Neuropilin-1 Expression Characterizes T Follicular Helper (Tfh) Cells Activated during B Cell Differentiation in Human Secondary Lymphoid Organs

<div><p>T follicular helper (Tfh) cells play an essential role in the development of antigen-specific B cell immunity. Tfh cells regulate the differentiation and survival of activated B cells outside and inside germinal centers (GC) of secondary lymphoid organs. They act through cognate contacts with antigen-presenting B cells, but there is no current marker to specifically identify those Tfh cells which productively interact with B cells. Here we show that neuropilin 1 (Nrp1), a cell surface receptor, is selectively expressed by a subset of Tfh cells in human secondary lymphoid organs. Nrp1 expression on Tfh cells correlates with B cell differentiation <i>in vivo</i> and <i>in vitro</i>, is transient, and can be induced upon co-culture with autologous memory B cells in a cell contact-dependent manner. Comparative analysis of <i>ex vivo</i> Nrp1⁺ and Nrp1^- Tfh cells reveals gene expression modulation during activation. Finally, Nrp1 is expressed by malignant Tfh-like cells in a severe case of angioimmunoblastic T-cell lymphoma (AITL) associated with elevated terminal B cell differentiation. Thus, Nrp1 is a specific marker of Tfh cells cognate activation in humans, which may prove useful as a prognostic factor and a therapeutic target in neoplastic diseases associated with Tfh cells activity. </p> </div

Gene expression profiles of Nrp1^- and Nrp1⁺ Tfh cells.

<p>Nrp1^- and Nrp1⁺ Tfh cells were sorted from 5 distinct tonsils and analyzed for gene expression as described in Materials and Methods. Gene expression (normalized to ACTB) of a selection of relevant transcription factors (A), cytokines and chemokines (B), co-stimulatory, homing and cytokine receptors (C) in Nrp1^- and Nrp1⁺ Tfh cells is shown here. Numbers above bars indicate fold change and p-value (paired student’s t test). </p

Nrp1 expression by Tfh cells requires cognate B cell contact and reflects Tfh activity <i>in</i><i>vitro</i>.

<p>(A-C) Nrp1^- Tfh, Nrp1⁺ Tfh and non-Tfh cells were sorted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085589#pone-0085589-g001" target="_blank">Figure 1</a> and cultured with autologous B cells in the absence of exogenous stimuli. (A) Representative expression of CD25 and Nrp1 on T cells (top) and B cells (bottom) after 5 days of culture. (B) Percentage of Nrp1⁺ cells among T or B cells after 5 days of culture. (C) Percentage of Nrp1⁺ cells among T cells after 24, 48 and 72 hours of culture. In B and C, representative data from one out of three distinct experiments are shown. (D) Nrp1 expression on sorted Nrp1⁺ Tfh cells after 5 days of culture alone (dark line) or with autologous B cells (shaded histogram). (E-F) Nrp1^- Tfh cells were cultured with autologous B cells in the absence or presence of a transwell membrane separating the two cell types. (E) Representative Nrp1 expression on T cells after 5 days of culture. (F) Number of live B cells per well after 5 days of culture. (G-J) Autologous or allogeneic cocultures were performed using Nrp1^- Tfh and B cells sorted from two distinct tonsils (a) and (b). (G) Nrp1 expression on T cells after 5 days of culture. Data represent one experiment with triplicate wells and are representative of three distinct experiments. (I-J) IgG production in culture supernatant after 10 days of culture. (K-L) Sorted naive, GC or memory B cells were cultured in the absence or presence of autologous CXCR5^- Nrp1^- non-Tfh cells or CXCR5⁺ Nrp1^- Tfh cells. (K) Nrp1 expression on non-Tfh cells and Nrp1^- Tfh cells after 4 days of culture. (L) IgG production in culture supernatants after 14 days of culture. Data were compared using Student’s impaired t-test (*: p≤0.05, **: p≤0.01).</p

Correlation between Nrp1 expression and germinal center activity in secondary lymphoid organs.

