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Kidney transplantation: analysis of the expression and T cell-mediated activation of latent TGF-β.

Willet JD, Pichitsiri W, Jenkinson SE, Brain JG, Wood K, Alhasan AA, Spielhofer J, Robertson H, Ali S, Kirby JA - J. Leukoc. Biol. (2012)

Bottom Line: A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-β by pSmad 3.However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-β associated with TEC.Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-β.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Medicine, The Medical School, Newcastle University, Newcastle upon Tyne, UK.

ABSTRACT
Activated T cells infiltrate a renal allograft during rejection and can respond to TGF-β within the tubules, causing local differentiation and expression of the αE(CD103)β7 integrin. This study was performed to examine the expression of latent TGF-β within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF-β complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-β by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-β associated with TEC. Although activated T cells expressed little cell-surface TSP-1, this was increased by culture with fibronectin or fibronectin-expressing renal TEC. Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-β. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell-surface TSP-1, allowing activation of latent TGF-β sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.

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Examination of T cell expression of the β 6 integrin, NRP-1, and TSP-1.Immunofluorescence flow cytometry of activated T cells showing no significant cell surface of expression of the β6 integrin (A). Subpopulations of activated T cells expressed cell-surface NRP-1 (B) and TSP-1 (C). Each shaded histogram shows the result of labeling with isotype-matched control antibodies; the open histograms show experimental labeling. (D) The mean expression of cell-surface TSP-1 by duplicate samples of activated T cells from three different volunteer blood donors (D1, D2, D3) was increased by coculture with TEC for 1 h prior to flow cytometry (P<0.05); pretreatment of the TEC with TGF-β1 further increased the expression of TSP-1 (P<0.005). (E) Representative immunofluorescence showing extracellular expression of fibronectin (FITC, green) by resting, immortalized TEC (DAPI-stained nuclei, blue). (F) T cells show markedly increased cell-surface expression of TSP-1 after addition to plate-bound fibronectin for 1 h. The bars show mean results ± sem; two separate experiments.
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Figure 4: Examination of T cell expression of the β 6 integrin, NRP-1, and TSP-1.Immunofluorescence flow cytometry of activated T cells showing no significant cell surface of expression of the β6 integrin (A). Subpopulations of activated T cells expressed cell-surface NRP-1 (B) and TSP-1 (C). Each shaded histogram shows the result of labeling with isotype-matched control antibodies; the open histograms show experimental labeling. (D) The mean expression of cell-surface TSP-1 by duplicate samples of activated T cells from three different volunteer blood donors (D1, D2, D3) was increased by coculture with TEC for 1 h prior to flow cytometry (P<0.05); pretreatment of the TEC with TGF-β1 further increased the expression of TSP-1 (P<0.005). (E) Representative immunofluorescence showing extracellular expression of fibronectin (FITC, green) by resting, immortalized TEC (DAPI-stained nuclei, blue). (F) T cells show markedly increased cell-surface expression of TSP-1 after addition to plate-bound fibronectin for 1 h. The bars show mean results ± sem; two separate experiments.

Mentions: Activated human T cells did not express cell-surface β6 integrin (Fig. 4A), and neither TSP-1 nor NRP-1 was expressed by resting T cells (not shown). However, a small proportion of T cells did express cell-surface NRP-1 after activation for 48 h (17.3±2.3%; n=8 T cell donors; mean±sem; representative result shown in Fig. 4B) and TSP-1 after 72 h (15.9±1.3%; n=19 T cell donors; representative result shown in Fig. 4C); the majority of activated T cells expressed intracellular TSP-1 (not shown). The proportion of activated T cells expressing cell-surface TSP-1 was increased significantly by 1 h coculture with resting TEC (P<0.05; n=3 separate T cell donors; Fig. 4D); culture with TGF-β-pretreated epithelial cells further increased cell-surface TSP-1 expression (P<0.005; Fig. 4D). Immunofluorescence analysis of cultured TEC demonstrated deposition of fibronectin between the cells (Fig. 4E). Treatment of activated T cells with plate-bound fibronectin (Fig. 4F) increased the proportion expressing cell-surface TSP-1 within a 1-h culture period (P<0.001 for all tested concentrations of fibronectin); an increase in TSP-1 expression (P<0.05) was observed between T cells treated with 5 μg/ml and 10 μg/ml fibronectin, suggesting a dose-dependent response.


Kidney transplantation: analysis of the expression and T cell-mediated activation of latent TGF-β.

