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Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking.

Mertens J, Fabri M, Zingarelli A, Kubacki T, Meemboor S, Groneck L, Seeger J, Bessler M, Hafke H, Odenthal M, Bieler JG, Kalka C, Schneck JP, Kashkar H, Kalka-Moll WM - PLoS Pathog. (2009)

Bottom Line: The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR).In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population.The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne Medical Center, Köln, Germany.

ABSTRACT
Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an alpha-helix configuration. In this paper we look at the immune response of CD8(+) T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8(+)CD28(-) T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8(+)CD28(-) T lymphocytes. The Sp1-induced CD8(+)CD28(-) T lymphocytes are CD122(low)CTLA-4(+)CD39(+). They synthesize IL-10 and TGF-beta. The Sp1-induced CD8(+)CD28(-) T cells exhibit immunosuppressive properties on CD4(+) T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8(+) T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8(+) T cell activation by enhancing crosslinking of TCR. The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8(+)CD28(-) T lymphocytes in vivo and in vitro. The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

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Sp1 induces peptide-specific CD8+CD28− T lymphocytes and enhances TCR cross-linking on CD8+ T cells.A. Sp1 induces peptide-specific CD8+CD28− T lymphocytes. Total spleen cells from OT-1 mice and purified OT-1 CD8+ T cells alone (purity >95%) were treated with SIINFEKL (10−9 M). SIINFEKL-loaded MHC class I dimers were coincubated with purified OT-1 CD8+ T cells. Cells were treated for 16 h in vitro with Sp1 (100 µg/ml), mSp1 (100 µg/ml), or medium alone. Cells were stained for CD8 and CD28 surface expression and prepared for flow cytometry. The percentages (±SD; n = 4) of the respective CD8+CD28− cells are given in one representative histogram out of three experiments performed independently. B. Sp1 enhances TCR cross-linking on CD8+ T cells. CD8+ T cells from P14 mice were incubated with FITC-labeled and GP33–41- or ASN-loaded (nonspecific control) Db-Ig dimers in different concentrations in the presence or absence of Sp1. The mean fluorescence intensity was measured by flow cytometry. Using ASN as a control for nonspecific binding, the specific binding of GP33–41 (open circle) and GP33–41 plus Sp1 (filled circles) was calculated as described. A scatchard plot of the specific binding data is shown. The model fit parameters are summarized in the upper right box. Kd is the single site equilibrium dissociation constant. Kx is the equilibrium cross-linking constant with units of inverse number for receptors per cell. Rt is the total number of receptors available for binding in mean channel fluorescence (MCF) unit equivalents. Kv is the avidity at low dimer concentrations, which is estimated as Kv∼Kd/KxRt.
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ppat-1000596-g007: Sp1 induces peptide-specific CD8+CD28− T lymphocytes and enhances TCR cross-linking on CD8+ T cells.A. Sp1 induces peptide-specific CD8+CD28− T lymphocytes. Total spleen cells from OT-1 mice and purified OT-1 CD8+ T cells alone (purity >95%) were treated with SIINFEKL (10−9 M). SIINFEKL-loaded MHC class I dimers were coincubated with purified OT-1 CD8+ T cells. Cells were treated for 16 h in vitro with Sp1 (100 µg/ml), mSp1 (100 µg/ml), or medium alone. Cells were stained for CD8 and CD28 surface expression and prepared for flow cytometry. The percentages (±SD; n = 4) of the respective CD8+CD28− cells are given in one representative histogram out of three experiments performed independently. B. Sp1 enhances TCR cross-linking on CD8+ T cells. CD8+ T cells from P14 mice were incubated with FITC-labeled and GP33–41- or ASN-loaded (nonspecific control) Db-Ig dimers in different concentrations in the presence or absence of Sp1. The mean fluorescence intensity was measured by flow cytometry. Using ASN as a control for nonspecific binding, the specific binding of GP33–41 (open circle) and GP33–41 plus Sp1 (filled circles) was calculated as described. A scatchard plot of the specific binding data is shown. The model fit parameters are summarized in the upper right box. Kd is the single site equilibrium dissociation constant. Kx is the equilibrium cross-linking constant with units of inverse number for receptors per cell. Rt is the total number of receptors available for binding in mean channel fluorescence (MCF) unit equivalents. Kv is the avidity at low dimer concentrations, which is estimated as Kv∼Kd/KxRt.

