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CD152 (CTLA-4) determines CD4 T cell migration in vitro and in vivo.

Knieke K, Hoff H, Maszyna F, Kolar P, Schrage A, Hamann A, Debes GF, Brunner-Weinzierl MC - PLoS ONE (2009)

Bottom Line: We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner.Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo.We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration.

View Article: PubMed Central - PubMed

Affiliation: Experimentelle Pädiatrie, Universitätskinderklinik, Otto-von-Guericke Universität, Magdeburg, Germany.

ABSTRACT

Background: Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration.

Methodology/principal findings: We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo.

Conclusions/significance: We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge.

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CD152-enhanced migration is primarily mediated by DCs.(A) Surface expression of CD152 on CD4+ cells is up-regulated by stimulation with DCs. Naϊve CD4+CD62L+ TCRtg T cells were stimulated with 1 µg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152+ CD4+ based on total CD4+ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Naϊve CD4+CD62L+ TCRtg T cells were labeled with CFSE and antigen-specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc-stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4+ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4+ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4+CD62L+ TCRtg T cells were stimulated under Th1 conditions with 1 µg/ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4+ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments.
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pone-0005702-g005: CD152-enhanced migration is primarily mediated by DCs.(A) Surface expression of CD152 on CD4+ cells is up-regulated by stimulation with DCs. Naϊve CD4+CD62L+ TCRtg T cells were stimulated with 1 µg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152+ CD4+ based on total CD4+ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Naϊve CD4+CD62L+ TCRtg T cells were labeled with CFSE and antigen-specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc-stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4+ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4+ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4+CD62L+ TCRtg T cells were stimulated under Th1 conditions with 1 µg/ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4+ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments.

Mentions: To examine the role of CD152 in T cell migration, we stimulated CD152-deficient (CD152−/−) monoclonal OVA-specific TCRtg CD4+ T cells from OTII mice (TCRtgCD152−/−) and monoclonal OVA-specific TCRtg CD4+ T cells from CD152+/+ OTII mice (TCRtgCD152+/+) with cognate antigen in vitro and tested chemotaxis to the CXCR4 ligand CXCL12, the CCR7 ligand CCL19, and the CCR5 ligand CCL4 in a Transwell chemotaxis assay (Fig. 1A, and data not shown). Similar baseline migration, which was routinely 5–10% in our Transwell migration assays (Figs. 1–5), was detectable for TCRtgCD152+/+ T cells and TCRtgCD152−/− T cells. Under these conditions, no migration towards the CCR5 ligand CCL4 was seen in either population (data not shown). In contrast, TCRtgCD152+/+ T cells migrated 2-fold better in response to CXCL12 than did TCRtgCD152−/− T cells (Fig. 1A). Similarly, migration towards the CCR7 ligand CCL19 was diminished by 70% in TCRtgCD152−/− T cells compared with TCRtgCD152+/+ cells.


CD152 (CTLA-4) determines CD4 T cell migration in vitro and in vivo.

Knieke K, Hoff H, Maszyna F, Kolar P, Schrage A, Hamann A, Debes GF, Brunner-Weinzierl MC - PLoS ONE (2009)

CD152-enhanced migration is primarily mediated by DCs.(A) Surface expression of CD152 on CD4+ cells is up-regulated by stimulation with DCs. Naϊve CD4+CD62L+ TCRtg T cells were stimulated with 1 µg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152+ CD4+ based on total CD4+ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Naϊve CD4+CD62L+ TCRtg T cells were labeled with CFSE and antigen-specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc-stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4+ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4+ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4+CD62L+ TCRtg T cells were stimulated under Th1 conditions with 1 µg/ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4+ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682661&req=5

