Cytokine-secreting follicular T cells shape the antibody repertoire

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.

Figure 1. Kinetics and identification of IL-4-producing cells in the draining lymph nodes after L. major infection

(a) Sections of the draining popliteal lymph nodes from KN2 × 4get dual reporter mice infected with L. major were stained for GFP (green, IL-4-competent cells), and IgD (red, B cell follicles) (top) and human CD2 (green, IL-4-secreting cells) and PNA (red, germinal centers) (bottom) at the indicated times post-infection. Individual 40x images were merged to form the composite picture of the lymph node. In bottom panel green staining was increased by 5 pixels. Time points are representative of at least two independent experiments (n = 2–4 lymph nodes). (b) The expression of human CD2 (IL-4-secreting cells) was analyzed in situ on sections from the draining lymph nodes 14 and 21 days after L. major infection. 100x images from slides stained for human CD2 (green), CD23 (red, follicular dendritic cell), and IgD (blue, B cell follicles). GC light and dark zones were identified by the presence and absence of CD23 staining, respectively. Sections are representative of two lymph nodes per time point.

Figure 2. IL-4 producing cells in the lymph node coexpress ICOS, CXCR5, and human CD2⁺ in KN2 × 4get mice after L. major infection

(a) Flow cytometry of draining popliteal lymph node cells from KN2 × 4get dual reporter mice infected with L. major 21 days previously. Plots represent CD4⁺, DAPI⁻, B220⁻, CD8⁻ lymphocytes. The numbers in each quadrant represent the percent of cells expressing CXCR5 plotted against GFP or huCD2. Plots are representative of at least two independent experiments (n = 5–7 mice). (b) Flow cytometry of CD4⁺ T cells from L. major-infected lymph nodes. Numbers represent the percentage of CD4⁺ T cells expressing ICOS and GFP or ICOS and huCD2 among total CD4⁺ T cells. Plots are representative of 10 mice from 4 independent experiments. (c) KN2 × 4get mice were infected with 0.5 × 10⁶ L. major promastigotes and the draining lymph nodes were examined at the indicated times. Popliteal lymph nodes were sectioned and stained for human CD2 (green), ICOS (red), B220 (blue).

Figure 3. IL-4-producing CD4 T cells in lymph nodes are T _FH cells and are functionally distinct from canonical T _H2 cells

(a) Relative expression of mRNA transcripts plotted against housekeeping gene HPRT for the indicated genes from CD4⁺ T cells isolated from the lymph nodes and lungs of mice infected with N. brasiliensis 9 days previously. Data are representative of at least two independent experiments. (b) ELISPOT assay for IL-21-secreting cells from tissues of mice infected with N. brasiliensis 10 days previously. GFP⁺, CD4⁺ T cells were isolated and sorted from the indicated organs and cultured in the presence or absence of PMA plus ionomycin. Data are representative of at least 3 independent experiments. (c) Eosinophil recruitment into the lung 10 days post-N. brasiliensis infection of combined IL-4 and 13-deficient mice adoptively transferred with 50,000 GFP⁺, huCD2⁺, CD4⁺ T cells from the lymph node (huCD2⁺ LN) or lung (huCD2⁺ Lung) compared to infected mice that received no T cell transfer (No Tx) or uninfected naïve (Naïve) combined IL-4 and 13-deficient controls. Total Siglec-F^hi tissue eosinophils ± S.E.M. (n = 5–9 mice per group combined from 3 independent experiments). *(P < 0.01)

Figure 4. ICOS regulates IL-4 production by follicular T helper cells

KN2 × 4get dual reporter mice were infected with L. major and injected every other day with 100 µg of anti-ICOS-L antibody or isotype control. The draining popliteal lymph nodes were isolated 14 days after infection. (a)Sections from draining popliteal lymph nodes were stained as indicated. Top, anti-GFP (green, IL-4-competent cells), PNA (red, GC) and anti-IgD (blue, B cell follicles). Bottom, anti-human CD2 (green, IL-4-secreting cells), anti-IgD (red, B cell follicles) and anti-BCL6 (blue, GC). Individual 40x images were merged to form the composite picture of the whole lymph node. Images are representative of lymph nodes from at least two independent experiments (n = 2–4 mice). (b) 100x images taken from popliteal lymph nodes of L. major-infected mice stained with anti-human CD2 (green), anti-IgD (red, B cell follicles) and anti-CD23 (blue, follicular DC). (c) Graphs represent the percentage of GFP or huCD2-expressing CD4⁺ T cells or CD95⁺, GL7⁺ germinal center B cells among total lymphocytes in draining lymph nodes of OVA-NP(15)-challenged mice after treatment with anti-ICOS-L (white bars) or isotype control (black bars). *(P < 0.01); **(P < 0.001). Data representative of at least two independent experiments (n = 4 mice).

