CD4⁺CD25⁺ T cells have been proposed as the principal regulators of both self-tolerance and transplantation tolerance. Although CD4⁺CD25⁺ T cells do have a suppressive role in transplantation tolerance, so do CD4⁺CD25⁻ T cells, although 10-fold less potent. Abs to CTLA-4, CD25, IL-10, and IL-4 were unable to abrogate suppression mediated by tolerant spleen cells so excluding any of these molecules as critical agents of suppression. CD4⁺CD25⁺ T cells from naive mice can also prevent rejection despite the lack of any previous experience of donor alloantigens. However, this requires many more naive than tolerized cells to provide the same degree of suppression. This suggests that a capacity to regulate transplant rejection pre-exists in naive mice, and may be amplified in “tolerized” mice. Serial analysis of gene expression confirmed that cells sorted into CD4⁺CD25⁺ and CD4⁺CD25⁻ populations were distinct in that they responded to TCR ligation with very different programs of gene expression. Further characterization of the differentially expressed genes may lead to the development of diagnostic tests to monitor the tolerant state.

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. Here, we report a combinatorial immunotherapy approach that uses a highly efficient and tumor-selective gene carrier to improve anticancer efficacy and circumvent the systemic toxicity. In this study, we engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that exhibit not only enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)–cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2–encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8⁺ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.

Chemokine CCL5/RANTES is highly expressed in cancer where it contributes to inflammation and malignant progression. In this study, we show that CCL5 plays a critical role in immune escape in colorectal cancer. We found that higher levels of CCL5 expression in human and murine colon tumor cells correlated with higher levels of apoptosis of CD8+ T cells and infiltration of T-regulatory cells (T_reg). In mouse cells, RNA interference (RNAi)-mediated knockdown of CCL5 delayed tumor growth in immunocompetent syngeneic hosts but had no effect on tumor growth in immunodeficient hosts. Reduced tumor growth was correlated with a reduction in T_reg infiltration and CD8⁺ T-cell apoptosis in tumors. Notably, we found that CCL5 enhanced the cytotoxicity of T_reg against CD8⁺ T cells. We also found tumor growth to be diminished in mice lacking CCR5, a CCL5 receptor, where a similar decrease in both T_reg cell infiltration and CD8⁺ T-cell apoptosis was noted. TGF-β signaling blockade diminished apoptosis of CD8⁺ T cells, implicating TGF-β as an effector of CCL5 action. In support of this concept, CCL5 failed to enhance the production of TGF-β by CCR5-deficient T_reg or to enhance their cytotoxic effects against CD8⁺ T cells. CCR5 signaling blockade also diminished the in vivo suppressive capacity of T_reg in inhibiting the antitumor responses of CD8⁺ T cells, in the same way as CCL5 signaling blockade. Together, our findings establish that CCL5/CCR5 signaling recruits T_reg to tumors and enhances their ability to kill antitumor CD8⁺ T cells, thereby defining a novel mechanism of immune escape in colorectal cancer. Cancer Res; 72(5); 1092–102. ©2012 AACR.

Glioma-associated microglia/macrophages (GAMs) comprise macrophages of peripheral origin and brain-intrinsic microglia, which support tumor progression. Chemokine C-C ligand 5 (CCL5) is an inflammatory mediator produced by immune cells and is involved in tumor growth and migration in several cancers, including glioma. However, the mechanisms detailing how CCL5 facilitates glioma invasion remain largely unresolved. Glioma migration and invasion were determined by wound healing, transwell assay, and 3D µ-slide chemotaxis assay. The expression levels of CCL5, CD68, matrix metalloproteinase 2 (MMP2), phosphorylated Ca²⁺/calmodulin-dependent protein kinase II (p-CaMKII), p-Akt, and phosphorylated proline-rich tyrosine kinase 2 were determined by cytokine array, quantitative PCR, western blot, or immunohistochemistry. Zymography and intracellular calcium assays were used to analyze MMP2 activity and intracellular calcium levels, respectively. CCL5 modulated the migratory and invasive activities of human glioma cells in association with MMP2 expression. In response to CCL5, glioma cells underwent a synchronized increase in intracellular calcium levels and p-CaMKII and p-Akt expression levels. CCL5-directed glioma invasion and increases in MMP2 were suppressed after inhibition of p-CaMKII. Glioma cells tended to migrate toward GAM-conditioned media activated by granulocyte-macrophage colony-stimulating factor (GM-CSF) in which CCL5 was abundant. This homing effect was associated with MMP2 upregulation, and could be ameliorated either by controlling intracellular and extracellular calcium levels or by CCL5 antagonism. Clinical results also revealed the associations between CCL5 and GAM activation. Our results suggest that modulation of glioma CaMKII may restrict the effect of CCL5 on glioma invasion and could be a potential therapeutic target for alleviating glioma growth.

