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+.

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.

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.

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).

(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.

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.

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.

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.

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.

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.

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.

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.

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.

3-(2-Acetyl-anilino)propanoic acid.

The title mol-ecule, C(11)H(13)NO(3), has its propanoic acid group in an extended conformation, such that the mol-ecule is nearly planar, with a mean deviation of 0.036 Š[the maxima being 0.106 (1) and 0.110 (1) Šfor the two methyl-ene C atoms]. The NH group forms an intra-molecular hydrogen bond with the acetyl group; in the crystal COOH group forms a centrosymmetric hydrogen-bonded dimer.

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.

Combined IL-21 and low-dose IL-2 therapy induces anti-tumor immunity and long-term curative effects in a murine melanoma tumor model.

BACKGROUND: In vivo studies have recently demonstrated that interleukin 21 (IL-21) enhances the anti-tumor function of T-cells and NK cells in murine tumor models, and the combined use of IL-21 and IL-15 has resulted in prolonged tumor regression and survival in mice with previously established tumors. However, the combined anti-tumor effects of IL-21 and low dose IL-2 have not been studied even though IL-2 has been approved for human use, and, at low dose administration, stimulates the proliferation of memory T cells, and does not significantly increase antigen-induced apoptosis or regulatory T cell (Treg) expansion. This study examined whether recombinant IL-21 alone or in combination with low-dose IL-2 could improve the in vivo anti-tumor function of naïve, tumor-antigen specific CD8+ T cells in a gp100(25-33) T cell receptor transgenic pmel murine melanoma model. METHODS: Congenic C57BL/6 (Ly5.2) mice bearing subcutaneous B16F10 melanoma tumors were sublethally irradiated to induce lymphopenia. After irradiation naive pmel splenocytes were adoptively transferred, and mice were immunized with bone marrow-derived dendritic cells pulsed with human gp100(25-33) (hgp100(25-33)). Seven days after vaccination groups of mice received 5 consecutive days of intraperitoneal administration of IL-2 alone (20 x 10(3) IU), IL-21 alone (20 microg) or IL-21 and IL-2. Control animals received no cytokine therapy. RESULTS: IL-21 alone and IL-2 alone both delayed tumor progression, but only IL-21 significantly augmented long-term survival (20%) compared to the control group. However, combination therapy with IL-21 and IL-2 resulted in the highest long-term (>150 days) tumor-free survival frequency of 46%. Animals that were tumor-free for > 150 days demonstrated tumor-specific protection after rechallenge with B16F10 melanoma cells. At peak expansion (21 days post vaccination), the combination of IL-21 plus IL-2 resulted in a 2- to 3-fold higher absolute number of circulating tumor antigen-specific pmel CD8+ T cells than was stimulated by IL-2 or IL-21 alone. Pmel CD8+ T cells were predominantly partitioned into central memory (CD62L+/CD127+) or effector-memory (CD62L-/CD127+) phenotypes by day 28-post vaccination in IL-21 + IL-2 treated mice. CONCLUSION: These observations support the potential use of IL-21 and low-dose IL-2 therapy in combination with a tumor-antigen vaccine and lymphopenic conditioning in future cancer clinical trials to maintain high numbers of anti-tumor memory CD8+ T cells with the potential to sustain long term tumor regression and survival.

Adjuvant immunization of HLA-A2-positive melanoma patients with a modified gp100 peptide induces peptide-specific CD8+ T-cell responses.

PURPOSE: To measure the CD8+ T-cell response to a melanoma peptide vaccine and to compare an every-2-weeks with an every-3-weeks vaccination schedule. PATIENTS AND METHODS: Thirty HLA-A2-positive patients with resected stage I to III melanoma were randomly assigned to receive vaccinations every 2 weeks (13 vaccines) or every 3 weeks (nine vaccines) for 6 months. The synthetic, modified gp100 peptide, g209-2M, and a control peptide, HPV16 E7, were mixed in incomplete Freund's adjuvant and injected subcutaneously. Peripheral blood mononuclear cells obtained before and after vaccination by leukapheresis were analyzed using a fluorescence-based HLA/peptide-tetramer binding assay and cytokine flow cytometry. RESULTS: Vaccination induced an increase in peptide-specific T cells in 28 of 29 patients. The median frequency of CD8+ T cells specific for the g209-2M peptide increased markedly from 0.02% before to 0.34% after vaccination (P <.0001). Eight patients (28%) exhibited peptide-specific CD8+ T-cell frequencies greater than 1%, including two patients with frequencies of 4.96% and 8.86%, respectively. Interferon alfa-2b-treated patients also had significant increases in tetramer-binding cells (P <.0001). No difference was observed between the every-2-weeks and the every-3-weeks vaccination schedules (P =.59). CONCLUSION: Flow cytometric analysis of HLA/peptide-tetramer binding cells was a reliable means of quantifying the CD8+ T-cell response to peptide immunization. This assay may be suitable for use in future trials to optimize different vaccination strategies. Concurrent interferon treatment did not inhibit the development of a peptide-specific immune response and vaccination every 2 weeks, and every 3 weeks produced similar results.

gp100(209-2M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells.