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.

Figure 6. Somatic hypermutation on day 14 in J_H4 intron and V _H 186.2 region among B–T conjugates and NP-specific B cells after OVA-NP(15) alum injection

(a) Analysis of J_H4 introns cloned from B–T conjugates and NP⁺ B cell singlets. The pie charts represent the number of unique clones (70 clones for B–T conjugates; 42 clones for NP-singlets) with nucleotide mutations in the J_H4 region. Pie segments are proportional to the number of sequences with the marked number of nucleotide changes. The mutation frequency is indicated below the pie chart and represents the number of mutations per base pair. Data compiled from two independent experiments. (b) Analysis of V_H186.2 heavy chains cloned from GFP⁺ B–T conjugates and NP⁺ B cell singlets 14 days post-injection of OVA-NP. The pie charts represent the number of unique clones (25 clones for B–T conjugates; 44 clones for NP-singlets) sequenced for nucleotide mutations in the V-region. Pie segments are proportional to the number of sequences with the marked number of nucleotide changes. The mutation frequency is indicated below the pie chart and represents the number of mutations per base pair. Data compiled from two independent experiments. (c) The graph represents the ratio of amino acid replacement mutations to silent mutations within the framework and complementarity-determining regions compared to the canonical V_H186.2 gene in GFP⁺ B–T conjugates (filled bars) and NP-specific B cells (open bars). Data compiled from two independent experiments.

Figure 7. Role of IL-4-producing T _FH cells in plasma cell generation and affinity maturation

(a) Mice homozygous or heterozygous for the KN2 knock-in allele were immunized with OVA-NP(15) precipitated in alum. 100x images of germinal centers in KN2/+ (IL-4-sufficient) and KN2/KN2 (IL-4-deficient) mice 14 days after immunization. Sections were stained with anti-human CD2 (green, IL-4-secreting cells), anti-PNA (red, GC) and anti-B220 (blue, B cell follicles). (b) The graphs represent the total number of huCD2⁺, CD4⁺ T cells and germinal center B cells ± S.E.M. (n = 6 mice per group combined from two independent experiments) at each time after OVA-NP(15) injection in KN2/+ (black bars) and KN2/KN2 (white bars). (c) The graphs represent the number of anti-NP IgG1-secreting cells in the draining lymph node and bone marrow and the concentration of NP specific IgG1 antibodies in the serum ± S.E.M. (n = 6 mice per group combined from two independent experiments) of infected KN2/+ (closed circles) and KN2/KN2 (open circles) mice following OVA-NP(15) challenge that are able to bind to BSA-NP(23). (d) The graph represents affinity maturation of serum anti-NP-specific IgG1 antibodies from KN2/+ mice (closed circles) and KN2/KN2 (open circles) following OVA-NP(15) challenge. Ratio depicts binding of serum IgG1 to BSA-NP(3)/BSA-NP(23) ± S.E.M. (n= 6 mice per group combined from two independent experiments). (e) CD4⁺, GFP⁺ T cells were isolated from KN2 × 4get dual reporter mice 14 days after OVA-NP(15) injection and 25,000–50,000 GFP⁺ (GFP⁺) or huCD2⁺ (huCD2⁺, GFP⁺) cells were transferred into combined IL-4 and 13-deficient hosts. Draining lymph nodes and serum were collected on day 14 after OVA-NP(15) challenge. Serum IgG1 antibodies and AFCs from transferred mice were compared to naïve (Naïve) and untransferred (No Tx) combined IL-4 and IL-13 deficient mice. The bars represent the number of anti-NP IgG1-secreting cells and concentration of high-affinity anti-NP-specific IgG1 antibodies in the serum ± S.E.M. (n= 6 mice per group combined from two independent experiments) that are able to bind BSA-NP(3). *(p<0.05); **(p<0.01) (f) Serum IgG1 was collected from OVA-NP(15) challenged combined IL-4 and 13-deficient mice. Combined IL-4 and 13-deficient mice were left untransferred (No Tx) or transferred with 25,000–50,000 GFP+ (GFP⁺) or huCD2+, GFP (huCD2⁺, GFP⁺), CD4+ T cells prior to challenge. Affinity maturation was measured as a ratio of serum anti-NP IgG1 antibodies bound to NP(3)-BSA/NP(23) BSA. *(p<0.05); **(p<0.001)