<p>Tonsils and non-malignant reactive lymph nodes were compared for their percentages of Tfh cells (A), Nrp1⁺ T cells (B) and Nrp1^- Tfh cells (D) in CD4⁺ T cells and for their percentage of Nrp1⁺ cells in Tfh cells (C) . (E-L) Correlation between the percentage of Tfh cells (E and I), Nrp1⁺ T cells (F and J), Nrp1⁺ cells in Tfh cells (G and K), and Nrp1^- Tfh cells (H and L) and the percentage of GC B cells (E-H) or plasmablasts (I-L) among CD19⁺ cells in tonsils (full circle) and non-malignant reactive lymph nodes (white circle). (M-P) Correlation between the percentage of Nrp1⁺ cells in Tfh cells (M and O), or the percentage of Nrp1^- Tfh cells (N and P), and the percentage of GC B cells (M-N) or plasmablasts (O-P) among CD19⁺ cells only in tonsils only. Data were compared using Student’s impaired t-test (*: p≤0.05, ***: p≤0.001) (A-D). For correlation analyses, the correlation coefficient r² and the associated p-value are shown.</p

Tonsillar Nrp1⁺ CD4⁺ T cells support survival and Ig production of B cells <i>in</i><i>vitro</i>.

<p>(A) Flow cytometry analysis of Nrp1^- Tfh (CXCR5⁺ Nrp1^-: i), Nrp1⁺ Tfh (CXCR5⁺ Nrp1⁺: ii) and non-Tfh cells (CXCR5^- Nrp1^-: iii). (B-C) These three T cell subsets were cultured with B cells without exogenous stimulation, and compared for their ability to maintain B-cell survival after 5 days (B) and to induce the production of IgG, IgA and IgM (C) after 10 days of culture. Representative data from one out of four experiments are shown. Data were compared using Student’s impaired t-test (ns: not significant, *: p≤0.05, **: p≤0.01).</p

Nrp1⁺ T cells are not proliferating and have no regulatory activity.

<p>(A) Ki67 expression in tonsillar CD3⁺ CD4⁺ Nrp1^- and Nrp1⁺ T cells. Five tonsils were analyzed and the expression data was compared with a Student’s paired t-test (**: p≤0.01). (B) The regulatory function of Nrp1⁺ Tfh cells was tested in coculture experiments with CFSE-labeled non-Tfh cells as described in Materials and Methods. The percentage of non-Tfh cells having diluted CFSE after 5 days of culture with various ratios of Nrp1⁺ Tfh cells was analyzed by flow cytometry. Data pooled from three distinct experiments are summarized here.</p

Tonsillar Nrp1⁺ CD4⁺ T cells have a Tfh phenotype <i>in</i><i>vivo</i>.

<p>(A) Representative flow cytometry analysis of Nrp1 and CD25, Foxp3, CD69, CCR7 and CD45RA co-expression on tonsillar CD3⁺ CD4⁺ T cells population. (B) CD25 and Foxp3 co-expression on tonsillar CD3⁺ CD4⁺ Nrp1⁺ and Nrp1^- T cell populations. (C-F) Nrp1 expression on tonsillar Tfh cells and non-Tfh cells, defined as CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ and CD3⁺ CD4⁺ CXCR5^- PD-1^- respectively (C-D), or CD3⁺ CD4⁺ CXCR5⁺ ICOS^hi and CD3⁺ CD4⁺ CXCR5^- ICOS^lo respectively (E-F). Numbers in flow cytometry plots indicate the mean percentage ± SD of Tfh cells in CD4⁺ T cells (left) and of Nrp1⁺ cells in Tfh cells (middle) and non-Tfh cells (right) (n=10 tonsils). (G-H) Nrp1 expression on tonsillar CD3⁺ CD4⁺ CXCR5⁺ CD57⁺ and CD3⁺ CD4⁺ CXCR5^- CD57^- Tfh cells. Data were compared using Student’s impaired t-test (**: p≤0.01, ***: p≤0.001).</p

