Accumulation of CCR4+ CTLA-4hi FOXP3+CD25hi Regulatory T Cells in Colon Adenocarcinomas Correlate to Reduced Activation of Conventional T Cells

BACKGROUND: Colorectal cancer usually gives rise to a specific anti-tumor immune response, but for unknown reasons the resulting immunity is not able to clear the tumor. Recruitment of activated effector lymphocytes to the tumor is important for efficient anti-tumor responses, while the presence of regulatory T cells (Treg) down-modulate tumor-specific immunity. We therefore aimed to determine homing mechanisms and activation stage of Treg and effector T cell infiltrating colon tumors compared to cells from the unaffected mucosa in patients suffering from colon adenocarcinoma. METHODOLOGY/PRINCIPAL FINDINGS: Lymphocytes were isolated from unaffected and tumor mucosa from patients with colon adenocarcinoma, and flow cytometry, immunohistochemistry, and quantitative PCR was used to investigate the homing mechanisms and activation stage of infiltrating Treg and conventional lymphocytes. We detected significantly higher frequencies of CD25(high)FOXP3⁺CD127(low) putative Treg in tumors than unaffected mucosa, which had a complete demethylation in the FOXP3 promotor. Tumor-associated Treg had a high expression of CTLA-4, and some appeared to be antigen experienced effector/memory cells based on their expression of αEβ7 (CD103). There were also significantly fewer activated T cells and more CTLA-4⁺ conventional T cells susceptible to immune regulation in the tumor-associated mucosa. In contrast, CD8⁺granzyme B⁺ putative cytotoxic cells were efficiently recruited to the tumors. The frequencies of cells expressing α4β7 and the Th1 associated chemokine receptor CXCR3 were significantly decreased among CD4⁺ T cells in the tumor, while frequencies of CD4⁺CCR4⁺ lymphocytes were significantly increased. CONCLUSIONS/SIGNIFICANCE: This study shows that CCR4⁺CTLA4(hi) Treg accumulate in colon tumors, while the frequencies of activated conventional Th1 type T cells are decreased. The altered lymphocyte composition in colon tumors will probably diminish the ability of the immune system to effectively attack tumor cells, and reducing the Treg activity is an important challenge for future immunotherapy protocols

Polymer Nanoparticle-Mediated Delivery of Oxidized Tumor Lysate-Based Cancer Vaccines

Cancer vaccination is a powerful strategy to combat cancer. A very attractive approach to prime the immune system against cancer cells involves the use of tumor lysate as antigen source. The immunogenicity of tumor lysate can be further enhanced by treatment with hypochlorous acid. This study explores poly(lactic-co-glycolic acid) (PLGA) nanoparticles to enhance the delivery of oxidized tumor lysate to dendritic cells. Using human donor-derived dendritic cells, it is found that the use of PLGA nanoparticles enhances antigen uptake and dendritic cell maturation, as compared to the use of the free tumor lysate. The ability of the activated dendritic cells to stimulate autologous peripheral blood mononuclear cells (PBMCs) is assessed in vitro by coculturing PBMCs with A375 melanoma cells. Live cell imaging analysis of this experiment highlights the potential of nanoparticle-mediated dendritic-cell-based vaccination approaches. Finally, the efficacy of the PLGA nanoparticle formulation is evaluated in vivo in a therapeutic vaccination study using B16F10 tumor-bearing C57BL/6J mice. Animals that are challenged with the polymer nanoparticle-based oxidized tumor lysate formulation survive for up to 50 days, in contrast to a maximum of 41 days for the group that receives the corresponding free oxidized tumor lysate-based vaccine.L

Rate of Freeze Impacts the Survival and Immune Responses Post Cryoablation of Melanoma

The emergence of ablative therapies has revolutionized the treatment of inoperable solid tumors. Cryoablation stands out for its uniqueness of operation based on hypothermia, and for its ability to unleash the native tumor antigens, resulting in the generation of anti-tumor immune responses. It is not clearly understood how alterations in the rate of freeze impact the immune response outcomes. In this study, we tested fast freeze and slow freeze rates for their locoregional effectiveness and their ability to elicit immune responses in a B16F10 mouse model of melanoma. Tumor bearing mice treated with fast freeze protocol survived better than the ones treated with slow freeze protocol. Fast freeze resulted in a higher magnitude of CD4+ and CD8+ T-cell responses, and a significantly extended survival post re-challenge. Thus, fast freeze rate should be applied in any future studies employing cryoablation as an in vivo vaccination tool in conjunction with targeted immunotherapies.</jats:p

CD4+ regulatory T cells in gastric cancer mucosa are proliferating and express high levels of IL-10 but little TGF-β

An increase of regulatory T cells, defined as CD25(high)- and/or FOXP3(+)-expressing CD4(+) T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ

Expression of MAdCAM-1 on endothelial cells in tumor and unaffected mucosa.

<p>The expression of MAdCAM-1 mRNA (A) and protein (B) was evaluated in the tumor and unaffected mucosa real time PCR and immunohistochemistry respectively. The mRNA concentrations were normalized against 18s mRNA. Graphs show paired individual values from 8 and 12 patients, and representative MAdCAM-1 staining in unaffected mucosa (above) and tumor tissue (below). * p<0.05, Wilcoxon signed rank test.</p

Chemokine receptor expression on T cells from tumor and unaffected mucosa.

<p>The expression of CXCR3 (A) and CCR4 (B) was evaluated on conventional CD4⁺ and CD8⁺ T cells and Treg from the tumor and unaffected mucosa using flow cytometry. The frequencies of positive cells are given as the percentage of all CD4⁺ or CD8⁺ cells, respectively. Graphs show paired individual values from 9 to 10 patients. * p<0.05, ** p<0.01, Wilcoxon signed rank test.</p

Immunofluorescence detection of T cell subsets in tumor and unaffected mucosa.

<p>The expression of surface CD4 (A and B) and CD8 (C and D) in combination with intracellular FOXP3 was determined in unaffected (A and C) and tumor mucosa (B and D). E and F show isotype control staining of the respective tissues, and G a close-up of FOXP3⁺ cells (green nucleus) in close association with CD8⁺ conventional T cells (red surface staining) in a section of tumor mucosa.</p