A single intratumoral injection of IL-12 and GM-CSF-loaded slow-release microspheres induces T cell-dependent eradication of established primary and metastatic tumors in a murine lung tumor model. To determine how the delivery of cytokines directly to the microenvironment of a tumor nodule induces local and systemic antitumor T cell activity, we characterized therapy-induced phenotypic and functional changes in tumor-infiltrating T cell populations. Analysis of pretherapy tumors demonstrated that advanced primary tumors were infiltrated by CD4+ and CD8+ T cells with an effector/memory phenotype and CD4+CD25+Foxp3+ T suppressor cells. Tumor-associated effector memory CD8+ T cells displayed impaired cytotoxic function, whereas CD4+CD25+Foxp3+ cells effectively inhibited T cell proliferation demonstrating functional integrity. IL-12/GM-CSF treatment promoted a rapid up-regulation of CD43 and CD69 on CD8+ effector/memory T cells, augmented their ability to produce IFN-γ, and restored granzyme B expression. Importantly, treatment also induced a concomitant and progressive loss of T suppressors from the tumor. Further analysis established that activation of pre-existing effector memory T cells was short-lived and that both the effector/memory and the suppressor T cells became apoptotic within 4 days of treatment. Apoptotic death of pre-existing effector/memory and suppressor T cells was followed by infiltration of the tumor with activated, nonapoptotic CD8+ effector T lymphocytes on day 7 posttherapy. Both CD8+ T cell activation and T suppressor cell purge were mediated primarily by IL-12 and required IFN-γ. This study provides important insight into how local IL-12 therapy alters the immunosuppressive tumor milieu to one that is immunologically active, ultimately resulting in tumor regression.

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A single intratumoral injection of IL-12 and GM-CSF-encapsulated microspheres induces the complete regression of advanced spontaneous tumors in her-2/neu transgenic mice. However, tumor regression in this model is transient and long-term cure is not achieved due to recurrence. Posttherapy molecular analysis of immune activation/suppression markers within the tumor microenvironment demonstrated a dramatic up-regulation of IFN-γ and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGFβ, and IL-10 expression. Therapy-induced reversion of immune suppression was transient since all three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting in tumor resurgence. Repeated treatment enhanced short-term tumor regression, but did not augment long-term survival. Serial long-term analysis demonstrated that although chronic stimulation enhanced the IFN-γ response, this was countered by a parallel increase in Foxp3, TGFβ, and IL-10 expression. Analysis of tumor-infiltrating T lymphocyte populations showed that the expression of Foxp3 and IL-10 was associated with CD4+CD25+ T cells. Repeated treatment resulted in a progressive increase in tumor-infiltrating CD4+CD25+Foxp3+ T suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-infiltrating CD8+ T cells demonstrated that although treatment enhanced IFN-γ production, antitumor cytotoxicity was diminished. Monitoring of CD8+ T cells that specifically recognized a dominant MHC class I her-2/neu peptide showed a dramatic increase in tetramer-specific CD8+ T cells after the first treatment; however, continuous therapy resulted in the loss of this population. These results demonstrate that both enhanced suppressor activity and deletion of tumor-specific T cells are responsible for the progressive loss of efficacy that is associated with chronic immune therapy.

A single intratumoral injection of interleukin-12 and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres induced the regression of advanced spontaneous mammary tumors, suppressed additional tumor development, and enhanced survival in her-2/neu transgenic mice. Posttherapy tumor eradication was dependent on both CD4+ and CD8+ T cells and correlated with the tumor infiltration kinetics of a transient effector T-cell response. Upon long-term monitoring, tumor regression was found to be temporary, and disease-free survival was not achieved despite the development of systemic anti-tumor cytotoxic T-cell memory and antibody responses. Repeated immunization of mice enhanced short-term tumor suppression, resulting in the complete regression of primary tumors in up to 40% of the mice, but did not improve long-term survival owing to recurrence. The failure of chronic therapy to achieve complete cure was associated with an inability to maintain the intensity of the posttherapy effector T-cell response in this model.

Progesterone pretreatment of ovariectomized rat uteri increases the number of synchronously proliferating stromal cells in response to estradiol 17-b. To identify the signals involved in stimulating synchronous proliferation, sexually mature ovariectomized rats were injected with progesterone (2 mg) for 3 consecutive days. Estradiol 17-b (0 . 2 mg) was administered to initiate cell cycle entry. Uterine samples were removed at various times after hormone administration and changes in wingless (Wnt) pathway effectors and gene targets were identified by microarray. Progesterone pretreatment decreased glycogen synthase kinase-3b (GSK-3b) and increased expression of T-cell factor/lymphoid enhancer factor (TCF/LEF). GSK-3b protein decreased markedly in the uterine stroma of progesterone-pretreated uteri with the concomitant appearance of b-catenin in these stromal cells. Translocation of b-catenin from the cytosol to the nuclei in progesterone-pretreated stromal cells was stimulated in response to estradiol. b-Catenin binding to TCF/LEF increased (P!0 . 05) in progesteronepretreated uteri in response to estradiol. Progesterone stimulated the expression of the Wnt target gene urokinase plasminogen activator receptor (uPA-R) in the periluminal uterine stromal cells. The expression of uPA-R increased in progesteronepretreated stromal cells in response to estradiol administration. Together, the results indicate that progesterone initiates Wnt signaling in the uterine stroma by down-regulating GSK-3b. However, nuclear translocation of b-catenin and sufficient complex formation with TCF/LEF to activate stromal cell cycle entry requires estradiol. Stimulation of a uterine stromal cell line to proliferate and differentiate resulted in b-catenin accumulation, suggesting that endocrine-dependent Wnt signaling controls proliferation and differentiation (decidualization).