TIM-3 functions to enforce CD8+ T cell exhaustion, a dysfunctional state associated with the tolerization of tumor microenvironment. Here we report apoptosis of IFN-γ competent TIM-3+ population of tumor-infiltrating CD8+ T cells in colon cancer. In humans suffering from colorectal cancer, TIM-3+ population is higher in cancer tissue-resident relative to peripheral blood CD8+ T cells. Both the TIM-3+ and TIM-3- cancer tissue-resident CD8+ T cells secrete IFN-γ of comparable levels, although apoptotic cells are more in TIM-3+ compared to TIM-3- population. In mouse CT26 colon tumor model, majority of tumor-infiltrating CD8+ T cells express TIM-3 and execute cytolysis function with higher effector cytokine secretion and apoptosis in TIM-3+ compared to TIM-3- population. The tumor cells secrete galectin-9, which increases apoptosis of tumor-infiltrating CD8+ T cells. Galectin-9/TIM-3 signaling blockade with anti-TIM-3 antibody reduces the apoptosis and in addition, inhibits tumor growth in mice. The blockade increases therapeutic efficacy of cyclophosphamide to treat tumor in mice as well. These results reveal a previously unexplored role of TIM-3 on tumor-infiltrating CD8+ T cells in vivo.

Interleukin-12 (IL-12) has long been considered to be effective in triggering an anticancer immune response, however, the dosage has been limited by potential systemic immunotoxicity. Since focused ultrasound (FUS) has been confirmed to temporally and locally open the blood–brain barrier (BBB), the purpose of this study was to elucidate the possibility of combining FUS-induced BBB opening with IL-12 delivery to enhance the anticancer immunological response for glioma treatment. FUS energy combined with microbubble administration was delivered transcranially to open BBB, and C-6 glioma rats were used in this study. The efficacy in inducing BBB opening and the corresponding immunological response were primarily evaluated in normal animals. The anticancer immune-triggering chemokine, IL-12, was intraperitoneally administered during the treatment phase to evaluate the effect of immunological response on tumor progression. Glioma animals were sub-grouped to evaluate the effect of the immune response in suppressing glioma when IL-12 was combined with FUS-induced BBB opening. We performed flow cytometry to verify consequent immune cell population changes of peripheral/tissue lymphocytes as well as macrophages from the animals. Brain sections of sacrificed animals were also used for histological and immunohistochemical analysis. IL-12 level among experimental groups were measured via ELISA analysis. We also analyzed survival and followed tumor progression in vivo via T2-weighted magnetic resonance imaging. FUS-induced BBB opening had no obvious effect on the T lymphocytes population in normal animals, either in the brain or systemically. Yet, it triggered mild changes in the tumor-infiltrating lymphocyte (TIL) population, particularly in numbers of CD3+CD8+ cytotoxic T lymphocytes (CTLs) in the tumor region. IL-12 administration triggered a profound increase in all TIL populations, including CD3+CD4+ T helper cells (Th), CTL, and CD4+CD25+ regulatory T cells (Treg), but combined FUS-BBB opening with IL-12 administration produced the most significant IL-12 increase, CTL increase and CTL/Treg ratio increase, thus contributing to the most significant suppression of tumor progression and increased animal survival. This study provides evidence that FUS-BBB opening can enhance immune-modulating agent delivery to the brain, which improve the anticancer immune response in brain tumor treatment.