A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular DC

Abstract

Nectins and Nectin-like molecules (Necl) play a critical role in cell polarity within epithelia and in the nervous and reproductive systems. Recently, immune receptors specific for Nectins/Necl have been described. Since the expression and distribution of Nectins/Necl is often subverted during tumorigenesis, it has been suggested that the immune system may use these receptors to recognize and eliminate tumors. Here we describe a novel immunoreceptor, Washington University Cell Adhesion Molecule, which is expressed on human follicular B helper T cells (TFH) and binds a Nectin/Necl family member, the poliovirus receptor (PVR), under both static and flow conditions. Furthermore, we demonstrate that PVR is abundantly expressed by follicular DC (FDC) within the germinal center. These results reveal a novel molecular interaction that mediates adhesion of TFH to FDC and provide the first evidence that immune receptors for Nectins/Necl may be involved the generation of T cell-dependent antibody responses.

Figure 1

WUCAM binds to PVR. (A) Baf3 cells transfected with WUCAM (WUCAM/Baf3) form conjugates with Daudi cells transfected with PVR (PVR/Daudi) but not mock-transfected-Baf3 or Daudi cells. One experiment representative of 3 is illustrated. (B) Soluble forms of human PVR (PVR-hFc) and mouse Tage4 (Tage4-hFc) bind WUCAM/Baf3 (grey profiles), but not mock-transfected Baf3 (empty profiles). A soluble form of WUCAM (WUCAM-mFc) binds PVR/Daudi (grey profiles), but not control Daudi cells (empty profiles). Moreover, WUCAMmFc does not bind WUCAM/Baf3 or Baf3 (grey and empty profiles respectively). (C) The anti-WUCAM antibody 4E1.2 (black thin dotted line) and the anti-PVR antibody SKII.4 (grey dotted line) block binding of a soluble form of PVR (PVR-hFc) to WUCAM/Baf3 cells to background levels (black thin line). An isotype control antibody does not affect the binding (black solid line). Experiments in (B) and (C) were performed three times with identical results. (D) Detachment profile of WUCAM-Baf3 cells interacting with surface-immobilized PVR-hFc. The percentage of WUCAM/Baf3 (black squares) or mock transfected-Baf3 (empty circles) bound to PVR-hFc is shown as a function of increasing wall shear stress. Cells were allowed to settle onto the indicated surface-immobilized protein substrates for 10 min prior to the application of flow (0.5 to 16 dyn cm-2). Values represent the mean ± SD for two experiments performed in duplicate.

Figure 2

WUCAM is expressed upon cell activation. (A) PBMC stained ex-vivo (upper panels) or after in-vitro stimulation for 6 days with PHA and IL-2 (lower panels). Cells were stained with anti-WUCAM and counterstained with anti-CD3, anti-CD56, anti-CD4 and anti-CD8. One donor representative of 3 donors tested is shown. (B) Time course analysis of WUCAM expression on CD4⁺ T cells, CD8⁺ T cells and CD56⁺CD3⁻ NK cells. Cells were stained as in (A). Bars represent the percentage of WUCAM⁺ cells within the indicated lineage at different time points. One donor representative of 3 donors tested is shown. (C) Most of the CD4⁺ T cells expressing WUCAM also express the chemokine receptor CXCR5 (insert). The percentage of WUCAM⁺ cells is reduced in comparison to panels B and C as staining was performed at day 8 after stimulation, when WUCAM expression begins to decline.

Figure 3

WUCAM expression on purified Tfh cells. (A) Tonsil cells were analyzed ex-vivo for WUCAM expression and counterstained with the lineage markers CD3, CD4, CD8, CD56 and CD19. The vast majority of cells expressing WUCAM are confined within the CD4⁺ T cell subset. However, WUCAM expression was repeatedly observed on some CD3⁺CD8^low/int. (B) WUCAM expression is mostly confined to CXCR5^high/ICOS^high CD4⁺ T cells that correspond to Tfh cells. Although the level of WUCAM and the percentages of WUCAM⁺ CD4⁺ T cells varied in different donors, only CXCR5^high/ICOS^high cells within CD4⁺ T cells expressed WUCAM in every sample analyzed. (C) WUCAM⁺ CD4⁺ T cells express high levels of CD69. One specimen representative of three tonsil specimens analyzed is shown. (D) ICOS^high CD4⁺ T cells from tonsil bind soluble PVR (PVR-hFc) but not a control soluble molecule (ctr-hFc). This binding is significantly reduced by the anti-WUCAM antibody 4E1.2, but not by an irrelevant isotypematched control (ctr-IgG3). One experiment representative of two is shown.

Figure 4

PVR is highly expressed by FDC. Immunohistochemical analysis of human lymph node (A, B, E, F) and spleen (C, D) sections reveals expression of PVR on a vast network of cells within the germinal centers that for location, shape and distribution closely resemble the FDC network (A, B, C and D). (E) PVR is also brightly expressed by some IDC in the T cell area of lymph nodes. Other IDC express lower level of PVR. (F) HEV also express PVR. PVR was detected with mAb SKII.4, followed by a biotinylated anti-Ig secondary antibody and streptavidin-immunoperoxidase (brown) or streptavidine-alkaline phosphatase (red). Magnifications: 400×(b and d), 200× (e and f) and 100× (a and c). Scale bars: 50 microns (b and d), 100 microns (e and f) and 200 microns (a and c).