Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression identifies Klf2 as a novel repressor of TFH cell differentiation. (A) OT-II T cells were retrovirally transduced with Klf2 cDNA or an empty control vector encoding GFP only. 24 h after infection, the expression of CD62L, CD44, OX-40, and 4-1BB on Thy-1.1+ GFP+ cells was analyzed by flow cytometry in vitro (representative results from two independent experiments with quadruplicate wells). In addition, GFP+ cells were sorted and expression of S1pr1, Bcl-6, c-Maf, Prdm1, and Ascl2 was measured by quantitative RT-PCR (relative to β2-microglobulin; mean ± SEM of two independent experiments with three technical replicates each). (B) OT-II x CreERT2 x Klf2wt/wt or Klf2fl/fl splenocytes were stimulated with OVA peptide in the presence of tamoxifen (TMX). T cells were sorted after 24 h and analyzed by quantitative RT-PCR (relative to Hprt; mean ± SEM of two independent experiments with three technical replicates each) for expression of Klf2, S1pr1, Bcl-6, Prdm1, Tbx21, Gata3, c-Maf, and Ascl2. (C) OT-II T cells were retrovirally transfected with plasmids encoding Ascl2 (hu CD4 as reporter) and Klf2 (GFP reporter) or empty vector controls. After 20 and 44 h, expression of CXCR5 was analyzed by flow cytometry. Representative experiment out of two with quadruplicates. (D and E) OT-II T cells were retrovirally transduced with Klf2 cDNA or control vector and transferred into C57BL/6 recipient mice immunized on the same day with NP-OVA. After 42 h (D) or 6 d (E), antigen-specific GFP+ CD4+ T cells were analyzed for the TFH phenotype by flow cytometry. Representative contour plots for CXCR5/PD-1 expression on day 6 and bar graphs with expression of PD-1, CXCR5, PSGL-1, and CD62L are shown. Representative experiment out of three with six to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4322049&req=5

fig8: Overexpression identifies Klf2 as a novel repressor of TFH cell differentiation. (A) OT-II T cells were retrovirally transduced with Klf2 cDNA or an empty control vector encoding GFP only. 24 h after infection, the expression of CD62L, CD44, OX-40, and 4-1BB on Thy-1.1+ GFP+ cells was analyzed by flow cytometry in vitro (representative results from two independent experiments with quadruplicate wells). In addition, GFP+ cells were sorted and expression of S1pr1, Bcl-6, c-Maf, Prdm1, and Ascl2 was measured by quantitative RT-PCR (relative to β2-microglobulin; mean ± SEM of two independent experiments with three technical replicates each). (B) OT-II x CreERT2 x Klf2wt/wt or Klf2fl/fl splenocytes were stimulated with OVA peptide in the presence of tamoxifen (TMX). T cells were sorted after 24 h and analyzed by quantitative RT-PCR (relative to Hprt; mean ± SEM of two independent experiments with three technical replicates each) for expression of Klf2, S1pr1, Bcl-6, Prdm1, Tbx21, Gata3, c-Maf, and Ascl2. (C) OT-II T cells were retrovirally transfected with plasmids encoding Ascl2 (hu CD4 as reporter) and Klf2 (GFP reporter) or empty vector controls. After 20 and 44 h, expression of CXCR5 was analyzed by flow cytometry. Representative experiment out of two with quadruplicates. (D and E) OT-II T cells were retrovirally transduced with Klf2 cDNA or control vector and transferred into C57BL/6 recipient mice immunized on the same day with NP-OVA. After 42 h (D) or 6 d (E), antigen-specific GFP+ CD4+ T cells were analyzed for the TFH phenotype by flow cytometry. Representative contour plots for CXCR5/PD-1 expression on day 6 and bar graphs with expression of PD-1, CXCR5, PSGL-1, and CD62L are shown. Representative experiment out of three with six to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To test whether suppression of Klf2 is the major mechanism by which ICOS regulates TFH cell differentiation, we overexpressed Klf2 in antigen-specific T cells. OT-II T cells were retrovirally transduced with a vector coding for Klf2 and GFP or a control vector with GFP only. 1 d after infection, overexpression of Klf2 in vitro resulted in a 1.7- and 2.5-fold increased expression of its known target genes CD62L and S1pr1, respectively (Fig. 8 A). At the same time, expression levels of CD44 and other activation markers, like OX-40 and 4-1BB, were unaltered or even increased, which shows that overexpression of Klf2 did not result in a general block of T cell activation. Importantly, Bcl-6, c-Maf, Prdm1, and Ascl2 were expressed equally in both groups, demonstrating that overexpression of Klf2 did not directly regulate key factors of TFH cell differentiation. To substantiate these results, we stimulated T cells from inducible Klf2 KO mice. As expected, Klf2 mRNA and the known downstream target S1pr1 were strongly reduced, whereas expression of the TFH-related transcription factors Bcl-6, c-Maf, and Ascl-2 remained unchanged (Fig. 8 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)

