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Cytokine-secreting follicular T cells shape the antibody repertoire.

Reinhardt RL, Liang HE, Locksley RM - Nat. Immunol. (2009)

Bottom Line: High-affinity antibodies are critical for host protection and underlie successful vaccines.The generation of such antibodies requires T cell-dependent help, which mediates germinal center reactions in which mutation and selection of B cells occurs.Our findings support a model in which B cells compete for cytokines produced by follicular helper T cells that shape the affinity and isotype of the antibody response.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Departments of Medicine and Microbiology & Immunology, University of California San Francisco, USA.

ABSTRACT
High-affinity antibodies are critical for host protection and underlie successful vaccines. The generation of such antibodies requires T cell-dependent help, which mediates germinal center reactions in which mutation and selection of B cells occurs. Using an interleukin 4-reporter system, we show here that CD4(+) follicular helper T cells constituted essentially all of the cytokine-secreting T cells in lymph nodes and were functionally distinct from T cells secreting the same cytokine in peripheral tissues. Follicular helper T cells with different cytokine profiles could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin class switching with evidence of somatic hypermutation. Our findings support a model in which B cells compete for cytokines produced by follicular helper T cells that shape the affinity and isotype of the antibody response.

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B–T conjugates regulate immunoglobulin class-switching(a) Mice were infected with L. major and draining lymph nodes were harvested 21 days after infection. HuCD2 (IL-4 secretion) expression on GFP+ CD4+ T cells or B–T cell conjugates gated in leftmost panel are indicated by arrows in the four panels of which the first panels denote staining with anti-human CD2 and the right panels denote staining with an isotype control antibody. Plots are representative of at least six experiments (n = 2–4 mice per experiment).(b) Size as marked by forward-size scatter of GFP+, CD4+, B220+ co-expressing conjugates compared to GFP+ singlet or GFP− doublet cells in the draining lymph node of KN2 × 4get mice.(c) Gating scheme of CD4+ T cell and B cell doublets in the draining lymph node of L. major-infected KN2 × 4get mice prior to sorting. All GFP+,CD4+, B220+ cells express the T cell receptor-associated CD3 marker.(d) GFP+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA post-sort and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(e) huCD2+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(f) B–T cell conjugates from KN2 × 4get dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID and IgG1-post-switch transcripts. cDNA from GFP+; GFP+, huCD2+; or GFP−, huCD2− CD4+ B–T cell conjugates was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction. Gels are representative of at least two independent experiments with cDNA at 3 serial dilutions.(g) 200x image from section of the draining popliteal lymph node from KN2 x great mice 14 days after L. major infection. huCD2 (Red, IL-4-secretion), YFP (green, IFN-γ-competence), PNA (blue, germinal centers). Image is representative of 6 lymph nodes from at least two independent experiments.(h) B–T cell conjugates from KN2 x great dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID, IgG1 and IgG2a switch and germline transcripts. cDNA from YFP+ or huCD2+ CD4+, CD19+ B–T cell conjugates or GFP−, huCD2−, CD4−, CD19+ singlets was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction and gels were loaded with 3 serial dilutions of cDNA. Gel is representative of at least two independent experiments with cDNA used at three serial dilutions.
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Figure 5: B–T conjugates regulate immunoglobulin class-switching(a) Mice were infected with L. major and draining lymph nodes were harvested 21 days after infection. HuCD2 (IL-4 secretion) expression on GFP+ CD4+ T cells or B–T cell conjugates gated in leftmost panel are indicated by arrows in the four panels of which the first panels denote staining with anti-human CD2 and the right panels denote staining with an isotype control antibody. Plots are representative of at least six experiments (n = 2–4 mice per experiment).(b) Size as marked by forward-size scatter of GFP+, CD4+, B220+ co-expressing conjugates compared to GFP+ singlet or GFP− doublet cells in the draining lymph node of KN2 × 4get mice.(c) Gating scheme of CD4+ T cell and B cell doublets in the draining lymph node of L. major-infected KN2 × 4get mice prior to sorting. All GFP+,CD4+, B220+ cells express the T cell receptor-associated CD3 marker.(d) GFP+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA post-sort and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(e) huCD2+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(f) B–T cell conjugates from KN2 × 4get dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID and IgG1-post-switch transcripts. cDNA from GFP+; GFP+, huCD2+; or GFP−, huCD2− CD4+ B–T cell conjugates was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction. Gels are representative of at least two independent experiments with cDNA at 3 serial dilutions.(g) 200x image from section of the draining popliteal lymph node from KN2 x great mice 14 days after L. major infection. huCD2 (Red, IL-4-secretion), YFP (green, IFN-γ-competence), PNA (blue, germinal centers). Image is representative of 6 lymph nodes from at least two independent experiments.(h) B–T cell conjugates from KN2 x great dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID, IgG1 and IgG2a switch and germline transcripts. cDNA from YFP+ or huCD2+ CD4+, CD19+ B–T cell conjugates or GFP−, huCD2−, CD4−, CD19+ singlets was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction and gels were loaded with 3 serial dilutions of cDNA. Gel is representative of at least two independent experiments with cDNA used at three serial dilutions.

