) FOXP3(-) non-Treg cells. However, this study reports that the majority of resting human memory CD4(+) FOXP3(-) T cells expresses intermediate levels of CD25 and that CD25 expression can be used to delineate a functionally distinct memory subpopulation. The CD25(NEG) memory T-cell population contains the vast majority of late differentiated cells that respond to antigens associated with chronic immune responses and are increased in patients with systemic lupus erythematosus (SLE). In contrast, the CD25(INT) memory T cells respond to antigens associated with recall responses, produce a greater array of cytokines, and are less dependent on costimulation for effector responses due to their expression of CD25. Lastly, compared to the CD25(NEG) and Treg-cell populations, the CD25(INT) memory population is lost to a greater degree from the blood of cancer patients treated with IL-2. Collectively, these results show that in humans, a large proportion of CD4(+) memory T cells express intermediate levels of CD25, and this CD25(INT) FOXP3(-) subset is a functionally distinct memory population that is uniquely affected by IL-2.

The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer.

BACKGROUND: HER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity. METHODS: In this study we examined the effect of α-TEA plus HER2/neu-specific antibody treatment on HER2/neu-expressing breast cancer cells in vitro and in a HER2/neu positive human xenograft tumor model in vivo. RESULTS: We show in vitro that α-TEA plus anti-HER2/neu antibody has an increased cytotoxic effect against murine mammary tumor cells and human breast cancer cells and that the anti-tumor effect of α-TEA is independent of HER2/neu status. More importantly, in a human breast cancer xenograft model, the combination of α-TEA plus trastuzumab resulted in faster tumor regression and more tumor-free animals than trastuzumab alone. CONCLUSION: Due to the cancer cell selectivity of α-TEA, and because α-TEA kills both HER2/neu positive and HER2/neu negative breast cancer cells, it has the potential to be effective and less toxic than existing chemotherapeutic drugs when used in combination with HER2/neu antibody.

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.

Monitoring cytokine profiles during immunotherapy.

Measuring cytokine production is an integral part of measuring immune response during immunotherapy. Current technologies allow the simultaneous quantification of multiple cytokines in a variety of tissues. Patterns of cytokine response can be referred to as cytokine profiles. This article discusses the experimental design and data analysis of a number of studies that examined cytokine profiles in humans. We highlight potential sources of variability, both due to assay nuances and the diversity of human populations. We present strategies for analyzing data, emphasizing both multidimensional analysis and the value of treating each donor as his or her own control.

Exhaustive expansion: A novel technique for analyzing complex data generated by higher-order polychromatic flow cytometry experiments.

BACKGROUND: The complex data sets generated by higher-order polychromatic flow cytometry experiments are a challenge to analyze. Here we describe Exhaustive Expansion, a data analysis approach for deriving hundreds to thousands of cell phenotypes from raw data, and for interrogating these phenotypes to identify populations of biological interest given the experimental context. METHODS: We apply this approach to two studies, illustrating its broad applicability. The first examines the longitudinal changes in circulating human memory T cell populations within individual patients in response to a melanoma peptide (gp100209-2M) cancer vaccine, using 5 monoclonal antibodies (mAbs) to delineate subpopulations of viable, gp100-specific, CD8+ T cells. The second study measures the mobilization of stem cells in porcine bone marrow that may be associated with wound healing, and uses 5 different staining panels consisting of 8 mAbs each. RESULTS: In the first study, our analysis suggests that the cell surface markers CD45RA, CD27 and CD28, commonly used in historical lower order (2-4 color) flow cytometry analysis to distinguish memory from naïve and effector T cells, may not be obligate parameters in defining central memory T cells (TCM). In the second study, we identify novel phenotypes such as CD29+CD31+CD56+CXCR4+CD90+Sca1-CD44+, which may characterize progenitor cells that are significantly increased in wounded animals as compared to controls. CONCLUSIONS: Taken together, these results demonstrate that Exhaustive Expansion supports thorough interrogation of complex higher-order flow cytometry data sets and aids in the identification of potentially clinically relevant findings.

CD122+CD8+ Treg suppress vaccine-induced antitumor immune responses in lymphodepleted mice.

Lymphodeleption prior to adoptive transfer of tumor-specific T cells greatly improves the clinical efficacy of adoptive T-cell therapy for patients with advanced melanoma, and increases the therapeutic efficacy of cancer vaccines in animal models. Lymphodepletion reduces competition between lymphocytes, and thus creates "space" for enhanced expansion and survival of tumor-specific T cells. Within the lymphodepleted host, Ag-specific T cells still need to compete with other lymphocytes that undergo lymphopenia-driven proliferation. Herein, we describe the relative capacity of naïve T cells, Treg, and NK cells to undergo lymphopenia-driven proliferation. We found that the major population that underwent lymphopenia-driven proliferation was the CD122+ memory-like T-cell population (CD122+CD8+ Treg), and these cells competed with Ag-driven proliferation of melanoma-specific T cells. Removal of CD122+CD8+ Treg resulted in a greater expansion of tumor-specific T cells and tumor infiltration of functional effector/memory T cells. Our results demonstrate the lymphopenia-driven proliferation of CD122+CD8+ Treg in reconstituted lymphodepleted mice limited the antitumor efficacy of DC vaccination in conjunction with adoptive transfer of tumor-specific T cells.

