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Batf is important for IL-4 expression in T follicular helper cells.

Sahoo A, Alekseev A, Tanaka K, Obertas L, Lerman B, Haymaker C, Clise-Dwyer K, McMurray JS, Nurieva R - Nat Commun (2015)

Bottom Line: Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus.In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells.Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Apart from T helper (Th)-2 cells, T follicular helper (Tfh) cells are a major class of IL-4-producing T cells, required for regulation of type 2 humoral immunity; however, transcriptional control of IL-4 production in Tfh cells remains mainly unknown. Here, we show that the basic leucine zipper transcription factor ATF-like, Batf is important for IL-4 expression in Tfh cells rather than in canonical Th2 cells. Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus. In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells. Batf deficiency impairs the generation of IL-4-producing Tfh cells that results in protection against allergic asthma. Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

No MeSH data available.


Related in: MedlinePlus

Batf-dependent regulation of IL-4 in Tfh cells.(a) Male WT and Batf KO mice (6–8 weeks old, n=5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum. Seven days later, splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells were then sorted as described in the Methods section and expression of the indicated cytokines was analysed by qRT–PCR and ELISA. (b) FACS-sorted naive CD4+ T cells from male WT and Batf KO (6–8 weeks old) mice were intravenously transferred into TCR-β KO (6–8 weeks old, 10 million cells per mouse, n=4 per group) mice followed by i.p. injection with Ova in Alum. Seven days later splenocytes were restimulated with Ova ex vivo and Tfh and nTfh cells from the spleen were sorted and effector cytokine levels were analysed as in (a). (c) Naive CD4+ T cells from male WT OTII and Batf KO OTII (6–8 weeks old) mice were transferred to male B6.SJL (CD45.1+) mice (6–8 weeks old, 10 million cells per mouse; n=5 per group) followed by immunization with Ova in Alum 24 h post transfer. Seven days post immunization splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. Tfh and nTfh cells were sorted and analysed as in (a). (d) Mixed bone marrow chimeric mice (n=10) were generated by co-transfer of bone marrow cells from male WT (CD45.1+) and Batf KO (CD45.2+) mice into sublethally irradiated male Rag1 KO (6–8 weeks old) mice. Eight weeks later, mice were immunized with Ova in Alum and Tfh and non-Tfh cells from the spleen of immunized mice were sorted and analysed as in (a). The qRT–PCR data shown in (a–d) were normalized by the expression of a reference gene Actb. Results shown are mean±s.e.m. and representative of at least three independent experiments. P values: *<0.05 and **<0.01. Student's t-test was performed to detect between-group differences.
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f1: Batf-dependent regulation of IL-4 in Tfh cells.(a) Male WT and Batf KO mice (6–8 weeks old, n=5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum. Seven days later, splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells were then sorted as described in the Methods section and expression of the indicated cytokines was analysed by qRT–PCR and ELISA. (b) FACS-sorted naive CD4+ T cells from male WT and Batf KO (6–8 weeks old) mice were intravenously transferred into TCR-β KO (6–8 weeks old, 10 million cells per mouse, n=4 per group) mice followed by i.p. injection with Ova in Alum. Seven days later splenocytes were restimulated with Ova ex vivo and Tfh and nTfh cells from the spleen were sorted and effector cytokine levels were analysed as in (a). (c) Naive CD4+ T cells from male WT OTII and Batf KO OTII (6–8 weeks old) mice were transferred to male B6.SJL (CD45.1+) mice (6–8 weeks old, 10 million cells per mouse; n=5 per group) followed by immunization with Ova in Alum 24 h post transfer. Seven days post immunization splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. Tfh and nTfh cells were sorted and analysed as in (a). (d) Mixed bone marrow chimeric mice (n=10) were generated by co-transfer of bone marrow cells from male WT (CD45.1+) and Batf KO (CD45.2+) mice into sublethally irradiated male Rag1 KO (6–8 weeks old) mice. Eight weeks later, mice were immunized with Ova in Alum and Tfh and non-Tfh cells from the spleen of immunized mice were sorted and analysed as in (a). The qRT–PCR data shown in (a–d) were normalized by the expression of a reference gene Actb. Results shown are mean±s.e.m. and representative of at least three independent experiments. P values: *<0.05 and **<0.01. Student's t-test was performed to detect between-group differences.