Mentions: Activation of CD8+CD28− T lymphocytes is mediated through TCR signaling. To test whether the induction of the CD8+CD28− T cell population depends on peptide specificity and TCR specificity, we incubated spleen cells from OT-1 mice carrying a transgenic TCR on their CD8+ T cell surface for the specific recognition of the ovalbumin sequence SIINFEKL with Sp1, mSp1, or medium alone as controls, in the presence of the SIINFEKL peptide (10−9 M). Fig. 7A demonstrates that irrespective of the TCR specificity, Sp1 induces an increase of the CD28− T cell population within the SIINFEKL-stimulated CD8+ T lymphocytes (84% versus 30% and 31% of medium- and mSp1-treated cells, respectively). We further analyzed Ts induction of CD8+ T cells with an artificial APC system that lacks APCs but contains MHC class I molecules loaded with SIINFEKL peptide. Compared to the negative control, Sp1 induced an increased expression in the CD28− phenotype in 24% of CD8+ T cells. We also tested whether the induction of the CD8+CD28− T cells requires MHC class I molecules at all and cultured purified CD8+ OT-1 T cells in the presence of SIINFEKL in medium alone, and in medium containing mSp1 or Sp1. Compared to the medium and mSp1 control, Sp1-treated CD8+ T cells show an CD8+CD28− T cell increase of 32% and 27%, respectively. These results demonstrate an APC- and MHC class I-independent induction of the CD28− phenotype by Sp1 in peptide-specific CD8+ T cells. At the same time, they allowed us to study the effect of Sp1 on the TCR membrane organisation using a MHC-Ig dimer model. Quantification of the TCR membrane organisation was achieved by fitting the binding data into a model of equilibrium dimerization of homogeneous monovalent receptors by divalent ligand [49] (Fig. 7B; upper right box). In this model, the first monovalent binding binds with single site affinity [50]. If there is another receptor nearby then the dimer may bind with both “arms” creating an “apparent binding affinity,” i.e., the avidity. Since the receptors are identical, increases in avidity can be attributed to increases in local concentration of receptors. In this model the single site dissociation constant, Kd, characterizes the binding of one site on the MHC to one TCR and the dimensionless cross-linking constant, KxRt, characterizes the ability of the MHC/TCR complex to recruit another TCR. The binding avidity, Kv, is approximately the ratio Kd/KxRt [51]. When there is minimal cross-linking potential, KxRt is close to 1 and Kd and Kv are similar. If the cross linking potential Kx is high, there is an enhancement of binding due to dimerization, resulting in a stronger measured avidity than the intrinsic single site affinity. Analysis of the binding data indicated that the overall avidity, Kv, of the dimeric MHC in the presence of Sp1 was ∼2.5-fold higher than in the absence of Sp1 (Fig. 7B). The increased avidity of MHC-Ig binding is due to an increased cross-linking constant (Kx = 0.58 cells/# in the absence of Sp1 vs. Kx = 1.25 cells in the presence of Sp1), rather than an increased intrinsic dissociation constant (Kd = 1.7 nM vs. Kd = 1.2 nM), or to changes in the total amount of receptors (Rt = 9.9 # mean channel fluorescence (MCF) vs. Rt = 11.9 # MCF). Thus, the enhanced avidity results from an increase in the cross-linking potential in the presence of Sp1.


Streptococcus pneumoniae serotype 1 capsular polysaccharide induces CD8CD28 regulatory T lymphocytes by TCR crosslinking.

Mertens J, Fabri M, Zingarelli A, Kubacki T, Meemboor S, Groneck L, Seeger J, Bessler M, Hafke H, Odenthal M, Bieler JG, Kalka C, Schneck JP, Kashkar H, Kalka-Moll WM - PLoS Pathog. (2009)