2-Amino-1-(2-carboxyl-atoeth-yl)pyrimidin-1-ium monohydrate.

In the title structure, C(7)H(9)N(3)O(2)·H(2)O, there are two formula units in the asymmetric unit. The mol-ecule is a zwitterion, containing a quaternary N atom and a deprotonated carboxyl group, with C-O distances in the range 1.256 (2)-1.266 (3) Å. The two independent mol-ecules form a hydrogen-bonded R(2) (2)(16) dimer about an approximate inversion center via N-H⋯O hydrogen bonds, with N⋯O distances of 2.766 (2) and 2.888 (2) Å. O-H⋯O hydro-gen bonds involving the water mol-ecules and additional N-H⋯O hydrogen bonds link these dimers, forming double chains.

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.

Disruption of TGF-beta 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-beta 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-beta dependent and evaluated whether TGF-beta 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 TGFbetaRII in T cells. This loss of suppression was a result of the crucial role of TGF-beta 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-beta. We conclude that blocking TGF-beta 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-beta blockade may limit generation of new tumor-sensitized Treg cells and improve the generation of therapeutic immune responses in patients with cancer.

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.

Characterization of the class I-restricted gp100 melanoma peptide-stimulated primary immune response in tumor-free vaccine-draining lymph nodes and peripheral blood.

PURPOSE: The aim of this study was to characterize the primary gp100(209-2M)-specific T-cell response in vaccine-draining, metastases-free lymph nodes and peripheral blood of peptide-vaccinated stage I to III melanoma patients. EXPERIMENTAL DESIGN: After two or three gp100(209-2M) vaccinations, sentinel lymph nodes that drained both the primary tumor and adjacent vaccine sites were excised concomitant with wide excision of the tumor. Comparative 7-color flow cytometry phenotype analysis was done on gp100 tetramer-positive CD8(+) T cells from sentinel lymph nodes, closely proximate time-related peripheral blood mononuclear cells (PBMC) collected 2 to 4 weeks after sentinel lymph node excision, and on PBMC collected 6 months later after 7 or 11 more immunizations. Lymph node and peripheral blood T cells were tested for proliferative response, functional avidity, and tumor cell-induced CD107 mobilization. RESULTS: The frequencies of gp100-specific CD8(+) T cells from time-related PBMC and sentinel lymph nodes were comparable and were similar to those reported for virus-specific memory T cells. Their respective in vitro proliferation responses were also equivalent but statistically higher than proliferation responses of peripheral blood T cells collected after completion of the entire vaccine regimen. By contrast, functional avidity and CD107 responses were significantly higher in circulating T cells. Sentinel lymph node-derived, gp100-specific CD8(+) T cells predominantly expressed central and effector memory phenotype signatures, whereas there were higher frequencies of effector T cells in the peripheral blood. CONCLUSION: Priming immunization with gp100(209-2M) without coadministration of CD4(+) helper T cell-restricted antigens induced the effective expansion of peptide-specific central and effector memory CD8(+) T cells with high proliferation potential in vaccine-draining lymph nodes of stage I to III melanoma patients. Lymph node memory T cells gave rise to circulating gp100-specific effector T cells exhibiting increased functional maturation.

Phenotype and functional characterization of long-term gp100-specific memory CD8+ T cells in disease-free melanoma patients before and after boosting immunization.

PURPOSE: Effective cancer vaccines must both drive a strong CTL response and sustain long-term memory T cells capable of rapid recall responses to tumor antigens. We sought to characterize the phenotype and function of gp100 peptide-specific memory CD8+ T cells in melanoma patients after primary gp100(209-2M) immunization and assess the anamnestic response to boosting immunization. EXPERIMENTAL DESIGN: Eight-color flow cytometry analysis of gp100-specific CD8+ T cells was done on peripheral blood mononuclear cells collected shortly after the primary vaccine regimen, 12 to 24 months after primary vaccination, and after boosting immunization. The anamnestic response was assessed by comparing the frequency of circulating gp100-specific T cells before and after boosting. Gp100 peptide-induced in vitro functional avidity and proliferation responses and melanoma-stimulated T-cell CD107 mobilization were compared for cells from all three time points for multiple patients. RESULTS: The frequency of circulating gp100-specific memory CD8+ T cells was comparable with cytomegalovirus-specific and FLU-specific T cells in the same patients, and the cells exhibited anamnestic proliferation after boosting. Their phenotypes were not unique, and individual patients exhibited one of two distinct phenotype signatures that were homologous to either cytomegalovirus-specific or FLU-specific memory T cells. Gp100-specific memory T cells showed some properties of competent memory T cells, such as heightened in vitro peptide-stimulated proliferation and increase in central memory (TCM) differentiation when compared with T-cell responses measured after the primary vaccine regimen. However, they did not acquire enhanced functional avidity usually associated with competent memory T-cell maturation. CONCLUSIONS: Although vaccination with class I-restricted melanoma peptides alone can break tolerance to self-tumor antigens, it did not induce fully competent memory CD8+ T cells--even in disease-free patients. Data presented suggest other vaccine strategies will be required to induce functionally robust long-term memory T cells.

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.