Mentions: The unexpected predominance of IL-4 secretion in and around the follicles and the germinal centers led us to hypothesize that competition among GC B cells for rare cytokine-producing GC T cells may underlie antibody isotype class switching and possibly affinity maturation. Among IL-4-producing TFH cells, a small fraction of GFP+ or huCD2+ cells co-expressed the B cell marker CD19, suggesting that these may represent stable B–T cell conjugates (Fig. 5a; Supplementary Fig. 3 online) 9, 25. Corroborating further their identification as B–T conjugates, GFP+ cells that expressed both T and B cell markers were twice the size of the single cells (Fig. 5b). Although isolated GC B cells can form conjugates with T cells in vitro and real-time studies have imaged B–T conjugates in vivo, no physiological relevance has been established for these conjugates25, 26. To confirm this relevance in vivo, we infected mice with L. major and isolated B–T conjugates after 3 weeks. As compared to non-IL-4-expressing conjugates, the IL-4-expressing conjugates were greatly enriched for expression of germinal center markers, consistent with localization of cytokine expression to regions of active T cell help (Supplementary Fig. 4 online). B–T conjugates represent a rare population (0.2 to 1% of total lymphocytes) in the lymph node during the course of infection (Fig. 5c; data not shown). Furthermore, IL-4-secreting (huCD2+, GFP+) and IL-4-competent (huCD2−, GFP+) conjugates represent only 1 to 3% of total conjugates, respectively (Fig. 5a,c). Indeed, all CD4+B220+ cells that expressed GFP co-expressed the TCR marker CD3 and represented B–T conjugates (Fig. 5c). To analyze stable conjugates, the small percentage of GFP+ or huCD2+ cells that co-expressed the T and B cell markers CD4 and B220 were sorted to high purity and then treated with EDTA to dissociate cell-cell contacts. The resulting single cells segregated into approximately equal numbers of CD4 and B220 single-positive populations, consistent with their original purification as B–T conjugates (Fig. 5d,e). Importantly, all of the GFP+ or huCD2+ cells were identified as CD4+ T cells rather than B cells, consistent with IL-4 production by TFH cells and not by B cells.


Cytokine-secreting follicular T cells shape the antibody repertoire.

Reinhardt RL, Liang HE, Locksley RM - Nat. Immunol. (2009)

B–T conjugates regulate immunoglobulin class-switching(a) Mice were infected with L. major and draining lymph nodes were harvested 21 days after infection. HuCD2 (IL-4 secretion) expression on GFP+ CD4+ T cells or B–T cell conjugates gated in leftmost panel are indicated by arrows in the four panels of which the first panels denote staining with anti-human CD2 and the right panels denote staining with an isotype control antibody. Plots are representative of at least six experiments (n = 2–4 mice per experiment).(b) Size as marked by forward-size scatter of GFP+, CD4+, B220+ co-expressing conjugates compared to GFP+ singlet or GFP− doublet cells in the draining lymph node of KN2 × 4get mice.(c) Gating scheme of CD4+ T cell and B cell doublets in the draining lymph node of L. major-infected KN2 × 4get mice prior to sorting. All GFP+,CD4+, B220+ cells express the T cell receptor-associated CD3 marker.(d) GFP+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA post-sort and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(e) huCD2+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(f) B–T cell conjugates from KN2 × 4get dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID and IgG1-post-switch transcripts. cDNA from GFP+; GFP+, huCD2+; or GFP−, huCD2− CD4+ B–T cell conjugates was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction. Gels are representative of at least two independent experiments with cDNA at 3 serial dilutions.(g) 200x image from section of the draining popliteal lymph node from KN2 x great mice 14 days after L. major infection. huCD2 (Red, IL-4-secretion), YFP (green, IFN-γ-competence), PNA (blue, germinal centers). Image is representative of 6 lymph nodes from at least two independent experiments.(h) B–T cell conjugates from KN2 x great dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID, IgG1 and IgG2a switch and germline transcripts. cDNA from YFP+ or huCD2+ CD4+, CD19+ B–T cell conjugates or GFP−, huCD2−, CD4−, CD19+ singlets was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction and gels were loaded with 3 serial dilutions of cDNA. Gel is representative of at least two independent experiments with cDNA used at three serial dilutions.
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Related In: Results  -  Collection

