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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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ICOS down-regulates Klf2 to maintain TFH cell homing markers. (A) OT-II splenocytes were stimulated in vitro with OVA323-339 peptide. T cells were sorted from cultures at the indicated times and Klf2 mRNA was quantified by RT-PCR. (B) Recipients of Thy-1.1+ OT-II T cells were immunized with cognate antigen and antigen-specific Thy-1.1+ CXCR5/PD-1 double-positive (TFH) or double-negative cells (non-TFH) were sorted from draining lymph nodes on day 8. Klf2 expression was analyzed by quantitative RT-PCR. For comparison, Klf2 expression in naive OT-II T cells is shown. (C–E) Thy-1.1+ OT-II T cells were transferred into C57BL/6 recipients immunized subcutaneously with NP-OVA. On day 7 after immunization, recipients were treated with anti–ICOS-L, CTLA-4–Ig, or control reagents (CTRL). Antigen-specific TFH cells (Thy-1.1+ CXCR5+ PD-1+) from draining lymph nodes were sorted for preparation of RNA 6 h after blockade and analyzed by flow cytometry 20 h after blockade, respectively. (C) Klf2 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (D) CD62L and CD69 expression analyzed by flow cytometry (MFI) and S1pr1 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (E) Expression of Klf2 and S1pr1 by RT-PCR and CD62L and CD69 by flow cytometry after CTLA-4–Ig blockade. All quantitative RT-PCR data are mean values (± SEM) from three experiments with three technical replicates each and β2-microglobulin as housekeeping gene. Flow cytometry data are a representative result from three (ICOS-L blockade) or two (B7 blockade) independent experiments with seven animals per group. (F) WT, ICOS KO, or CD28 KO OT-II T cells were transferred into C57BL/6 recipients or ICOS KO OT-II T cells were transferred into CD80/86 (B7) KO recipients. Transgenic T cells were sorted from pools of draining lymph nodes 3 d after immunization with NP-OVA. Klf2 mRNA was quantified by RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). Dots represent individual mice, bars indicate the mean, and error bars represent the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig6: ICOS down-regulates Klf2 to maintain TFH cell homing markers. (A) OT-II splenocytes were stimulated in vitro with OVA323-339 peptide. T cells were sorted from cultures at the indicated times and Klf2 mRNA was quantified by RT-PCR. (B) Recipients of Thy-1.1+ OT-II T cells were immunized with cognate antigen and antigen-specific Thy-1.1+ CXCR5/PD-1 double-positive (TFH) or double-negative cells (non-TFH) were sorted from draining lymph nodes on day 8. Klf2 expression was analyzed by quantitative RT-PCR. For comparison, Klf2 expression in naive OT-II T cells is shown. (C–E) Thy-1.1+ OT-II T cells were transferred into C57BL/6 recipients immunized subcutaneously with NP-OVA. On day 7 after immunization, recipients were treated with anti–ICOS-L, CTLA-4–Ig, or control reagents (CTRL). Antigen-specific TFH cells (Thy-1.1+ CXCR5+ PD-1+) from draining lymph nodes were sorted for preparation of RNA 6 h after blockade and analyzed by flow cytometry 20 h after blockade, respectively. (C) Klf2 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (D) CD62L and CD69 expression analyzed by flow cytometry (MFI) and S1pr1 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (E) Expression of Klf2 and S1pr1 by RT-PCR and CD62L and CD69 by flow cytometry after CTLA-4–Ig blockade. All quantitative RT-PCR data are mean values (± SEM) from three experiments with three technical replicates each and β2-microglobulin as housekeeping gene. Flow cytometry data are a representative result from three (ICOS-L blockade) or two (B7 blockade) independent experiments with seven animals per group. (F) WT, ICOS KO, or CD28 KO OT-II T cells were transferred into C57BL/6 recipients or ICOS KO OT-II T cells were transferred into CD80/86 (B7) KO recipients. Transgenic T cells were sorted from pools of draining lymph nodes 3 d after immunization with NP-OVA. Klf2 mRNA was quantified by RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). Dots represent individual mice, bars indicate the mean, and error bars represent the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Mentions: To investigate which transcription factors are regulated by ICOS-mediated signaling and might play a role for the maintenance of the TFH phenotype, we performed a transcriptome analysis. Antigen-specific TFH cells were sorted from draining lymph nodes after 6 h of blocking ICOS-L. Comparing the ICOS-L blockade with the control group, we observed no difference for any of the transcription factors known to be important for TFH cell differentiation including Bcl-6, Blimp-1, Irf4, Ascl2, and c-Maf. However, the zinc-finger transcription factor Klf2 showed up as one of the most significantly differentially regulated genes. This transcription factor is highly expressed in naive T cells, strongly down-regulated shortly after T cell receptor triggering, and maintained on a lower level in activated T cells (Fig. 6 A). The sixfold lower expression of Klf2 in TFH versus non-TFH cells made it a particularly interesting target for further analyses (Fig. 6 B). Upon blocking ICOS-L, Klf2 was rapidly up-regulated by a factor of 3.5, approaching the expression levels of non-TFH cells (Fig. 6 C). Klf2 was originally described as a factor controlling thymocyte egress via up-regulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CD62L (Carlson et al., 2006). Therefore, we analyzed these target genes on antigen-specific TFH cells after ICOS-L blockade. CD62L and S1PR1 were up-regulated 1.6- and 2.8-fold, respectively, whereas CD69, which is inversely regulated to S1PR1 (Shiow et al., 2006), was reduced by 30% (Fig. 6 D). In contrast, blockade of the CD28 pathway did not change Klf2 expression or expression of any of the latter receptors (Fig. 6 E).