Sp1 induces peptide-specific CD8+CD28− T lymphocytes and enhances TCR cross-linking on CD8+ T cells.A. Sp1 induces peptide-specific CD8+CD28− T lymphocytes. Total spleen cells from OT-1 mice and purified OT-1 CD8+ T cells alone (purity >95%) were treated with SIINFEKL (10−9 M). SIINFEKL-loaded MHC class I dimers were coincubated with purified OT-1 CD8+ T cells. Cells were treated for 16 h in vitro with Sp1 (100 µg/ml), mSp1 (100 µg/ml), or medium alone. Cells were stained for CD8 and CD28 surface expression and prepared for flow cytometry. The percentages (±SD; n = 4) of the respective CD8+CD28− cells are given in one representative histogram out of three experiments performed independently. B. Sp1 enhances TCR cross-linking on CD8+ T cells. CD8+ T cells from P14 mice were incubated with FITC-labeled and GP33–41- or ASN-loaded (nonspecific control) Db-Ig dimers in different concentrations in the presence or absence of Sp1. The mean fluorescence intensity was measured by flow cytometry. Using ASN as a control for nonspecific binding, the specific binding of GP33–41 (open circle) and GP33–41 plus Sp1 (filled circles) was calculated as described. A scatchard plot of the specific binding data is shown. The model fit parameters are summarized in the upper right box. Kd is the single site equilibrium dissociation constant. Kx is the equilibrium cross-linking constant with units of inverse number for receptors per cell. Rt is the total number of receptors available for binding in mean channel fluorescence (MCF) unit equivalents. Kv is the avidity at low dimer concentrations, which is estimated as Kv∼Kd/KxRt.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2742891&req=5

ppat-1000596-g007: Sp1 induces peptide-specific CD8+CD28− T lymphocytes and enhances TCR cross-linking on CD8+ T cells.A. Sp1 induces peptide-specific CD8+CD28− T lymphocytes. Total spleen cells from OT-1 mice and purified OT-1 CD8+ T cells alone (purity >95%) were treated with SIINFEKL (10−9 M). SIINFEKL-loaded MHC class I dimers were coincubated with purified OT-1 CD8+ T cells. Cells were treated for 16 h in vitro with Sp1 (100 µg/ml), mSp1 (100 µg/ml), or medium alone. Cells were stained for CD8 and CD28 surface expression and prepared for flow cytometry. The percentages (±SD; n = 4) of the respective CD8+CD28− cells are given in one representative histogram out of three experiments performed independently. B. Sp1 enhances TCR cross-linking on CD8+ T cells. CD8+ T cells from P14 mice were incubated with FITC-labeled and GP33–41- or ASN-loaded (nonspecific control) Db-Ig dimers in different concentrations in the presence or absence of Sp1. The mean fluorescence intensity was measured by flow cytometry. Using ASN as a control for nonspecific binding, the specific binding of GP33–41 (open circle) and GP33–41 plus Sp1 (filled circles) was calculated as described. A scatchard plot of the specific binding data is shown. The model fit parameters are summarized in the upper right box. Kd is the single site equilibrium dissociation constant. Kx is the equilibrium cross-linking constant with units of inverse number for receptors per cell. Rt is the total number of receptors available for binding in mean channel fluorescence (MCF) unit equivalents. Kv is the avidity at low dimer concentrations, which is estimated as Kv∼Kd/KxRt.
Mentions: Activation of CD8+CD28− T lymphocytes is mediated through TCR signaling. To test whether the induction of the CD8+CD28− T cell population depends on peptide specificity and TCR specificity, we incubated spleen cells from OT-1 mice carrying a transgenic TCR on their CD8+ T cell surface for the specific recognition of the ovalbumin sequence SIINFEKL with Sp1, mSp1, or medium alone as controls, in the presence of the SIINFEKL peptide (10−9 M). Fig. 7A demonstrates that irrespective of the TCR specificity, Sp1 induces an increase of the CD28− T cell population within the SIINFEKL-stimulated CD8+ T lymphocytes (84% versus 30% and 31% of medium- and mSp1-treated cells, respectively). We further analyzed Ts induction of CD8+ T cells with an artificial APC system that lacks APCs but contains MHC class I molecules loaded with SIINFEKL peptide. Compared to the negative control, Sp1 induced an increased expression in the CD28− phenotype in 24% of CD8+ T cells. We also tested whether the induction of the CD8+CD28− T cells requires MHC class I molecules at all and cultured purified CD8+ OT-1 T cells in the presence of SIINFEKL in medium alone, and in medium containing mSp1 or Sp1. Compared to the medium and mSp1 control, Sp1-treated CD8+ T cells show an CD8+CD28− T cell increase of 32% and 27%, respectively. These results demonstrate an APC- and MHC class I-independent induction of the CD28− phenotype by Sp1 in peptide-specific CD8+ T cells. At the same time, they allowed us to study the effect of Sp1 on the TCR membrane organisation using a MHC-Ig dimer model. Quantification of the TCR membrane organisation was achieved by fitting the binding data into a model of equilibrium dimerization of homogeneous monovalent receptors by divalent ligand [49] (Fig. 7B; upper right box). In this model, the first monovalent binding binds with single site affinity [50]. If there is another receptor nearby then the dimer may bind with both “arms” creating an “apparent binding affinity,” i.e., the avidity. Since the receptors are identical, increases in avidity can be attributed to increases in local concentration of receptors. In this model the single site dissociation constant, Kd, characterizes the binding of one site on the MHC to one TCR and the dimensionless cross-linking constant, KxRt, characterizes the ability of the MHC/TCR complex to recruit another TCR. The binding avidity, Kv, is approximately the ratio Kd/KxRt [51]. When there is minimal cross-linking potential, KxRt is close to 1 and Kd and Kv are similar. If the cross linking potential Kx is high, there is an enhancement of binding due to dimerization, resulting in a stronger measured avidity than the intrinsic single site affinity. Analysis of the binding data indicated that the overall avidity, Kv, of the dimeric MHC in the presence of Sp1 was ∼2.5-fold higher than in the absence of Sp1 (Fig. 7B). The increased avidity of MHC-Ig binding is due to an increased cross-linking constant (Kx = 0.58 cells/# in the absence of Sp1 vs. Kx = 1.25 cells in the presence of Sp1), rather than an increased intrinsic dissociation constant (Kd = 1.7 nM vs. Kd = 1.2 nM), or to changes in the total amount of receptors (Rt = 9.9 # mean channel fluorescence (MCF) vs. Rt = 11.9 # MCF). Thus, the enhanced avidity results from an increase in the cross-linking potential in the presence of Sp1.

