CD318 is a ligand for CD6

Abstract

It has been proposed that CD6, an important regulator of T cells, functions by interacting with its currently identified ligand, CD166, but studies performed during the treatment of autoimmune conditions suggest that the CD6-CD166 interaction might not account for important functions of CD6 in autoimmune diseases. The antigen recognized by mAb 3A11 has been proposed as a new CD6 ligand distinct from CD166, yet the identity of it is hitherto unknown. We have identified this CD6 ligand as CD318, a cell surface protein previously found to be present on various epithelial cells and many tumor cells. We found that, like CD6 knockout (KO) mice, CD318 KO mice are also protected in experimental autoimmune encephalomyelitis. In humans, we found that CD318 is highly expressed in synovial tissues and participates in CD6-dependent adhesion of T cells to synovial fibroblasts. In addition, soluble CD318 is chemoattractive to T cells and levels of soluble CD318 are selectively and significantly elevated in the synovial fluid from patients with rheumatoid arthritis and juvenile inflammatory arthritis. These results establish CD318 as a ligand of CD6 and a potential target for the diagnosis and treatment of autoimmune diseases such as multiple sclerosis and inflammatory arthritis.

Keywords: CD318; CD6; T cell; autoimmunity; ligand.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

CD318 as the potential antigen recognized by mAb 3A11. (A) Probing the HBL-100 cell lysates with a commercial anti-CD318 Ab. Cell lysate were separated by SDS/PAGE and probed with a commercial anti-CD318 Ab in Western blot, showing a ∼135-kDa band (arrow). Data are representative of two independent experiments. (B) CD318 up-regulation in response to IFN-γ stimulation. HBL-100 cells were stimulated with human IFN-γ for 72 h, and CD318 expression levels were analyzed by flow cytometry following staining with a commercial anti-CD318 mAb (Clone CUB1). Thin shaded line, isotype control; thick line, without stimulation; dotted line, with stimulation. Data are representative of three independent experiments. (C) Probing the mAb 3A11 immunoprecipitates with the anti-CD318 Ab. HBL-100 cell lysates were immunoprecipitated with the same concentrations of mAb 3A11 or mIgG1 control, then the immunoprecipitates were separated by SDS/PAGE and probed with a commercial anti-CD318 Ab. The arrows indicate the full length and different isoforms of CD318 that immunoprecipitated. Data are representative of two independent experiments. (D) Three different concentrations (2.5, 5, 10 μg) of rCD318 or BSA control were separated by SDS/PAGE and probed with either mAb 3A11 (Lower) or a commercial anti-CD318 Ab (Upper).

Fig. 2.

The anti-CD318 mAb and mAb 3A11 have an identical staining pattern on cells previously known to express or lack CD318 expression on the cell surface. Reported CD318-positive cells (HBL-100, A549, and Caco-2) and negative cells (MCF-7, Molt-4, and Raji) were stained either with the anti-CD318 mAb (A) or mAb 3A11 (B) and analyzed by flow cytometry. Data are representative of three independent experiments. Shaded histogram: isotype controls (mIgG2b for CD318 staining and mIgG1 for mAb 3A11 staining). Open histogram represents CD318 (A) or mAb 3A11 (B) staining.

Fig. 3.

The anti-CD318 mAb and mAb 3A11 have an identical staining pattern on cells engineered to up-regulate or down-regulate CD318 expression. (A) The anti-CD318 mAb and mAb 3A11 staining on cells overexpressing CD318. MDA-468 cells transfected with vector alone (control) or a doxycycline-inducible CD318 expressing construct were incubated with doxycycline overnight, then stained either with CD318 Ab (Upper) or mAb 3A11 (Lower) and analyzed by flow cytometry. Shaded histograms, isotype controls; thin open histograms, basal expression level of anti-CD318 mAb/mAb 3A11 staining before doxycycline induction; thick open histograms, level of anti-CD318 mAb/mAb 3A11 staining after doxycycline induction. Data are representative of three independent experiments. (B) The anti-CD318 mAb and mAb 3A11 staining on cells with CD318 knocked down. MDA-468 WT and CD318 knockdown cells were stained with either CD318 mAb (Upper) or mAb 3A11 (Lower) and analyzed by flow cytometry. Thin lines, isotype controls; dashed lines, WT cells stained with the anti-CD318 mAb or mAb 3A11; thick lines, CD318 knocked down cells stained with the anti-CD318 mAb and mAb 3A11. Data are representative of three independent experiments.

Fig. 4.