Mentions: According to a recent study, Tfh cells serve as a alternative source of IL-4 in a helminth infection model2. Since Batf deficiency did not affect IL-4 expression in Th2 cells in vitro (Supplementary Fig. 2a), the dramatic decrease in IL-4 expression in vivo in Batf KO mice could be potentially attributed to Tfh cells211. To address this possibility, we stimulated splenocytes from Ova-immunized WT and Batf KO mice with Ova ex vivo for 3 days and sorted and analysed CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells as described in the Methods section (Supplementary Fig. 3; Fig. 1a). Consistent with in vitro-differentiated Th2 cells, Batf deficiency in nTfh cells led to decreased expression of IL-5 and IL-13 as well as Gata3, but not IL-4 (Fig. 1a). However, the decrease in IL-4 level was prominent in Batf KO Tfh cells both at mRNA and protein levels (Fig. 1a). To further demonstrate whether this profound defect in IL-4 production by Batf-deficient Tfh cells is T-cell intrinsic, we sorted and analysed Tfh and nTfh cells from TCR-β KO mice reconstituted with naive WT and Batf KO CD4+ T cells and subjected to Ova in Alum immunization (Fig. 1b). Tfh cells from mice reconstituted with Batf-deficient CD4+ T cells showed a consistent defect in IL-4 expression compared with Tfh cells from mice, which received WT CD4+ T cells, while IL-4 level remained unaltered in WT and Batf KO nTfh cells (Fig. 1b). To confirm that the acquired Tfh cell phenotype was truly antigen specific, we adoptively transferred naive WT and Batf KO Ova transgenic (OT) II cells into B6.SJL (CD45.1+) mice and immunized them with Ova in Alum. Seven days post immunization donor WT and Batf KO Tfh and nTfh cells were sorted from the spleen of these mice and IL-4, IL-5 and IL-13 levels were analysed by quantitative reverse-transcription PCR (qRT–PCR) and enzyme-linked immunosorbent assay (ELISA) (Fig. 1c). Similar to our above observations, Batf KO OTII Tfh cells showed lower IL-4 level compared with WT cells, while IL-4 level was comparable between WT and KO OTII nTfh cells (Fig. 1c).


Batf is important for IL-4 expression in T follicular helper cells.

Sahoo A, Alekseev A, Tanaka K, Obertas L, Lerman B, Haymaker C, Clise-Dwyer K, McMurray JS, Nurieva R - Nat Commun (2015)

Batf-dependent regulation of IL-4 in Tfh cells.(a) Male WT and Batf KO mice (6–8 weeks old, n=5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum. Seven days later, splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells were then sorted as described in the Methods section and expression of the indicated cytokines was analysed by qRT–PCR and ELISA. (b) FACS-sorted naive CD4+ T cells from male WT and Batf KO (6–8 weeks old) mice were intravenously transferred into TCR-β KO (6–8 weeks old, 10 million cells per mouse, n=4 per group) mice followed by i.p. injection with Ova in Alum. Seven days later splenocytes were restimulated with Ova ex vivo and Tfh and nTfh cells from the spleen were sorted and effector cytokine levels were analysed as in (a). (c) Naive CD4+ T cells from male WT OTII and Batf KO OTII (6–8 weeks old) mice were transferred to male B6.SJL (CD45.1+) mice (6–8 weeks old, 10 million cells per mouse; n=5 per group) followed by immunization with Ova in Alum 24 h post transfer. Seven days post immunization splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. Tfh and nTfh cells were sorted and analysed as in (a). (d) Mixed bone marrow chimeric mice (n=10) were generated by co-transfer of bone marrow cells from male WT (CD45.1+) and Batf KO (CD45.2+) mice into sublethally irradiated male Rag1 KO (6–8 weeks old) mice. Eight weeks later, mice were immunized with Ova in Alum and Tfh and non-Tfh cells from the spleen of immunized mice were sorted and analysed as in (a). The qRT–PCR data shown in (a–d) were normalized by the expression of a reference gene Actb. Results shown are mean±s.e.m. and representative of at least three independent experiments. P values: *<0.05 and **<0.01. Student's t-test was performed to detect between-group differences.
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Related In: Results  -  Collection

