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ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2.

Weber JP, Fuhrmann F, Feist RK, Lahmann A, Al Baz MS, Gentz LJ, Vu Van D, Mages HW, Haftmann C, Riedel R, Grün JR, Schuh W, Kroczek RA, Radbruch A, Mashreghi MF, Hutloff A - J. Exp. Med. (2015)

Bottom Line: While CD28 regulated early expression of the master transcription factor Bcl-6, ICOS co-stimulation was essential to maintain the phenotype by regulating the novel TFH transcription factor Klf2 via Foxo1.Klf2 directly binds to Cxcr5, Ccr7, Psgl-1, and S1pr1, and low levels of Klf2 were essential to maintain this typical TFH homing receptor pattern.Our study describes for the first time the exclusive role of ICOS and its downstream signaling in the maintenance of TFH cells by controlling their anatomical localization in the B cell follicle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chronic Immune Reactions, Cell Biology, and Bioinformatics, German Rheumatism Research Centre, a Leibniz Institute, 10117 Berlin, Germany Molecular Immunology, Robert Koch Institute, 13353 Berlin, Germany.

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Induced overexpression of Klf2 results in loss of TFH cells and termination of the GC response. (A and B) Thy-1.1+ OT-II x CreERT2 T cells were transduced with either an empty (control) retroviral vector or a vector containing a loxP-flanked dsRed stop cassette in front of the Klf2 coding region, preventing expression until the cassette is removed by (tamoxifen-induced) Cre-mediated excision. After a 20-h in vitro culture, dsRed-expressing cells were sorted and transferred together with NP-specific CD45.1+ B1-8i B cells into B6 WT (A) or B6 CD28 KO (B) recipients (to exclude effects from endogenous TFH cells). Recipients were immunized with NP-OVA on the same day. Four days later, Klf2 overexpression was induced by tamoxifen (TMX)-mediated excision of the dsRed cassette. (A) Flow cytometric analysis of OT-II T cells (Thy-1.1+) for expression of CXCR5, PD-1, PSGL-1, and CD62L on day 6. Shown are representative plots for PD-1/CXCR5 expression and bar graphs indicating percentage of PD-1+ CXCR5+ OT-II cells and geoMFI expression of PSGL-1 and CD62L. (B) Analysis of antigen-specific (CD45.1+) B cells on day 8. Representative plots for transgenic (CD45.1+) B cells with a GC phenotype (PNA+ GL7+) and bar graphs indicating the absolute number of GC B cells are shown. (C) OT-II x CreERT2 x Klf2fl/fl T cells were transferred into B6 mice subsequently immunized with NP-OVA. On day 4 after immunization, Klf2 knockdown was induced by tamoxifen. On day 6 and 7, ICOS-L was blocked using a monoclonal antibody. OT-II T cells from draining lymph nodes were analyzed on day 8 for expression of PD-1/CXCR5, CD62L, CCR7, and PSGL-1. Dots represent individual mice and bars indicate the mean. Representative experiments out of two, with five to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig9: Induced overexpression of Klf2 results in loss of TFH cells and termination of the GC response. (A and B) Thy-1.1+ OT-II x CreERT2 T cells were transduced with either an empty (control) retroviral vector or a vector containing a loxP-flanked dsRed stop cassette in front of the Klf2 coding region, preventing expression until the cassette is removed by (tamoxifen-induced) Cre-mediated excision. After a 20-h in vitro culture, dsRed-expressing cells were sorted and transferred together with NP-specific CD45.1+ B1-8i B cells into B6 WT (A) or B6 CD28 KO (B) recipients (to exclude effects from endogenous TFH cells). Recipients were immunized with NP-OVA on the same day. Four days later, Klf2 overexpression was induced by tamoxifen (TMX)-mediated excision of the dsRed cassette. (A) Flow cytometric analysis of OT-II T cells (Thy-1.1+) for expression of CXCR5, PD-1, PSGL-1, and CD62L on day 6. Shown are representative plots for PD-1/CXCR5 expression and bar graphs indicating percentage of PD-1+ CXCR5+ OT-II cells and geoMFI expression of PSGL-1 and CD62L. (B) Analysis of antigen-specific (CD45.1+) B cells on day 8. Representative plots for transgenic (CD45.1+) B cells with a GC phenotype (PNA+ GL7+) and bar graphs indicating the absolute number of GC B cells are shown. (C) OT-II x CreERT2 x Klf2fl/fl T cells were transferred into B6 mice subsequently immunized with NP-OVA. On day 4 after immunization, Klf2 knockdown was induced by tamoxifen. On day 6 and 7, ICOS-L was blocked using a monoclonal antibody. OT-II T cells from draining lymph nodes were analyzed on day 8 for expression of PD-1/CXCR5, CD62L, CCR7, and PSGL-1. Dots represent individual mice and bars indicate the mean. Representative experiments out of two, with five to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To investigate whether Klf2 overexpression in fully matured TFH cells is able to reverse the TFH phenotype, we developed a tamoxifen-inducible expression system. 48 h after induction of Klf2 overexpression, half of the TFH cells had lost their phenotype according to CXCR5/PD-1, PSGL-1, and CD62L expression (Fig. 9 A). In addition, the number of antigen-specific GC B cells was reduced more than twofold two days later (Fig. 9 B).


ICOS maintains the T follicular helper cell phenotype by down-regulating Krüppel-like factor 2.

