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

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

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

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

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

CD8+ T cells from PCC immunized mice protect naïve mice from EAE.

<p>A. Naïve C57BL/6 mice were immunized with PBS or 100µg of 50V PCC in CFA. Three weeks later, CD8+ T cells purified from the draining lymph nodes and spleen of the PBS immunized (CD8 PBS, squares) or PCC immunized (CD8 PCC, triangles) mice were adoptively transferred into naïve C57BL/6 recipients (CD8 PBS n = 10, CD8 PCC n = 10). Sixteen hours later, EAE was induced in the recipients upon subcutaneous immunization with MOG peptide accompanied by intravenous pertussis toxin on the day of EAE induction and 2 days later. B. Similar protocol was carried out with a second transfer of CD8+ T cells 9 days after EAE induction. The clinical course of EAE was analyzed using a paralysis grading score. Mean ± SEM. * indicates that the p-value<0.05 as assessed using ANOVA Fisher's PLSD. Arrows indicate the time points of CD8+ T cell transfer.</p

Qa-1-binding molecular pattern.

<p>A. The alignment of previously described 9 amino acid-long Qa-1-binding peptides (Qdm, pre-proinsulin, HSP60 signal sequence derived peptide) using the Geneious software (ClustalW analysis). B. A consensus sequence with conserved amino acids in position (P) 1, 2, 5, 7 and 9 was revealed from this alignement. C. The search of such a consensus sequence in mouse Vß chains derived from the IMGT database revealed the presence of consenting 9 amino acid long peptides in the leader sequences of all mouse Vß chains. * Described as being a Qa-1-binding peptide in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021628#pone.0021628-Panoutsakopoulou1" target="_blank">[12]</a>.</p

Regulatory CD8+ T cells prevent lymphocyte infiltration and CNS inflammation.

<p>Naïve mice were adoptively transferred with CD8+ T cells from PBS-immunized (CD8 PBS, n = 4) or PCC-immunized (CD8 PCC, n = 4) mice followed by EAE induction 16 hours later. Thirteen days after EAE induction, the mice were sacrificed and the lymphocyte populations in the brain and spinal cord were examined by flow cytometry. A. Absolute numbers of lymphocytes in the CNS gated according to their forward and side-scatter characteristics. B. MOG-specific CD4+ T cells were stained using MOG_38–49-I-Ab tetramers. Represented is the percentage of tetramer-positive cells among total CD4+ T cells in the CNS. C. Absolute numbers of CD4+ T cells that produce IL-17 in the CNS. D. Absolute numbers of IFNγ-producing CD4+ T cells in the CNS. Mean ± SEM. * indicates that the p-value<0.05 as assessed using Mann-Whitney non-parametric analysis.</p