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f1: Batf-dependent regulation of IL-4 in Tfh cells.(a) Male WT and Batf KO mice (6–8 weeks old, n=5 per group) were injected i.p. with 0.2 ml saline containing 100 μg Ova in Alum. Seven days later, splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells were then sorted as described in the Methods section and expression of the indicated cytokines was analysed by qRT–PCR and ELISA. (b) FACS-sorted naive CD4+ T cells from male WT and Batf KO (6–8 weeks old) mice were intravenously transferred into TCR-β KO (6–8 weeks old, 10 million cells per mouse, n=4 per group) mice followed by i.p. injection with Ova in Alum. Seven days later splenocytes were restimulated with Ova ex vivo and Tfh and nTfh cells from the spleen were sorted and effector cytokine levels were analysed as in (a). (c) Naive CD4+ T cells from male WT OTII and Batf KO OTII (6–8 weeks old) mice were transferred to male B6.SJL (CD45.1+) mice (6–8 weeks old, 10 million cells per mouse; n=5 per group) followed by immunization with Ova in Alum 24 h post transfer. Seven days post immunization splenocytes from immunized mice were isolated and stimulated with Ova (100 μg ml−1) for 72 h ex vivo. Tfh and nTfh cells were sorted and analysed as in (a). (d) Mixed bone marrow chimeric mice (n=10) were generated by co-transfer of bone marrow cells from male WT (CD45.1+) and Batf KO (CD45.2+) mice into sublethally irradiated male Rag1 KO (6–8 weeks old) mice. Eight weeks later, mice were immunized with Ova in Alum and Tfh and non-Tfh cells from the spleen of immunized mice were sorted and analysed as in (a). The qRT–PCR data shown in (a–d) were normalized by the expression of a reference gene Actb. Results shown are mean±s.e.m. and representative of at least three independent experiments. P values: *<0.05 and **<0.01. Student's t-test was performed to detect between-group differences.
Mentions: According to a recent study, Tfh cells serve as a alternative source of IL-4 in a helminth infection model2. Since Batf deficiency did not affect IL-4 expression in Th2 cells in vitro (Supplementary Fig. 2a), the dramatic decrease in IL-4 expression in vivo in Batf KO mice could be potentially attributed to Tfh cells211. To address this possibility, we stimulated splenocytes from Ova-immunized WT and Batf KO mice with Ova ex vivo for 3 days and sorted and analysed CD4+CD44hiCXCR5hiPD1hi (Tfh) and CD4+CD44hiCXCR5− (nTfh) cells as described in the Methods section (Supplementary Fig. 3; Fig. 1a). Consistent with in vitro-differentiated Th2 cells, Batf deficiency in nTfh cells led to decreased expression of IL-5 and IL-13 as well as Gata3, but not IL-4 (Fig. 1a). However, the decrease in IL-4 level was prominent in Batf KO Tfh cells both at mRNA and protein levels (Fig. 1a). To further demonstrate whether this profound defect in IL-4 production by Batf-deficient Tfh cells is T-cell intrinsic, we sorted and analysed Tfh and nTfh cells from TCR-β KO mice reconstituted with naive WT and Batf KO CD4+ T cells and subjected to Ova in Alum immunization (Fig. 1b). Tfh cells from mice reconstituted with Batf-deficient CD4+ T cells showed a consistent defect in IL-4 expression compared with Tfh cells from mice, which received WT CD4+ T cells, while IL-4 level remained unaltered in WT and Batf KO nTfh cells (Fig. 1b). To confirm that the acquired Tfh cell phenotype was truly antigen specific, we adoptively transferred naive WT and Batf KO Ova transgenic (OT) II cells into B6.SJL (CD45.1+) mice and immunized them with Ova in Alum. Seven days post immunization donor WT and Batf KO Tfh and nTfh cells were sorted from the spleen of these mice and IL-4, IL-5 and IL-13 levels were analysed by quantitative reverse-transcription PCR (qRT–PCR) and enzyme-linked immunosorbent assay (ELISA) (Fig. 1c). Similar to our above observations, Batf KO OTII Tfh cells showed lower IL-4 level compared with WT cells, while IL-4 level was comparable between WT and KO OTII nTfh cells (Fig. 1c).

Bottom Line: Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus.In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells.Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, MD Anderson Cancer Center, Houston, Texas 77030, USA.

ABSTRACT
Apart from T helper (Th)-2 cells, T follicular helper (Tfh) cells are a major class of IL-4-producing T cells, required for regulation of type 2 humoral immunity; however, transcriptional control of IL-4 production in Tfh cells remains mainly unknown. Here, we show that the basic leucine zipper transcription factor ATF-like, Batf is important for IL-4 expression in Tfh cells rather than in canonical Th2 cells. Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus. In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells. Batf deficiency impairs the generation of IL-4-producing Tfh cells that results in protection against allergic asthma. Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

No MeSH data available.


Related in: MedlinePlus