Recombinant Tp0965 protein, purified using Ni-NTA resin, analysed by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis(SDS-PAGE).

<p>The electrophoresis showed a 40 kDa protein band. M: protein markers (lanes concentrations: 0.1∼0.2 mg/ml). Arrow indates the recombinant Tp0965 protein.</p

Effects of rTp0965 on chemoattraction of monocytes to HUVECs.

<p>(A) Confluent HUVECs in the wells were preincubated with rTp0965 (800 ng/ml) for 24 h. Monocyte THP-1 cells stained with calcein AM were then added in the inserts for 2 h and the percentage of THP-1 cells in the wells were counted under the fluorescence microscope. (B and C) Confluent HUVECs were incubated with various concentrations of the rTp0965 for increasing time intervals. The levels of MCP-1 in the supernatants were determined by ELISA. (D and E) Confluent HUVECs were incubated with various concentrations of rTp0965 for increasing time intervals. The levels of MCP-1 mRNA in HUVECs were measured by RT-PCR. (* = <i>P</i><0.05, compared to a control). Results shown are those of one experiment representative of three similar experiments.</p

Effects of rTp0965 on F-actin reorganization. Confluent HUVECs were left untreated (a) or were stimulated with rTp0965 (800 ng/ml; b) for 24 h.

<p>(<b>A</b>). Other HUVECs were preincubated with Y-27632 for 30 mins and then stimulated with rTp0965 (800 ng/ml; c). Cells were then fixed and were stained with rhodamine-phalloidin. (<b>B</b>) Effect of rTp0965 on HUVECs expression of claudin-1. Confluent HUVECs were stimulated with rTp0965 (800 ng/ml) or preincubated with Y-27632 for 30 mins and then stimulated with rTp0965 (800 ng/ml) for 2 h. The expression of claudin-1 in total cell lysates were evaluated by Western blot. A representative of three experiments is shown.</p

Effect of rTp0965 on adherence of monocytes to HUVECs.

<p>(A) Confluent HUVECs were stimulated with rTp0965 (800 ng/ml) for 24 h. Monocyte THP-1 cells stained with calcein AM were then added and incubated for 6 h. The percentage of THP-1 cells were counted under the fluorescence microscope. (B–E) Confluent HUVECs were incubated with various concentrations of the rTp0965 for increasing time intervals. The levels of ICAM-1 and E-selectin mRNA in HUVECs were assayed by RT-PCR. (F-I) Confluent HUVECs were incubated with various concentrations of rTp0965 for increasing time intervals. The expression levels of ICAM-1 and E-selectin were measured by cell ELISA. (* = <i>P</i><0.05, compared to a control). Results shown are those of one experiment representative of three similar experiments.</p

Effects of rTp0965 on the permeability of HUVEC monolayers. Confluent HUVECs in the wells were preincubated with rTp0965 (800 ng/ml) for 24 h.

<p>(<b>A</b>). HRP was then added to the transwell inserts and the flux of HRP in supernatants was calculated at 1 and 4h. (<b>B</b>) Effects of rTp0965 on migration of monocytes across HUVECs. Confluent monolayers of HUVECs cultured in Transwell inserts were pretreated with Y-27632 for 30 mins and HUVECs were then stimulated with rTp0965 (800 ng/ml) for 24 h. Monocyte THP-1 cells stained with calcein AM were then added in the inserts and the percentage of THP-1 cells in the wells was counted at 4 h under the fluorescence microscope.</p

Tim-3 Expression Defines Regulatory T Cells in Human Tumors

<div><p>Tim-3, a member of the novel Tim (T cell immunoglobulin and mucin domain) family, has been reported to negatively regulate the immune responses against viral infection and had implications for autoimmune disease. However, the nature and role of Tim-3⁺ CD4 T cells in human tumors remain largely unknown. In the present study, we characterized Tim-3⁺ CD4 T cells in 100 specimens from human hepatocellular, cervical, colorectal and ovarian carcinoma patients. Compared with peripheral blood and nontumor-infiltrating lymphocytes, the lymphocytes isolated from the corresponding tumor tissues of hepatocellular, cervical, colorectal and ovarian carcinoma patients contained significantly greater proportion of Tim-3⁺ CD4 T cells. The majority of tumor-derived Tim-3⁺ CD4 T cells exhibited an impaired capacity to produce IFN-γ and IL-2, but expressed higher levels of CD25, Foxp3, CTLA-4 and GITR than their Tim-3⁻ CD4 T cell counterparts. In contrast, most Tim-3⁺ CD4 T cells isolated from the paired nontumor tissues and peripheral blood did not express these molecules. Moreover, tumor-derived Tim-3⁺ CD4 T cells, but not tumor-derived Tim-3⁻ CD4 T cells, significantly suppressed the proliferation of autologous CD8⁺ T cells <i>in vitro</i>. Notably, multi-color immunofluorescence and confocal microscopy demonstrated that Tim-3⁺Foxp3⁺CD4⁺ cells were preferentially distributed in the tumor nest rather than the peritumoral stroma of hepatocellular carcinoma. Together, our data indicate that Tim-3-expressing CD4 T cells in human tumors could represent the functional regulatory T cells which contribute to the formation of the immune-suppressive tumor micromilieu.</p> </div