Thirty-five HLA-A2(+) patients with completely resected stage I-III melanoma were vaccinated multiple times over 6 months with a modified melanoma peptide, gp100(209-2M), emulsified in Montanide adjuvant. Direct ex vivo gp100(209-2M) tetramer analysis of pre- and postvaccine peripheral blood mononuclear cells (PBMCs) demonstrated significant increases in the frequency of tetramer(+) CD8(+) T cells after immunization for 33 of 35 evaluable patients (median, 0.36%; range, 0.05-8.9%). Ex vivo IFN-gamma cytokine flow cytometry analysis of postvaccine PBMCs after brief gp100(209-2M) in vitro activation showed that for all of the patients studied tetramer(+) CD8(+) T cells produced IFN-gamma; however, some patients had significant numbers of tetramer(+) IFN-gamma(-) CD8(+)T cells suggesting functional anergy. Additionally, 8 day gp100(209-2M) in vitro stimulation (IVS) of pre- and postvaccine PBMCs resulted in significant expansion of tetramer(+) CD8(+) T cells from postvaccine cells for 34 patients, and these IVS tetramer(+) CD8(+) T cells were functionally responsive by IFN-gamma cytokine flow cytometry analysis after restimulation with either native or modified gp100 peptide. However, correlated functional and phenotype analysis of IVS-expanded postvaccine CD8(+) T cells demonstrated the proliferation of functionally anergic gp100(209-2M)- tetramer(+) CD8(+) T cells in several patients and also indicated interpatient variability of gp100(209-2M) stimulated T-cell proliferation. Flow cytometry analysis of cryopreserved postvaccine PBMCs from representative patients showed that the majority of tetramer(+) CD8+ T cells (78.1 +/- 4.2%) had either an "effector" (CD45 RA(+)/CCR7(-)) or an "effector-memory" phenotype (CD45RA(-)/CCR7(-)). Notably, analysis of PBMCs collected 12-24 months after vaccine therapy demonstrated the durable presence of gp100(209-2M)-specific memory CD8(+) T cells with high proliferation potential. Overall, this report demonstrates that after vaccination with a MHC class I-restricted melanoma peptide, resected nonmetastatic melanoma patients can mount a significant antigen-specific CD8(+) T-cell immune response with a functionally intact memory component. The data further support the combined use of tetramer binding and functional assays in correlated ex vivo and IVS settings as a standard for immunomonitoring of cancer vaccine patients.

Vaccination of women with metastatic breast cancer, using a costimulatory gene (CD80)-modified, HLA-A2-matched, allogeneic, breast cancer cell line: clinical and immunological results.

MDA-MB-231, an HLA-A2(+), HER2/neu(+) allogeneic breast cancer cell line genetically modified to express the costimulatory molecule CD80 (B7-1), was used to vaccinate 30 women with previously treated stage IV breast cancer. Expression of CD80 conferred the ability to deliver a costimulatory signal and thereby improved the antigen presentation capability of the tumor cells to patient T cells in vitro. Patients were vaccinated with 10(7) or 10(8) irradiated gene-modified tumor cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) or BCG, three times at 2-week intervals and then monthly until progressive disease developed. GM-CSF-related flulike symptoms and minor injection site reactions were observed frequently. Prolonged disease stabilization was observed in four patients but no objective tumor regressions were seen. Immune responses were measured in matched peripheral blood samples collected before and after treatment from 9 of 15 patients treated at the 10(8) tumor cell dose. Four patients exhibited MHC class I-restricted cytokine production in response to the parental breast cancer cell line. One patient maintained an increased number of circulating tumor-specific, interferon gamma-secreting CD8(+) T cells for 24 months after the last vaccination. One patient exhibited a tumor-specific interleukin 5 response to an autologous tumor cell line. This immunization strategy proved to be safe and feasible, and induced tumor-specific immune responses in a minority of patients; however, no objective tumor regressions were observed.

Monitoring immune responses in cancer patients receiving tumor vaccines.

Clinical evaluation of therapeutic tumor vaccines has resulted in examination and comparison of the types of immune function assays required to monitor tumor antigen-stimulated T cell effector function in immunized patients. Three of the most commonly used assays include ELISPOT, tetramer assay, and cytokine flow cytometry (CFC). Discussed are the method and principles for each assay and an assessment of important methodological, reagent, and data acquisition issues that are relevant for the accurate and effective use of the assays. The sensitivity and utility of the assays and present arguments advocating their integrated use in future immunomonitoring studies are also discussed.