Overexpression identifies Klf2 as a novel repressor of TFH cell differentiation. (A) OT-II T cells were retrovirally transduced with Klf2 cDNA or an empty control vector encoding GFP only. 24 h after infection, the expression of CD62L, CD44, OX-40, and 4-1BB on Thy-1.1+ GFP+ cells was analyzed by flow cytometry in vitro (representative results from two independent experiments with quadruplicate wells). In addition, GFP+ cells were sorted and expression of S1pr1, Bcl-6, c-Maf, Prdm1, and Ascl2 was measured by quantitative RT-PCR (relative to β2-microglobulin; mean ± SEM of two independent experiments with three technical replicates each). (B) OT-II x CreERT2 x Klf2wt/wt or Klf2fl/fl splenocytes were stimulated with OVA peptide in the presence of tamoxifen (TMX). T cells were sorted after 24 h and analyzed by quantitative RT-PCR (relative to Hprt; mean ± SEM of two independent experiments with three technical replicates each) for expression of Klf2, S1pr1, Bcl-6, Prdm1, Tbx21, Gata3, c-Maf, and Ascl2. (C) OT-II T cells were retrovirally transfected with plasmids encoding Ascl2 (hu CD4 as reporter) and Klf2 (GFP reporter) or empty vector controls. After 20 and 44 h, expression of CXCR5 was analyzed by flow cytometry. Representative experiment out of two with quadruplicates. (D and E) OT-II T cells were retrovirally transduced with Klf2 cDNA or control vector and transferred into C57BL/6 recipient mice immunized on the same day with NP-OVA. After 42 h (D) or 6 d (E), antigen-specific GFP+ CD4+ T cells were analyzed for the TFH phenotype by flow cytometry. Representative contour plots for CXCR5/PD-1 expression on day 6 and bar graphs with expression of PD-1, CXCR5, PSGL-1, and CD62L are shown. Representative experiment out of three with six to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4322049&req=5

fig8: Overexpression identifies Klf2 as a novel repressor of TFH cell differentiation. (A) OT-II T cells were retrovirally transduced with Klf2 cDNA or an empty control vector encoding GFP only. 24 h after infection, the expression of CD62L, CD44, OX-40, and 4-1BB on Thy-1.1+ GFP+ cells was analyzed by flow cytometry in vitro (representative results from two independent experiments with quadruplicate wells). In addition, GFP+ cells were sorted and expression of S1pr1, Bcl-6, c-Maf, Prdm1, and Ascl2 was measured by quantitative RT-PCR (relative to β2-microglobulin; mean ± SEM of two independent experiments with three technical replicates each). (B) OT-II x CreERT2 x Klf2wt/wt or Klf2fl/fl splenocytes were stimulated with OVA peptide in the presence of tamoxifen (TMX). T cells were sorted after 24 h and analyzed by quantitative RT-PCR (relative to Hprt; mean ± SEM of two independent experiments with three technical replicates each) for expression of Klf2, S1pr1, Bcl-6, Prdm1, Tbx21, Gata3, c-Maf, and Ascl2. (C) OT-II T cells were retrovirally transfected with plasmids encoding Ascl2 (hu CD4 as reporter) and Klf2 (GFP reporter) or empty vector controls. After 20 and 44 h, expression of CXCR5 was analyzed by flow cytometry. Representative experiment out of two with quadruplicates. (D and E) OT-II T cells were retrovirally transduced with Klf2 cDNA or control vector and transferred into C57BL/6 recipient mice immunized on the same day with NP-OVA. After 42 h (D) or 6 d (E), antigen-specific GFP+ CD4+ T cells were analyzed for the TFH phenotype by flow cytometry. Representative contour plots for CXCR5/PD-1 expression on day 6 and bar graphs with expression of PD-1, CXCR5, PSGL-1, and CD62L are shown. Representative experiment out of three with six to seven animals per group. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To test whether suppression of Klf2 is the major mechanism by which ICOS regulates TFH cell differentiation, we overexpressed Klf2 in antigen-specific T cells. OT-II T cells were retrovirally transduced with a vector coding for Klf2 and GFP or a control vector with GFP only. 1 d after infection, overexpression of Klf2 in vitro resulted in a 1.7- and 2.5-fold increased expression of its known target genes CD62L and S1pr1, respectively (Fig. 8 A). At the same time, expression levels of CD44 and other activation markers, like OX-40 and 4-1BB, were unaltered or even increased, which shows that overexpression of Klf2 did not result in a general block of T cell activation. Importantly, Bcl-6, c-Maf, Prdm1, and Ascl2 were expressed equally in both groups, demonstrating that overexpression of Klf2 did not directly regulate key factors of TFH cell differentiation. To substantiate these results, we stimulated T cells from inducible Klf2 KO mice. As expected, Klf2 mRNA and the known downstream target S1pr1 were strongly reduced, whereas expression of the TFH-related transcription factors Bcl-6, c-Maf, and Ascl-2 remained unchanged (Fig. 8 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