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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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CD28 but not ICOS regulates early key events of TFH cell differentiation. WT (black) and CD28 KO (blue) or ICOS KO (red) OT-II cells were either co-transferred (A and B) or transferred separately (C and D) into C57BL/6 recipients subcutaneously immunized with NP-OVA. Draining lymph nodes were analyzed on day 3 after immunization. (A) Expression of Bcl-6, CXCR5, and CCR7 analyzed by flow cytometry and displayed as representative histograms and as bar graph with the geometric mean fluorescence intensity (geoMFI). Endogenous CD4+ T cells are shown in gray. Dots represent individual mice and bars indicate the mean. (B) Expression of CD40L was evaluated ex vivo by intracellular staining without restimulation and IL-21 and IL-4 were assessed after short-term restimulation with OVA peptide. Representative flow cytometry data and geoMFI or percentage of positive cells are shown. (C) WT or KO OT-II T cells were sorted from draining lymph nodes (pool from 20 animals) by magnetic and flow sorting for Thy-1.1+ cells for the preparation of RNA. Expression of Ascl2, c-Maf, Prdm1, Tbx21, and Gata3 mRNA was measured by quantitative RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). (D) Representative histological pictures of draining lymph nodes showing the T cell zone (CD4+, green), B cell follicle (B220+, magenta), and antigen-specific T cells (Thy-1.1+, white). The yellow dashed line demarcates the border between the T cell zone and the B cell follicle. Bars, 100 µm. Representative data from two (mRNA, cytokines, and histology) or four (all other) experiments. **, P < 0.01; ***, P < 0.001.
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fig2: CD28 but not ICOS regulates early key events of TFH cell differentiation. WT (black) and CD28 KO (blue) or ICOS KO (red) OT-II cells were either co-transferred (A and B) or transferred separately (C and D) into C57BL/6 recipients subcutaneously immunized with NP-OVA. Draining lymph nodes were analyzed on day 3 after immunization. (A) Expression of Bcl-6, CXCR5, and CCR7 analyzed by flow cytometry and displayed as representative histograms and as bar graph with the geometric mean fluorescence intensity (geoMFI). Endogenous CD4+ T cells are shown in gray. Dots represent individual mice and bars indicate the mean. (B) Expression of CD40L was evaluated ex vivo by intracellular staining without restimulation and IL-21 and IL-4 were assessed after short-term restimulation with OVA peptide. Representative flow cytometry data and geoMFI or percentage of positive cells are shown. (C) WT or KO OT-II T cells were sorted from draining lymph nodes (pool from 20 animals) by magnetic and flow sorting for Thy-1.1+ cells for the preparation of RNA. Expression of Ascl2, c-Maf, Prdm1, Tbx21, and Gata3 mRNA was measured by quantitative RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). (D) Representative histological pictures of draining lymph nodes showing the T cell zone (CD4+, green), B cell follicle (B220+, magenta), and antigen-specific T cells (Thy-1.1+, white). The yellow dashed line demarcates the border between the T cell zone and the B cell follicle. Bars, 100 µm. Representative data from two (mRNA, cytokines, and histology) or four (all other) experiments. **, P < 0.01; ***, P < 0.001.

Mentions: To understand the molecular mechanisms, we first quantified the expression of key molecules for TFH cell development and function at an early point in time. 3 d after immunization (similar data were obtained for days 2 and 4; not depicted), only a small fraction of CD28-deficient T cells expressed the TFH master transcription factor Bcl-6 (Fig. 2 A). Unexpectedly, we found that ICOS KO T cells up-regulated Bcl-6 to a similar extent as WT T cells, which seems to be in contrast to published data (Choi et al., 2011), as discussed below. In CD28- and ICOS-deficient T cells, the regulation of the two major TFH homing chemokine receptors was significantly impaired with an incomplete down-regulation of CCR7 and reduced up-regulation of CXCR5 (Fig. 2 A). However, when we analyzed the migration of developing TFH cells by immunohistology, ICOS-deficient T cells migrated to a similar extent as WT T cells toward the T/B cell border (Fig. 2 D). In stark contrast, CD28-deficient T cells remained equally distributed in the T cell zone. In the WT group, some T cells had already moved deeper into the B cell zone. These cells were reduced by 50% in the ICOS KO group (unpublished data), which is in line with findings by Xu et al. (2013), demonstrating that at later times (beyond day 4), a substantial difference regarding follicular localization of ICOS-deficient T cells develops.


