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1.
Figure 7

Figure 7. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Pan02 does not induce Foxp3 in dnTGFBetaRII CD4+25 T cells . A, Rag1−/− mice were reconstituted with CD4+25Foxp3 T cells taken from either CD57BL/6 (WT) or dnTGFBetaRII (TG) mice that cannot signal through the TGF-Beta receptor. At two days the mice were challenged with tumor and sacrificed after four weeks. The tumors were measured and TDLN were analyzed for expression of Foxp3 by real-time PCR.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
2.
Figure 4

Figure 4. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Tumor-derived CD4+ CD25+ cells are suppressive in vitro. To examine the suppressive capacity of tumor-infiltrating Treg, CD4+ CD25+ cells were isolated from the spleen and tumor of C57BL/6 (thy1.2+) mice 5 weeks after inoculation with Pan02. These tumor-induced CD4+25+ T cells were then placed in co-culture with CFSE-labeled thy1.1+ CD4+ CD25− T effector cells under activating conditions. Figure is representative FACS analysis and gating for CD4+ Thy1.1+ T cells.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
3.
Figure 3

Figure 3. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Foxp3+ T cells are prevalent in the Pan02 but not Eso2 tumor microenvironment. Immunohistochemical staining of Pan02 and Eso2 tumors grown in C57BL/6 mice for four weeks. In A, a and b, Hematoxylin and Eosin (H&E) staining of Pan02 and Eso2. In c and d, Immunohistochemical staining (Alexa Fluor 488) for Foxp3 in Pan02 and Eso2 tumors. In e and f, negative control sections processed identically to the test sections but stained without primary antibody. In B, the Pan02 tumors were stained for co-localization of Foxp3 and CD4 to further phenotype the tumor infiltrating lymphocytes.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
4.
Figure 1

Figure 1. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

In vitro conversion of naive CD4+25Foxp3 T cells into Foxp3+ T cells. A, Purified CD4+25 T cells were co-cultured in the presence or absence of TGF-Beta, irradiated splenocytes (APC), anti-CD3, or anti-CD28. The data shown is representative of three experiments. The expression of Foxp3 in CD4+25 T cells was normalized to 1.0. B, Induction of Foxp3 by TGF-Beta is concentration dependent. CD4+25 T cells were stimulated with anti-CD3 + anti-CD28 in the presence of various concentrations of TGF-Beta. Foxp3 expression in cells cultured without TGF-Beta was normalized to 1.00. The data represents the average of 3 experiments shown as fold change in expression relative to negative control.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
5.
Figure 5

Figure 5. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

In vivo induction of Foxp3 in CD4+25Foxp3 naive T cells by Pan02. All but control Rag1−/− mice were reconstituted with a purified population of CD4+25Foxp3 T cells on day -2. On day 0, all mice were either challenged with 0.25×106 Pan02 or Eso2. After four weeks the mice were sacrificed and the tumor draining lymph node (TDLN) and spleen removed. Foxp3 expression was measured by real-time PCR and is expressed as fold increase normalized to the initial CD4+25 naive T cell population.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
6.
Figure 8

Figure 8. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Tumor burden in reconstituted mice is not affected by TGF-Beta receptor signaling. A, Tumor burden of Rag1−/− mice reconstituted with WT versus dnTGFBetaRII CD4+25 naive T cells and subsequently challenged with Pan02 tumor. There was no statistically significant difference between the two groups. B, Pan02 tumor burden in normal C57BL/6 versus dnTGFBetaRII mice. Tumors in dnTGFBetaRII mice were significantly smaller than in wild type mice (p<0.02). The tumor burden is calculated as the tumor weight divided by the mouse total body weight expressed as a percentage.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
7.
Figure 2

Figure 2. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Pan02 pancreas adenocarcinoma produces elevated levels of TGF-Beta both in vitro and in vivo. A, Measured levels of TGF-Beta in the culture medium of Pan02 tumor cells. Pan02 and Eso2, a murine esophageal cancer line, were plated at 1.0×106 in RPMI/10% FBS and cultured for seven days. TGF-Beta levels were measured in the supernatant by ELISA. B, Elevated levels of TGF-Beta in the serum of Pan02 bearing mice. C57BL/6 mice were implanted with 0.25×106 Pan02 or Eso2 tumor cells. The tumors were allowed to grow for four weeks at which time the mice were sacrificed and the serum collected. Data is presented as the average of three experiments. Statistical significance is expressed as a p-value<0.001 as compared to the negative control.

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.
8.
Figure 6

Figure 6. From: Tumor derived TGF-Beta mediates conversion of CD4+Foxp3+ regulatory T cells in a murine model of pancreas cancer.

Blockade of TGF-Beta prevents induction of Foxp3 by Pan02 Rag1−/− mice were reconstituted on day -2 with CD4+25 T cells and challenged with tumor on day 0. The tumors were allowed to grow for four weeks after which the mice underwent Q7 injection with intravenous anti-TGF-Beta antibody (Ab7) or IgG isotype control antibody (IgG7). At six weeks the mice were sacrificed and the tumors and TDLN analyzed. A, Tumor-bearing mice that were treated with anti-TGF-Beta antibody had significantly smaller tumors. Tumor size is given as the multiplication of the diameter of the tumor in two dimensions which approximates the volume of the tumor. B, FACS analysis of TDLN for CD4 and CD25. The percentage CD4+25+ is indicated in each plot. C, Foxp3 expression in TDLN determined by real-time PCR. The expression in untreated Pan02-bearing mice was normalized to 1.0. The expression of Foxp3 after treatment with anti-TGF-Beta antibody was significantly lower than in untreated controls (p<0.001)

Tricia A. Moo-Young, et al. J Immunother. ;32(1):10.1097/CJI.0b013e318189f13c.

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