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)

ICOS down-regulates Klf2 to maintain TFH cell homing markers. (A) OT-II splenocytes were stimulated in vitro with OVA323-339 peptide. T cells were sorted from cultures at the indicated times and Klf2 mRNA was quantified by RT-PCR. (B) Recipients of Thy-1.1+ OT-II T cells were immunized with cognate antigen and antigen-specific Thy-1.1+ CXCR5/PD-1 double-positive (TFH) or double-negative cells (non-TFH) were sorted from draining lymph nodes on day 8. Klf2 expression was analyzed by quantitative RT-PCR. For comparison, Klf2 expression in naive OT-II T cells is shown. (C–E) Thy-1.1+ OT-II T cells were transferred into C57BL/6 recipients immunized subcutaneously with NP-OVA. On day 7 after immunization, recipients were treated with anti–ICOS-L, CTLA-4–Ig, or control reagents (CTRL). Antigen-specific TFH cells (Thy-1.1+ CXCR5+ PD-1+) from draining lymph nodes were sorted for preparation of RNA 6 h after blockade and analyzed by flow cytometry 20 h after blockade, respectively. (C) Klf2 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (D) CD62L and CD69 expression analyzed by flow cytometry (MFI) and S1pr1 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (E) Expression of Klf2 and S1pr1 by RT-PCR and CD62L and CD69 by flow cytometry after CTLA-4–Ig blockade. All quantitative RT-PCR data are mean values (± SEM) from three experiments with three technical replicates each and β2-microglobulin as housekeeping gene. Flow cytometry data are a representative result from three (ICOS-L blockade) or two (B7 blockade) independent experiments with seven animals per group. (F) WT, ICOS KO, or CD28 KO OT-II T cells were transferred into C57BL/6 recipients or ICOS KO OT-II T cells were transferred into CD80/86 (B7) KO recipients. Transgenic T cells were sorted from pools of draining lymph nodes 3 d after immunization with NP-OVA. Klf2 mRNA was quantified by RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). Dots represent individual mice, bars indicate the mean, and error bars represent the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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fig6: ICOS down-regulates Klf2 to maintain TFH cell homing markers. (A) OT-II splenocytes were stimulated in vitro with OVA323-339 peptide. T cells were sorted from cultures at the indicated times and Klf2 mRNA was quantified by RT-PCR. (B) Recipients of Thy-1.1+ OT-II T cells were immunized with cognate antigen and antigen-specific Thy-1.1+ CXCR5/PD-1 double-positive (TFH) or double-negative cells (non-TFH) were sorted from draining lymph nodes on day 8. Klf2 expression was analyzed by quantitative RT-PCR. For comparison, Klf2 expression in naive OT-II T cells is shown. (C–E) Thy-1.1+ OT-II T cells were transferred into C57BL/6 recipients immunized subcutaneously with NP-OVA. On day 7 after immunization, recipients were treated with anti–ICOS-L, CTLA-4–Ig, or control reagents (CTRL). Antigen-specific TFH cells (Thy-1.1+ CXCR5+ PD-1+) from draining lymph nodes were sorted for preparation of RNA 6 h after blockade and analyzed by flow cytometry 20 h after blockade, respectively. (C) Klf2 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (D) CD62L and CD69 expression analyzed by flow cytometry (MFI) and S1pr1 expression analyzed by quantitative RT-PCR after ICOS-L blockade. (E) Expression of Klf2 and S1pr1 by RT-PCR and CD62L and CD69 by flow cytometry after CTLA-4–Ig blockade. All quantitative RT-PCR data are mean values (± SEM) from three experiments with three technical replicates each and β2-microglobulin as housekeeping gene. Flow cytometry data are a representative result from three (ICOS-L blockade) or two (B7 blockade) independent experiments with seven animals per group. (F) WT, ICOS KO, or CD28 KO OT-II T cells were transferred into C57BL/6 recipients or ICOS KO OT-II T cells were transferred into CD80/86 (B7) KO recipients. Transgenic T cells were sorted from pools of draining lymph nodes 3 d after immunization with NP-OVA. Klf2 mRNA was quantified by RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). Dots represent individual mice, bars indicate the mean, and error bars represent the SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Mentions: To investigate which transcription factors are regulated by ICOS-mediated signaling and might play a role for the maintenance of the TFH phenotype, we performed a transcriptome analysis. Antigen-specific TFH cells were sorted from draining lymph nodes after 6 h of blocking ICOS-L. Comparing the ICOS-L blockade with the control group, we observed no difference for any of the transcription factors known to be important for TFH cell differentiation including Bcl-6, Blimp-1, Irf4, Ascl2, and c-Maf. However, the zinc-finger transcription factor Klf2 showed up as one of the most significantly differentially regulated genes. This transcription factor is highly expressed in naive T cells, strongly down-regulated shortly after T cell receptor triggering, and maintained on a lower level in activated T cells (Fig. 6 A). The sixfold lower expression of Klf2 in TFH versus non-TFH cells made it a particularly interesting target for further analyses (Fig. 6 B). Upon blocking ICOS-L, Klf2 was rapidly up-regulated by a factor of 3.5, approaching the expression levels of non-TFH cells (Fig. 6 C). Klf2 was originally described as a factor controlling thymocyte egress via up-regulation of sphingosine-1-phosphate receptor 1 (S1PR1) and CD62L (Carlson et al., 2006). Therefore, we analyzed these target genes on antigen-specific TFH cells after ICOS-L blockade. CD62L and S1PR1 were up-regulated 1.6- and 2.8-fold, respectively, whereas CD69, which is inversely regulated to S1PR1 (Shiow et al., 2006), was reduced by 30% (Fig. 6 D). In contrast, blockade of the CD28 pathway did not change Klf2 expression or expression of any of the latter receptors (Fig. 6 E).

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