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Figure 5: B–T conjugates regulate immunoglobulin class-switching(a) Mice were infected with L. major and draining lymph nodes were harvested 21 days after infection. HuCD2 (IL-4 secretion) expression on GFP+ CD4+ T cells or B–T cell conjugates gated in leftmost panel are indicated by arrows in the four panels of which the first panels denote staining with anti-human CD2 and the right panels denote staining with an isotype control antibody. Plots are representative of at least six experiments (n = 2–4 mice per experiment).(b) Size as marked by forward-size scatter of GFP+, CD4+, B220+ co-expressing conjugates compared to GFP+ singlet or GFP− doublet cells in the draining lymph node of KN2 × 4get mice.(c) Gating scheme of CD4+ T cell and B cell doublets in the draining lymph node of L. major-infected KN2 × 4get mice prior to sorting. All GFP+,CD4+, B220+ cells express the T cell receptor-associated CD3 marker.(d) GFP+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA post-sort and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(e) huCD2+, CD4+, B220+ doublets sorted from (a) were treated with 2 mM EDTA and reanalyzed by flow cytometry. Plots are representative of combined lymph nodes from at least two independent experiments (n = 2–3 mice).(f) B–T cell conjugates from KN2 × 4get dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID and IgG1-post-switch transcripts. cDNA from GFP+; GFP+, huCD2+; or GFP−, huCD2− CD4+ B–T cell conjugates was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction. Gels are representative of at least two independent experiments with cDNA at 3 serial dilutions.(g) 200x image from section of the draining popliteal lymph node from KN2 x great mice 14 days after L. major infection. huCD2 (Red, IL-4-secretion), YFP (green, IFN-γ-competence), PNA (blue, germinal centers). Image is representative of 6 lymph nodes from at least two independent experiments.(h) B–T cell conjugates from KN2 x great dual reporter mice were sorted 14 days after L. major infection from draining popliteal lymph nodes and semi-quantitative RT-PCR was performed for expression of AID, IgG1 and IgG2a switch and germline transcripts. cDNA from YFP+ or huCD2+ CD4+, CD19+ B–T cell conjugates or GFP−, huCD2−, CD4−, CD19+ singlets was normalized to Pax5 expression to ensure equivalent amounts of B cell cDNA was added per reaction and gels were loaded with 3 serial dilutions of cDNA. Gel is representative of at least two independent experiments with cDNA used at three serial dilutions.
Mentions: The unexpected predominance of IL-4 secretion in and around the follicles and the germinal centers led us to hypothesize that competition among GC B cells for rare cytokine-producing GC T cells may underlie antibody isotype class switching and possibly affinity maturation. Among IL-4-producing TFH cells, a small fraction of GFP+ or huCD2+ cells co-expressed the B cell marker CD19, suggesting that these may represent stable B–T cell conjugates (Fig. 5a; Supplementary Fig. 3 online) 9, 25. Corroborating further their identification as B–T conjugates, GFP+ cells that expressed both T and B cell markers were twice the size of the single cells (Fig. 5b). Although isolated GC B cells can form conjugates with T cells in vitro and real-time studies have imaged B–T conjugates in vivo, no physiological relevance has been established for these conjugates25, 26. To confirm this relevance in vivo, we infected mice with L. major and isolated B–T conjugates after 3 weeks. As compared to non-IL-4-expressing conjugates, the IL-4-expressing conjugates were greatly enriched for expression of germinal center markers, consistent with localization of cytokine expression to regions of active T cell help (Supplementary Fig. 4 online). B–T conjugates represent a rare population (0.2 to 1% of total lymphocytes) in the lymph node during the course of infection (Fig. 5c; data not shown). Furthermore, IL-4-secreting (huCD2+, GFP+) and IL-4-competent (huCD2−, GFP+) conjugates represent only 1 to 3% of total conjugates, respectively (Fig. 5a,c). Indeed, all CD4+B220+ cells that expressed GFP co-expressed the TCR marker CD3 and represented B–T conjugates (Fig. 5c). To analyze stable conjugates, the small percentage of GFP+ or huCD2+ cells that co-expressed the T and B cell markers CD4 and B220 were sorted to high purity and then treated with EDTA to dissociate cell-cell contacts. The resulting single cells segregated into approximately equal numbers of CD4 and B220 single-positive populations, consistent with their original purification as B–T conjugates (Fig. 5d,e). Importantly, all of the GFP+ or huCD2+ cells were identified as CD4+ T cells rather than B cells, consistent with IL-4 production by TFH cells and not by B cells.

Bottom Line: High-affinity antibodies are critical for host protection and underlie successful vaccines.The generation of such antibodies requires T cell-dependent help, which mediates germinal center reactions in which mutation and selection of B cells occurs.Our findings support a model in which B cells compete for cytokines produced by follicular helper T cells that shape the affinity and isotype of the antibody response.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Departments of Medicine and Microbiology & Immunology, University of California San Francisco, USA.

ABSTRACT
High-affinity antibodies are critical for host protection and underlie successful vaccines. The generation of such antibodies requires T cell-dependent help, which mediates germinal center reactions in which mutation and selection of B cells occurs. Using an interleukin 4-reporter system, we show here that CD4(+) follicular helper T cells constituted essentially all of the cytokine-secreting T cells in lymph nodes and were functionally distinct from T cells secreting the same cytokine in peripheral tissues. Follicular helper T cells with different cytokine profiles could be isolated as conjugates with B cells undergoing cytokine-specific immunoglobulin class switching with evidence of somatic hypermutation. Our findings support a model in which B cells compete for cytokines produced by follicular helper T cells that shape the affinity and isotype of the antibody response.

Show MeSH
Related in: MedlinePlus