pone-0005702-g005: CD152-enhanced migration is primarily mediated by DCs.(A) Surface expression of CD152 on CD4+ cells is up-regulated by stimulation with DCs. Naϊve CD4+CD62L+ TCRtg T cells were stimulated with 1 µg/ml OVA-peptide presented by matured DCs or by LPS-activated B cells. After 48 hours surface expression of CD152 was detected by liposome staining technique and subsequent FACS analysis. Left panels show CD152 staining, right panels show blocking controls. Numbers indicate the percentage of CD152+ CD4+ based on total CD4+ cells. (B) Equal proliferation of T cells stimulated with activated B cells or matured DCs. Naϊve CD4+CD62L+ TCRtg T cells were labeled with CFSE and antigen-specifically stimulated with activated B cells or matured DCs. Proliferation of T cells was determined by flow cytometry 48 hours (filled curve) and 72 hours later (black line). T cells cultured with different APCs but without antigenic stimulation are shown as heavy grey line. Numbers indicate the percentage of divided T cells 72 hours after stimulation relating to unstimulated controls (after 48 h 48% of Dc-stimulated T cells and 46% of Bc-stimulated T cells proliferated). (C) Kinetics of CD152 surface expression on CD4+ T cells. Indicated time after onset of stimulation cells cultured as described in 5A were stained with liposome staining technique for CD152. Percentages of surface expressing CD152 cells of total CD4+ cells are shown. (D) Chemotactic index of Th1 cells stimulated with different APCs. CD4+CD62L+ TCRtg T cells were stimulated under Th1 conditions with 1 µg/ml OVA peptide presented by activated B-cells or bone marrow derived DCs. On day 6 of primary stimulation, CD4+ cells were analyzed for migration capacity in chemotaxis assay. All data show representative data from at least two experiments.
Mentions: To examine the role of CD152 in T cell migration, we stimulated CD152-deficient (CD152−/−) monoclonal OVA-specific TCRtg CD4+ T cells from OTII mice (TCRtgCD152−/−) and monoclonal OVA-specific TCRtg CD4+ T cells from CD152+/+ OTII mice (TCRtgCD152+/+) with cognate antigen in vitro and tested chemotaxis to the CXCR4 ligand CXCL12, the CCR7 ligand CCL19, and the CCR5 ligand CCL4 in a Transwell chemotaxis assay (Fig. 1A, and data not shown). Similar baseline migration, which was routinely 5–10% in our Transwell migration assays (Figs. 1–5), was detectable for TCRtgCD152+/+ T cells and TCRtgCD152−/− T cells. Under these conditions, no migration towards the CCR5 ligand CCL4 was seen in either population (data not shown). In contrast, TCRtgCD152+/+ T cells migrated 2-fold better in response to CXCL12 than did TCRtgCD152−/− T cells (Fig. 1A). Similarly, migration towards the CCR7 ligand CCL19 was diminished by 70% in TCRtgCD152−/− T cells compared with TCRtgCD152+/+ cells.

Bottom Line: We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner.Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo.We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration.

View Article: PubMed Central - PubMed

Affiliation: Experimentelle Pädiatrie, Universitätskinderklinik, Otto-von-Guericke Universität, Magdeburg, Germany.

ABSTRACT

Background: Migration of antigen-experienced T cells to secondary lymphoid organs and the site of antigenic-challenge is a mandatory prerequisite for the precise functioning of adaptive immune responses. The surface molecule CD152 (CTLA-4) is mostly considered as a negative regulator of T cell activation during immune responses. It is currently unknown whether CD152 can also influence chemokine-driven T cell migration.

Methodology/principal findings: We analyzed the consequences of CD152 signaling on Th cell migration using chemotaxis assays in vitro and radioactive cell tracking in vivo. We show here that the genetic and serological inactivation of CD152 in Th1 cells reduced migration towards CCL4, CXCL12 and CCL19, but not CXCL9, in a G-protein dependent manner. In addition, retroviral transduction of CD152 cDNA into CD152 negative cells restored Th1 cell migration. Crosslinking of CD152 together with CD3 and CD28 stimulation on activated Th1 cells increased expression of the chemokine receptors CCR5 and CCR7, which in turn enhanced cell migration. Using sensitive liposome technology, we show that mature dendritic cells but not activated B cells were potent at inducing surface CD152 expression and the CD152-mediated migration-enhancing signals. Importantly, migration of CD152 positive Th1 lymphocytes in in vivo experiments increased more than 200% as compared to CD152 negative counterparts showing that indeed CD152 orchestrates specific migration of selected Th1 cells to sites of inflammation and antigenic challenge in vivo.

Conclusions/significance: We show here, that CD152 signaling does not just silence cells, but selects individual ones for migration. This novel activity of CD152 adds to the already significant role of CD152 in controlling peripheral immune responses by allowing T cells to localize correctly during infection. It also suggests that interference with CD152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge.

Show MeSH
Related in: MedlinePlus