Tumor-induced impairment of TCR signaling results in compromised functionality of tumor-infiltrating regulatory T cells - PubMed (original) (raw)

Tumor-induced impairment of TCR signaling results in compromised functionality of tumor-infiltrating regulatory T cells

M E Christine Lutsiak et al. J Immunol. 2008.

Abstract

This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4(+)CD25(+)FoxP3(+)GITR(high)CD27(low)CD62L(-). The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.

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Figures

Figure 1. Expansion and apoptosis of Treg cells in spleen and tumor infiltrate

(A) The level of in vivo proliferation of CD4+CD25+ Treg cells was measured by BrdU incorporation over 72 hours at various time points following tumor implant. The difference between spleen and tumor infiltrate is significant (P <0.001). Data are representative of three experiments. (B) The level of apoptosis of freshly recovered CD4+CD25+ Treg cells was measured using Annexin V staining at various time points following tumor implant. The difference between spleen and tumor infiltrate is significant (_P_ <0.05). Data are representative of three experiments. (C) Sorted CD4+CD25+ Treg cells (>96% FoxP3+) from the spleen and tumor infiltrate were cultured either in low serum media or with bound anti-CD3 in order to induce apoptosis. Apoptosis was measured using Annexin V staining after 24 hours. The experiment was repeated twice and one example is shown.

Figure 2. Functionality of Treg cells from spleens and tumor infiltrate of tumor-bearing mice

(A) Functionality of Treg cells from the spleen. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of splenic CD4+CD25+ Treg cells from tumor-bearing animals or non-tumor-bearing controls. The ratio of Treg cells:CD8+ cells was 1:1. Proliferation of CD8+ cells was measured by 3H-thymidine incorporation. *P<0.05; **P<0.005. Data are representative of six experiments. (B) Functionality of Treg cells from the spleen. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of splenic CD4+CD25+ Treg cells from tumor-bearing animals or non-tumor-bearing controls. The ratio of Treg cells:CD8+ cells was 1:1. IFN-γ production by CD8+ T cells was measured by cytokine bead array. *P<0.05. Data are representative of three experiments. (C) Functionality of Treg cells from the tumor infiltrate. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from tumor infiltrate or spleens of non-tumor-bearing controls. The ratio of Treg cells:CD8+ cells was 1:1. *P<0.05; **P<0.005. Data are representative of three experiments. (D) Functionality of Treg cells from the tumor infiltrate. CD8+ T cells from healthy animals were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from tumor infiltrate or spleens of non-tumor-bearing controls. Multiple ratios of Treg cells:CD8+ cells were used: 2:1, 1:1, 0.5:1, 0.25:1. **P<0.005. The experiment was repeated twice.

Figure 3. Capability of CD4+CD25+ Treg cells or CD8+ T cells to respond to stimulation through the TCR

(A) Cells from the tumor infiltrate or from spleen were stained for CD4, CD25, FoxP3 and either the TCR (left panel) or CD3 (right panel). During analysis, gates were placed around CD4+CD25+FoxP3+ cells and the level of expression of the TCR (left panel) or CD3 (right panel) was determined. The solid, filled histogram is the isotype control, the solid line histogram is the spleen, and the dotted line histogram is the tumor infiltrate. Data are representative of six experiments. (B) Calcium influx in Treg cells. Indo-labeled cells from either spleen (left panels) or tumor infiltrate (right panels) were loaded with 5 (top panels) or 1 (bottom panels) μg/mL biotinylated anti-CD3. In all cases, at 30 seconds a stimulus of 1 μg streptavidin was given. For analysis, gates were placed around CD4+CD25+ cells. Data are presented as the mean of 405 nm/510 nm versus time. Data are representative of three experiments. (C) Calcium influx in CD8+ T cells. Indo-labeled cells from either spleen (left panels) or tumor infiltrate (right panels) were loaded with 5 (top panels) or 1 (bottom panels) μg/mL biotinylated anti-CD3. In all cases, at 30 seconds a stimulus of 1 μg streptavidin was given. For analysis, gates were places around CD8+ cells. Data are presented as the mean of 405 nm/510 nm versus time. The experiment was repeated twice.

Figure 4. Phenotype and functionality of Treg cells from _Itk_-/- mice

(A) Expression of various cell surface markers on Treg cells from _Itk_-/- mice was examined by flow cytometry. CD25 expression histogram is gated on CD4+ cells. All other markers were examined on CD4+CD25+ cells. The solid line histogram represents the wild type controls, and the dashed line represents the _Itk_-/- mice. Data are representative of three experiments. (B) CD8+ T cells from C57BL/6 mice were stimulated with anti-CD3 and APC in the absence or presence of CD4+CD25+ Treg cells from C57BL/6 or _Itk_-/- mice. Proliferation of CD8+ cells was measured by 3H-thymidine incorporation. *P<0.05; **P<0.005 Data are representative of three experiments.

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Tumor-induced impairment of TCR signaling results in compromised functionality of tumor-infiltrating regulatory T cells - PubMed (original) (raw)