Chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counter-regulation.

Intratumoral delivery of IL-12 and GM-CSF induces local and systemic antitumor CD8(+) T cell activation and tumor kill. However, the effector response is transient and is rapidly countered by CD4(+) Foxp3(+) T suppressor cell expansion. To determine whether depletion of the pre-existing T suppressor cell pool prior to treatment could diminish posttherapy regulatory cell resurgence, FVBneuN mice bearing advanced spontaneous mammary tumors were treated with cyclophosphamide (CY) 1 d before IL-12/GM-CSF therapy. Administration of CY mediated a significant delay in the post-IL-12/GM-CSF T suppressor cell rebound, resulting in a 7-fold increase in the CD8(+) CTL/T suppressor cell ratio, a 3-fold enhancement of CTL cytotoxicity, and an extension of the effector window from 3 to 7 d. In long-term therapy studies, chronic chemoimmunotherapy promoted a dramatic enhancement of tumor regression, resulting in complete cure in 44% of the mice receiving CY plus IL-12/GM-CSF. Tumor eradication in the chronic therapy setting was associated with the ability to repeatedly rescue and maintain cytotoxic CD8(+) T cell activity. These findings demonstrated that chronic administration of CY in conjunction with immune therapy enhances the initial induction of antitumor T effector cells and, more importantly, sustains their cytotoxic activity over the long-term via persistent blockade of homeostatic counter-regulation.

Reversing tumor immune suppression with intratumoral IL-12: activation of tumor-associated T effector/memory cells, induction of T suppressor apoptosis, and infiltration of CD8+ T effectors.

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-gamma, 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-gamma. 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.

Chronic immune therapy induces a progressive increase in intratumoral T suppressor activity and a concurrent loss of tumor-specific CD8+ T effectors in her-2/neu transgenic mice bearing advanced spontaneous tumors.

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-gamma and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGFbeta, 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-gamma response, this was countered by a parallel increase in Foxp3, TGFbeta, 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-gamma 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.

IL-12 + GM-CSF microsphere therapy induces eradication of advanced spontaneous tumors in her-2/neu transgenic mice but fails to achieve long-term cure due to the inability to maintain effector T-cell activity.

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.

Characterization of cytokine-encapsulated controlled-release microsphere adjuvants.

Controlled-release, injectable polymer microspheres provide a clinically feasible alternative to gene-modification for the local, sustained delivery of cytokines to tumors for cancer immunotherapy. Long-term release kinetics, bioactivity profiles, and stability of interleukin-2 (IL-2), interleukin-12 (IL-12), and granulocyte- macrophage colony-stimulating factor (GM-CSF)-encapsulated microspheres prepared by phase inversion nanoencapsulation (PIN) were evaluated. While all formulations released physiologically relevant quantities of cytokine for up to 30 days, the individual release kinetics were different. Recovery of specific activity after encapsulation was 40%, 60%, and 90%-that of pre-encapsulation levels for IL-2, GM-CSF and IL-12, respectively. Upon storage, the IL-12 microspheres rapidly lost activity, whereas IL-2 and GM-CSF microspheres remained stable for at least 9 weeks. These studies demonstrate that biochemical properties of microsphere formulations vary depending on the cytokine, and rigorous characterization of formulations is a prerequisite to in vivo testing.

The flavonoid, quercetin, differentially regulates Th-1 (IFNgamma) and Th-2 (IL4) cytokine gene expression by normal peripheral blood mononuclear cells.

Flavonoids are plant metabolites that are dietary antioxidants and exert significant anti-tumor, anti-allergic, anti-inflammatory and anti-viral effects. It is generally accepted that Th-1 derived cytokines such as IL-2, IFNgamma and IL-12 promote cellular immunity while Th-2 derived cytokines such as IL-4, IL-5, IL-6 exert negative immunoregulatory effects on cellular immunity while upregulating humoral immunity. The molecular mechanisms underlying the biological activities of flavonoids have not been elucidated. We hypothesize that the flavonoid, quercetin, exert significant anti-viral and anti-tumor effects possibly by modulating the production of Th-1 and Th-2 derived cytokines. Peripheral blood mononuclear cells (PBMC, 1 x 10(6) cells/ml) from normal subjects were cultured with different concentrations of quercetin (0.5-50 microM) for 24-72 h and supernates were quantitated for IFN-gamma and IL-4 by ELISA and antiviral activity of IFNgamma by bioassay. FACS analysis was done to determine the number of IFN-gamma and IL-4 positive cells and RT-PCR was done to quantitate gene expression. Quercetin significantly induces the gene expression as well as the production of Th-1 derived IFNgamma and the downregulates Th-2 derived IL-4 by normal PBMC. Further, quercetin treatment increased the phenotypic expression of IFNgamma cells and decreased IL-4 positive cells by FACS analysis, which corroborate with protein secretion and gene expression studies. These results suggest that the beneficial immuno-stimulatory effects of quercetin may be mediated through the induction of Th-1 derived cytokine, IFNgamma, and inhibition of Th-2 derived cytokine, IL-4.