Weber JP, Fuhrmann F, Feist RK, Lahmann A, Al Baz MS, Gentz LJ, Vu Van D, Mages HW, Haftmann C, Riedel R, Grün JR, Schuh W, Kroczek RA, Radbruch A, Mashreghi MF, Hutloff A - J. Exp. Med. (2015)

Induced overexpression of Klf2 results in loss of TFH cells and termination of the GC response. (A and B) Thy-1.1+ OT-II x CreERT2 T cells were transduced with either an empty (control) retroviral vector or a vector containing a loxP-flanked dsRed stop cassette in front of the Klf2 coding region, preventing expression until the cassette is removed by (tamoxifen-induced) Cre-mediated excision. After a 20-h in vitro culture, dsRed-expressing cells were sorted and transferred together with NP-specific CD45.1+ B1-8i B cells into B6 WT (A) or B6 CD28 KO (B) recipients (to exclude effects from endogenous TFH cells). Recipients were immunized with NP-OVA on the same day. Four days later, Klf2 overexpression was induced by tamoxifen (TMX)-mediated excision of the dsRed cassette. (A) Flow cytometric analysis of OT-II T cells (Thy-1.1+) for expression of CXCR5, PD-1, PSGL-1, and CD62L on day 6. Shown are representative plots for PD-1/CXCR5 expression and bar graphs indicating percentage of PD-1+ CXCR5+ OT-II cells and geoMFI expression of PSGL-1 and CD62L. (B) Analysis of antigen-specific (CD45.1+) B cells on day 8. Representative plots for transgenic (CD45.1+) B cells with a GC phenotype (PNA+ GL7+) and bar graphs indicating the absolute number of GC B cells are shown. (C) OT-II x CreERT2 x Klf2fl/fl T cells were transferred into B6 mice subsequently immunized with NP-OVA. On day 4 after immunization, Klf2 knockdown was induced by tamoxifen. On day 6 and 7, ICOS-L was blocked using a monoclonal antibody. OT-II T cells from draining lymph nodes were analyzed on day 8 for expression of PD-1/CXCR5, CD62L, CCR7, and PSGL-1. Dots represent individual mice and bars indicate the mean. Representative experiments out of two, with five to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: Induced overexpression of Klf2 results in loss of TFH cells and termination of the GC response. (A and B) Thy-1.1+ OT-II x CreERT2 T cells were transduced with either an empty (control) retroviral vector or a vector containing a loxP-flanked dsRed stop cassette in front of the Klf2 coding region, preventing expression until the cassette is removed by (tamoxifen-induced) Cre-mediated excision. After a 20-h in vitro culture, dsRed-expressing cells were sorted and transferred together with NP-specific CD45.1+ B1-8i B cells into B6 WT (A) or B6 CD28 KO (B) recipients (to exclude effects from endogenous TFH cells). Recipients were immunized with NP-OVA on the same day. Four days later, Klf2 overexpression was induced by tamoxifen (TMX)-mediated excision of the dsRed cassette. (A) Flow cytometric analysis of OT-II T cells (Thy-1.1+) for expression of CXCR5, PD-1, PSGL-1, and CD62L on day 6. Shown are representative plots for PD-1/CXCR5 expression and bar graphs indicating percentage of PD-1+ CXCR5+ OT-II cells and geoMFI expression of PSGL-1 and CD62L. (B) Analysis of antigen-specific (CD45.1+) B cells on day 8. Representative plots for transgenic (CD45.1+) B cells with a GC phenotype (PNA+ GL7+) and bar graphs indicating the absolute number of GC B cells are shown. (C) OT-II x CreERT2 x Klf2fl/fl T cells were transferred into B6 mice subsequently immunized with NP-OVA. On day 4 after immunization, Klf2 knockdown was induced by tamoxifen. On day 6 and 7, ICOS-L was blocked using a monoclonal antibody. OT-II T cells from draining lymph nodes were analyzed on day 8 for expression of PD-1/CXCR5, CD62L, CCR7, and PSGL-1. Dots represent individual mice and bars indicate the mean. Representative experiments out of two, with five to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To investigate whether Klf2 overexpression in fully matured TFH cells is able to reverse the TFH phenotype, we developed a tamoxifen-inducible expression system. 48 h after induction of Klf2 overexpression, half of the TFH cells had lost their phenotype according to CXCR5/PD-1, PSGL-1, and CD62L expression (Fig. 9 A). In addition, the number of antigen-specific GC B cells was reduced more than twofold two days later (Fig. 9 B).

Bottom Line: While CD28 regulated early expression of the master transcription factor Bcl-6, ICOS co-stimulation was essential to maintain the phenotype by regulating the novel TFH transcription factor Klf2 via Foxo1.Klf2 directly binds to Cxcr5, Ccr7, Psgl-1, and S1pr1, and low levels of Klf2 were essential to maintain this typical TFH homing receptor pattern.Our study describes for the first time the exclusive role of ICOS and its downstream signaling in the maintenance of TFH cells by controlling their anatomical localization in the B cell follicle.

View Article: PubMed Central - HTML - PubMed

Affiliation: Chronic Immune Reactions, Cell Biology, and Bioinformatics, German Rheumatism Research Centre, a Leibniz Institute, 10117 Berlin, Germany Molecular Immunology, Robert Koch Institute, 13353 Berlin, Germany.

Show MeSH
Related in: MedlinePlus