Willet JD, Pichitsiri W, Jenkinson SE, Brain JG, Wood K, Alhasan AA, Spielhofer J, Robertson H, Ali S, Kirby JA - J. Leukoc. Biol. (2012)

Examination of T cell expression of the β 6 integrin, NRP-1, and TSP-1.Immunofluorescence flow cytometry of activated T cells showing no significant cell surface of expression of the β6 integrin (A). Subpopulations of activated T cells expressed cell-surface NRP-1 (B) and TSP-1 (C). Each shaded histogram shows the result of labeling with isotype-matched control antibodies; the open histograms show experimental labeling. (D) The mean expression of cell-surface TSP-1 by duplicate samples of activated T cells from three different volunteer blood donors (D1, D2, D3) was increased by coculture with TEC for 1 h prior to flow cytometry (P<0.05); pretreatment of the TEC with TGF-β1 further increased the expression of TSP-1 (P<0.005). (E) Representative immunofluorescence showing extracellular expression of fibronectin (FITC, green) by resting, immortalized TEC (DAPI-stained nuclei, blue). (F) T cells show markedly increased cell-surface expression of TSP-1 after addition to plate-bound fibronectin for 1 h. The bars show mean results ± sem; two separate experiments.
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Related In: Results  -  Collection

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Figure 4: Examination of T cell expression of the β 6 integrin, NRP-1, and TSP-1.Immunofluorescence flow cytometry of activated T cells showing no significant cell surface of expression of the β6 integrin (A). Subpopulations of activated T cells expressed cell-surface NRP-1 (B) and TSP-1 (C). Each shaded histogram shows the result of labeling with isotype-matched control antibodies; the open histograms show experimental labeling. (D) The mean expression of cell-surface TSP-1 by duplicate samples of activated T cells from three different volunteer blood donors (D1, D2, D3) was increased by coculture with TEC for 1 h prior to flow cytometry (P<0.05); pretreatment of the TEC with TGF-β1 further increased the expression of TSP-1 (P<0.005). (E) Representative immunofluorescence showing extracellular expression of fibronectin (FITC, green) by resting, immortalized TEC (DAPI-stained nuclei, blue). (F) T cells show markedly increased cell-surface expression of TSP-1 after addition to plate-bound fibronectin for 1 h. The bars show mean results ± sem; two separate experiments.
Mentions: Activated human T cells did not express cell-surface β6 integrin (Fig. 4A), and neither TSP-1 nor NRP-1 was expressed by resting T cells (not shown). However, a small proportion of T cells did express cell-surface NRP-1 after activation for 48 h (17.3±2.3%; n=8 T cell donors; mean±sem; representative result shown in Fig. 4B) and TSP-1 after 72 h (15.9±1.3%; n=19 T cell donors; representative result shown in Fig. 4C); the majority of activated T cells expressed intracellular TSP-1 (not shown). The proportion of activated T cells expressing cell-surface TSP-1 was increased significantly by 1 h coculture with resting TEC (P<0.05; n=3 separate T cell donors; Fig. 4D); culture with TGF-β-pretreated epithelial cells further increased cell-surface TSP-1 expression (P<0.005; Fig. 4D). Immunofluorescence analysis of cultured TEC demonstrated deposition of fibronectin between the cells (Fig. 4E). Treatment of activated T cells with plate-bound fibronectin (Fig. 4F) increased the proportion expressing cell-surface TSP-1 within a 1-h culture period (P<0.001 for all tested concentrations of fibronectin); an increase in TSP-1 expression (P<0.05) was observed between T cells treated with 5 μg/ml and 10 μg/ml fibronectin, suggesting a dose-dependent response.

Bottom Line: A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-β by pSmad 3.However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-β associated with TEC.Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-β.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular Medicine, The Medical School, Newcastle University, Newcastle upon Tyne, UK.

ABSTRACT
Activated T cells infiltrate a renal allograft during rejection and can respond to TGF-β within the tubules, causing local differentiation and expression of the αE(CD103)β7 integrin. This study was performed to examine the expression of latent TGF-β within renal allograft tissues and to define a mechanism by which T cells can activate and respond to this latent factor. Rejecting renal allograft biopsy tissues showed increased expression of the latent TGF-β complex, which was localized around the tubules by a mechanism that might involve interaction with heparan sulfate in the basement membrane. A cultured renal TEC line also expressed the latent complex, but these cells did not respond to this form of TGF-β by pSmad 3. However, coculture of these cells with activated T cells induced the expression of CD103, suggesting that T cells can activate and respond to the latent TGF-β associated with TEC. Although activated T cells expressed little cell-surface TSP-1, this was increased by culture with fibronectin or fibronectin-expressing renal TEC. Blockade of TSP-1 using LSKL peptides reduced the potential of activated T cells to differentiate in response to latent TGF-β. This study suggests that penetration of renal tubules by activated T cells leads to increased expression of T cell-surface TSP-1, allowing activation of latent TGF-β sequestered on heparan sulfate within the microenvironment. This mechanism may be important for localized phenotypic maturation of T cells that have infiltrated the kidney during allograft rejection.

Show MeSH
Related in: MedlinePlus