Semaphorin 3F and Neuropilin-2 Control the Migration of Human T-Cell Precursors

<div><p>Neuropilins and semaphorins are known as modulators of axon guidance, angiogenesis, and organogenesis in the developing nervous system, but have been recently evidenced as also playing a role in the immune system. Here we describe the expression and role of semaphorin 3F (SEMA3F) and its receptor neuropilin-2 (NRP2) in human T cell precursors. NRP2 and SEMA3F are expressed in the human thymus, in both lymphoid and non-lymphoid compartments. SEMA3F have a repulsive effect on thymocyte migration and inhibited CXCL12- and sphingosine-1-phosphate (S1P)-induced thymocyte migration by inhibiting cytoskeleton reorganization prior to stimuli. Moreover, NRP2 and SEMA3F are expressed in human T-cell acute lymphoblastic leukemia/lymphoma primary cells. In these tumor cells, SEMA3F also blocks their migration induced by CXCL12 and S1P. Our data show that SEMA3F and NRP2 are further regulators of human thymocyte migration in physiological and pathological conditions.</p></div

SEMA3F impairs the migratory response of human thymocytes towards S1P.

<p><b>a)</b> S1P₁ expression ascertained by flow cytometry in total human thymocytes and CD4/CD8-defined subsets as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103405#pone-0103405-g001" target="_blank">Figure 1c</a>. DN-1: CD4⁻CD8⁻ cells; DN-2: CD4^lowCD8^low; DP: CD4⁺CD8⁺; CD4-1: CD4^lowCD8⁻; CD4-2: CD4^highCD8⁻; CD8-1: CD4⁻CD8^low; CD8-2: CD4⁻CD8^high. n = 4. <b>b)</b> Bars represent migration of thymocytes in a transwell system. Results are represented by migration ratio, and controls without stimuli were normalized to 1.0. n = 4. Cells migrate towards S1P and when SEMA3F was combined with S1P, it inhibited S1P-induced thymocyte migration. <b>c)</b> Migration response of thymocytes from a representative experiment. Bottom panel show the migration of thymocyte subpopulations based on CD4/CD8 expression, showing that SEMA3F had effect and impaired S1P-induced migration in all thymocyte subpopulations. DP =  double-positive, CD4 =  CD4 single positive, CD8 =  CD8 single-positive. <b>d)</b> Bars show the numbers of migrating thymocytes. The black bar represent thymocyte migration of cells pre-treated with anti-NRP2 blocking antibody which partially abrogated SEMA3F action, as the difference observed between S1P and S1P+SEMA3F+anti-NRP2 is no longer significant (n = 3). <b>e)</b> F-actin modulation of human thymocytes (n = 4) was analyzed by flow cytometry and represented as [(MFI after addition of ligand)/(MFI before addition of ligand)]×100. MFI values obtained before addition of ligand were arbitrarily set at 100% that corresponded to time zero. Data are represented as means ± SEM.Results were analyzed by the unpaired Student's <i>t</i> test, comparing each time point with time zero. Differences were considered statistically significant when p<0.05 (*).</p

Expression of NRP2 and SEMA3F in the human thymus and thymocytes.

<p><b>a)</b> Upper panels show the expression of NRP2 and SEMA3F in the human thymus <i>in situ</i>, ascertained by immunofluorescence and confocal microscopy analysis. Lower panels show the expression of SEMA3F and cytokeratin, revealing that SEMA3F is expressed in the epithelial as well as in the non-epithelial compartments of the thymus. Colocalization analysis was performed with ImageJ software. Inserts show negative controls for each secondary antibody. C: cortex; M: medulla. Magnification: 400×. <b>b)</b> NRP2 and SEMA3F mRNA expression analyzed by real time quantitative PCR, compared with the control Abelson (Abl) gene in fresh thymocytes and the THPN thymic epithelial cell line. <b>c)</b> Cytofluorometric dot plot depicts the regions used to separate the CD4/CD8-defined thymocyte subpopulations. Graphs represent the expression of NRP2 and SEMA3F in total thymocytes and each subpopulation. n = 3–6. In the case of NRP2, mean fluorescence intensity (MFI) analyses are shown to illustrate differences in the expression among the thymocyte subpopulations. Data are represented as means ± SEM. Selected thymocyte subsets were analyzed by the unpaired Student's <i>t</i> test and differences were considered statistically significant when p<0.05 (*), p<0.01 (**) or p<0.001 (***). DN-1: CD4⁻CD8⁻ cells; DN-2: CD4^lowCD8^low; DP: CD4⁺CD8⁺; CD4-1: CD4^lowCD8⁻; CD4-2: CD4^highCD8⁻; CD8-1: CD4⁻CD8^low; CD8-2: CD4⁻CD8^high.</p