Tumor-infiltrating Tim-3⁺ CD4 T cells exhibit impaired ability to produce Th1-type cytokines. A

<p>. Representative FACS analysis illustrating the association between IFN-γ and Tim-3 expression in CD4 T cells isolated from the PBMCs of normal healthy controls (NC, <i>n</i> = 15) and HCC patients (HCC, <i>n</i> = 10), and nontumor-infiltrating lymphocytes (NILs, <i>n</i> = 6) and tumor-infiltrating lymphocytes (TILs, <i>n</i> = 6) from HCC patients. Statistical analysis showed the mean percentage of IFN-γ⁺ cells in the Tim-3 positive or negative CD4 T cell subsets (left), and the proportion of Tim-3⁺ cells in the IFN-γ positive or negative CD4 T cell subsets (right). <b>B</b>. Relationship between the expression of Tim-3 and IL-2 in CD4 T cells, At least 4 samples were tested in each group. Bars indicate the SEM; *, <i>P</i><0.05; **, <i>P</i><0.01; ***, <i>P</i><0.001. <b>C</b>. The majority of Tim-3⁺ CD4 T cells isolated from TILs did not express the Th1-specific transcription factor T-bet, <i>n = </i>4. <b>D</b>. Correlation of Tim-3 and PD-1 expression in tumor tissues, <i>n = </i>4.</p

Clinical and pathological characteristics of the HCC patients.

<p>Note: HBsAg, hepatitis B surface antigen; ALT, alanine aminotransferase; AFP, a-fetoprotein; TNM, tumor-node-metastasis.</p

Close interaction between Tim-3⁺CD4⁺ cells and galectin-9⁺ cells in HCC tumor tissue. A

<p>. Paraffin-embedded HCC tumor sections were subjected to multi-color immunofluorescence staining for CD4 (white), Tim-3 (green), galectin-9 (red) and nuclei (DAPI, blue). Confocal analysis showed that Tim-3⁺CD4⁺ cells were in close contact with galectin-9⁺ cells in the tumor nest. <b>B</b>. Paraffin-embedded HCC tumor sections were subjected to multi-color immunofluorescence staining for CD4 (green), galectin-9 (red) and nuclei (DAPI, blue). Confocal analysis indicated that galectin-9 was expressed by the tumor-infiltrating CD4⁺ cells. <b>C</b>. Paraffin-embedded HCC tumor sections were subjected to multi-color immunofluorescence staining for CD68 (green), galectin-9 (red) and nuclei (DAPI, blue). Confocal analysis indicated that galectin-9 was expressed by tumor-infiltrating CD68⁺ macrophages. White arrows in A–C indicate the magnified cells. Images are representative of 13 samples. For the quantification of CD4^+/−galectin-9^+/− cells <b>(D)</b> or CD68^+/−galectin-9^+/−<b>(E)</b> cells, eight CD4 or CD68 hot-spot fields per slide were taken with 40× oil-immersed objective lens. Numbers of each subset cells were counted manually by two independent, blinded observers. Gal-9 in D and E is short for galectin-9.</p

Tim-3 expression defines a population of CD4 T cells with Treg characteristics in TILs. A

<p>. Representative FACS analysis showing the association of CD25, CD127 and Tim-3 expression in CD4 T cells isolated from the PBMCs of normal healthy controls (NC, <i>n</i> = 7) and HCC patients (HCC, <i>n</i> = 6), and nontumor-infiltrating lymphocytes (NILs, <i>n</i> = 18) and tumor-infiltrating lymphocytes (TILs, <i>n</i> = 18) from HCC patients. <b>B</b>. FACS and statistical data showing that most tumor-infiltrating Tim-3⁺ CD4 T cells expressed the Treg-specific transcription factor Foxp3. Bars indicate the SEM; ***, <i>P</i><0.001. <b>C</b>. Paraffin-embedded HCC sections were subjected to multi-color immunofluorescence staining for CD4 (white), Tim-3 (green), Foxp3 (red) and nuclei (DAPI, blue). Confocal microscopic analysis revealing the different distribution of Tim-3⁺Foxp3⁺CD4⁺ cells in the peritumoral stroma and tumor nest of HCC. Images are representative of 13 samples. For the quantification of CD4⁺Tim-3^+/−Foxp3^+/− cells, eight CD4 hot-spot fields per slide were taken with 40× oil-immersed objective lens. Numbers of each subset cells were counted manually by two independent, blinded observers.</p