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)

CD28 but not ICOS regulates early key events of TFH cell differentiation. WT (black) and CD28 KO (blue) or ICOS KO (red) OT-II cells were either co-transferred (A and B) or transferred separately (C and D) into C57BL/6 recipients subcutaneously immunized with NP-OVA. Draining lymph nodes were analyzed on day 3 after immunization. (A) Expression of Bcl-6, CXCR5, and CCR7 analyzed by flow cytometry and displayed as representative histograms and as bar graph with the geometric mean fluorescence intensity (geoMFI). Endogenous CD4+ T cells are shown in gray. Dots represent individual mice and bars indicate the mean. (B) Expression of CD40L was evaluated ex vivo by intracellular staining without restimulation and IL-21 and IL-4 were assessed after short-term restimulation with OVA peptide. Representative flow cytometry data and geoMFI or percentage of positive cells are shown. (C) WT or KO OT-II T cells were sorted from draining lymph nodes (pool from 20 animals) by magnetic and flow sorting for Thy-1.1+ cells for the preparation of RNA. Expression of Ascl2, c-Maf, Prdm1, Tbx21, and Gata3 mRNA was measured by quantitative RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). (D) Representative histological pictures of draining lymph nodes showing the T cell zone (CD4+, green), B cell follicle (B220+, magenta), and antigen-specific T cells (Thy-1.1+, white). The yellow dashed line demarcates the border between the T cell zone and the B cell follicle. Bars, 100 µm. Representative data from two (mRNA, cytokines, and histology) or four (all other) experiments. **, P < 0.01; ***, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: CD28 but not ICOS regulates early key events of TFH cell differentiation. WT (black) and CD28 KO (blue) or ICOS KO (red) OT-II cells were either co-transferred (A and B) or transferred separately (C and D) into C57BL/6 recipients subcutaneously immunized with NP-OVA. Draining lymph nodes were analyzed on day 3 after immunization. (A) Expression of Bcl-6, CXCR5, and CCR7 analyzed by flow cytometry and displayed as representative histograms and as bar graph with the geometric mean fluorescence intensity (geoMFI). Endogenous CD4+ T cells are shown in gray. Dots represent individual mice and bars indicate the mean. (B) Expression of CD40L was evaluated ex vivo by intracellular staining without restimulation and IL-21 and IL-4 were assessed after short-term restimulation with OVA peptide. Representative flow cytometry data and geoMFI or percentage of positive cells are shown. (C) WT or KO OT-II T cells were sorted from draining lymph nodes (pool from 20 animals) by magnetic and flow sorting for Thy-1.1+ cells for the preparation of RNA. Expression of Ascl2, c-Maf, Prdm1, Tbx21, and Gata3 mRNA was measured by quantitative RT-PCR (expression relative to Hprt; mean ± SEM from triplicates and two independent experiments). (D) Representative histological pictures of draining lymph nodes showing the T cell zone (CD4+, green), B cell follicle (B220+, magenta), and antigen-specific T cells (Thy-1.1+, white). The yellow dashed line demarcates the border between the T cell zone and the B cell follicle. Bars, 100 µm. Representative data from two (mRNA, cytokines, and histology) or four (all other) experiments. **, P < 0.01; ***, P < 0.001.
Mentions: To understand the molecular mechanisms, we first quantified the expression of key molecules for TFH cell development and function at an early point in time. 3 d after immunization (similar data were obtained for days 2 and 4; not depicted), only a small fraction of CD28-deficient T cells expressed the TFH master transcription factor Bcl-6 (Fig. 2 A). Unexpectedly, we found that ICOS KO T cells up-regulated Bcl-6 to a similar extent as WT T cells, which seems to be in contrast to published data (Choi et al., 2011), as discussed below. In CD28- and ICOS-deficient T cells, the regulation of the two major TFH homing chemokine receptors was significantly impaired with an incomplete down-regulation of CCR7 and reduced up-regulation of CXCR5 (Fig. 2 A). However, when we analyzed the migration of developing TFH cells by immunohistology, ICOS-deficient T cells migrated to a similar extent as WT T cells toward the T/B cell border (Fig. 2 D). In stark contrast, CD28-deficient T cells remained equally distributed in the T cell zone. In the WT group, some T cells had already moved deeper into the B cell zone. These cells were reduced by 50% in the ICOS KO group (unpublished data), which is in line with findings by Xu et al. (2013), demonstrating that at later times (beyond day 4), a substantial difference regarding follicular localization of ICOS-deficient T cells develops.

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