Figure 1.
Tonsillar Nrp1+ CD4+ T cells have a Tfh phenotype in vivo.
(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+ ICOShi and CD3+ CD4+ CXCR5- ICOSlo 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).
Figure 2.
Tonsillar Nrp1+ CD4+ T cells support survival and Ig production of B cells in vitro.
(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).
Figure 3.
Nrp1+ T cells are not proliferating and have no regulatory activity.
(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.
Figure 4.
Correlation between Nrp1 expression and germinal center activity in secondary lymphoid organs.
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.
Figure 5.
Nrp1 expression by Tfh cells requires cognate B cell contact and reflects Tfh activity in vitro.
(A-C) Nrp1- Tfh, Nrp1+ Tfh and non-Tfh cells were sorted as in Figure 1 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).
Figure 6.
Gene expression profiles of Nrp1- and Nrp1+ Tfh cells.
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).
Figure 7.
Nrp1 expression in malignant Tfh-like cells in AITL.
Cell suspensions from malignant lymph node biopsies of 5 AITL patients (P1-P5) and one non-malignant reactive lymph node (LN 1) were analyzed for CXCR5 and PD-1 expression (A), and for Nrp1 and PD-1 expression (B) in gated CD3+ CD4+ T cells. (C) Bar graph display of percentage of Nrp1+ cells among PD1+ CD4+ T cells, serum gammaglobulin concentration and medullar plasmacytosis (percentage plasma cells among bone marrow cells) for each patient. Note that only P3 shows a significant proportion of Nrp1+ malignant T cells, high gammaglobulinaemia, and important medullar plasmacytosis.