Bottom Line: The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR).In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population.The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking.

View Article: PubMed Central - PubMed

Affiliation: Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne Medical Center, Köln, Germany.

ABSTRACT
Zwitterionic capsular polysaccharides (ZPS) of commensal bacteria are characterized by having both positive and negative charged substituents on each repeating unit of a highly repetitive structure that has an alpha-helix configuration. In this paper we look at the immune response of CD8(+) T cells to ZPSs. Intraperitoneal application of the ZPS Sp1 from Streptococcus pneumoniae serotype 1 induces CD8(+)CD28(-) T cells in the spleen and peritoneal cavity of WT mice. However, chemically modified Sp1 (mSp1) without the positive charge and resembling common negatively charged polysaccharides fails to induce CD8(+)CD28(-) T lymphocytes. The Sp1-induced CD8(+)CD28(-) T lymphocytes are CD122(low)CTLA-4(+)CD39(+). They synthesize IL-10 and TGF-beta. The Sp1-induced CD8(+)CD28(-) T cells exhibit immunosuppressive properties on CD4(+) T cells in vivo and in vitro. Experimental approaches to elucidate the mechanism of CD8(+) T cell activation by Sp1 demonstrate in a dimeric MHC class I-Ig model that Sp1 induces CD8(+) T cell activation by enhancing crosslinking of TCR. The expansion of CD8(+)CD28(-) T cells is independent, of direct antigen-presenting cell/T cell contact and, to the specificity of the T cell receptor (TCR). In CD8(+)CD28(-) T cells, Sp1 enhances Zap-70 phosphorylation and increasingly involves NF-kappaB which ultimately results in protection versus apoptosis and cell death and promotes survival and accumulation of the CD8(+)CD28(-) population. This is the first description of a naturally occurring bacterial antigen that is able to induce suppressive CD8(+)CD28(-) T lymphocytes in vivo and in vitro. The underlying mechanism of CD8(+) T cell activation appears to rely on enhanced TCR crosslinking. The data provides evidence that ZPS of commensal bacteria play an important role in peripheral tolerance mechanisms and the maintenance of the homeostasis of the immune system.

Show MeSH
Related in: MedlinePlus