CD6 interacts with CD318. (A) HT1080 CD166 KO cells are deficient of CD166. The CD166 KO cells were analyzed for CD166 expression by flow cytometry. Thin line, isotype control; thick line, CD166 KO cells stained with an anti-CD166 mAb; thin dash line, WT HT1080 cells stained with the anti-CD166 mAb. Data are representative of five independent experiments. (B) HT1080 CD166 KO cells express CD318. The CD166 KO cells were analyzed for CD318 expression by flow cytometry using the anti-CD318 mAb. Thin line, isotype control; thick line, CD166 KO cells stained with the anti-CD318 mAb. Data are representative of five independent experiments. (C) CD6 binds to WT and CD166 KO cells. WT and CD166 KO cells were incubated with the same concentrations of human IgG1 (control) or recombinant human CD6-Fc fusion protein (1 µM), followed by detecting the cell surface-bound CD6 using an Alexa488-anti-human IgG Ab. Thin line, isotype control; thick line, CD166 KO cells stained with CD6; thin dashed line, WT cells stained with CD6. Data are representative of six experiments. (D) Binding of CD6 onto CD166 KO cells is competitively inhibited by soluble CD318. CD166 KO cells were incubated with recombinant human CD6-Fc fusion protein (1 µM) in the presence of different concentrations of recombinant soluble CD318 (0, 1, and 3 µM), then level of cell surface-bound CD6 was quantitated by flow cytometric analysis after staining the cells with an Alexa488-anti-human IgG Ab. Thin shaded line: isotype control; thick dash line, no rCD318; thin dash line, 1 µM CD318; thin line, 3 µM rCD318. The numbers in parentheses represent the molar concentrations of either rHCD6 or rCD318. Data are representative of six independent experiments. (E) CD6 immunoprecipitates CD318 from the CD166 KO cell lysates. CD166 KO cell lysates were immunoprecipitated with the same concentrations of either soluble CD6 or human IgG1, then the immunoprecipitates were separated by SDS/PAGE and probed with the anti-CD318 Ab, showing that CD6 selectively pulled down CD318 (arrow). Data are representative of eight independent experiments. (F) Soluble CD318 binds to CHO cells that express CD6 on the surface. Control CHO cells (solid line) and CHO cells expressing human CD6 (dotted line) were incubated with recombinant CD318. After washing, the binding of CD318 on the cell surface was assessed by flow cytometry after staining the cells with the anti-CD318 mAb.

Fig. 5.

CD318 KO mice are protected in EAE. (A) WT and CD318 KO mice were immunized with MOG35–55 to induce EAE, and the development of EAE was monitored daily by clinical scoring. Combined results were from five individual experiments. WT, n = 21; CD318 KO, n = 25; data are mean ± SEM, *P < 0.05, (B and C). At the end of the experiments, splenocytes were collected and incubated with or without 10 μg/mL MOG35–55 peptide for 72 h. Levels of IFN-γ (B) and IL-17 (C) in the culture supernatants were measured by respective ELISA. Combined results were from three individual experiments, WT, n = 17; CD318 KO, n = 16; data are mean ± SEM, *P < 0.05. (D) Representative histology images of spinal cord sections from WT (Left) and CD318 KO (Right) mice in EAE, showing significantly reduced inflammation in the CD318 KO mouse spinal cords. Spinal cords were collected at the end of the experiment and processed for H&E staining. Squares in Upper show the areas that were amplified in Lower. (E) Flow cytometric analysis of infiltrated CD4+ T cells in spinal cord from WT and CD318 KO mice in EAE, showing significantly reduced CD4+ T-cell infiltration in the spinal cords of the CD318 KO mice in EAE. Spinal cords were collected at the end of the experiment, minced, and digested with collagenase. Single-cell suspension was prepared after Percoll centrifugation, stained with an anti-mouse CD4 mAb, and analyzed by a flow cytometer. (F) Mouse BMECs do not constitutively express CD318 but do after IFN-γ stimulation. Primary BMECs were isolated from WT mice, characterized, incubated without (Left) or with (Right) IFN-γ for 48 h, and stained with sheep anti-mouse CD318 IgG (dotted line) or control IgG (solid line), then analyzed on a flow cytometer.

Fig. S1.

Intact T-cell compartment in the lymph node and peripheral blood of CD318 KO mice. (A) A representative frequency of CD4+ and CD8+ T cells in WT and CD318 KO inguinal lymph nodes. Data are representative of 10 mice. (B) The summary of the frequency and total cell numbers of CD4+ and CD8+ T cells in WT and CD318 KO inguinal lymph node. n = 10 in each group. (C) A representative frequency of CD3+ T cells in WT and CD318 KO peripheral blood. (D) The summary of frequency of CD3+ T cells in the peripheral blood of WT and CD318 KO mice. n = 10 in each group.

Fig. 6.

CD318 is a potential biomarker for inflammatory arthritis and chemotactic for T cells. (A) CD318 is highly expressed in synovial tissues from RA patients. Synovial tissue sections from RA, OA, and nonrelevant controls (NL) were stained with either mAb 3A11 (mouse anti-human CD318) or the same amount of control IgGs, then slides were examined under a microscope. (B) Levels of total CD318 are elevated in synovial tissues from RA patients. Synovial tissue from patients with RA (n = 13), OA (n = 20), and normal synovial tissues (Ctrl, n = 17) were homogenized, and levels of total CD318 were analyzed by ELISA. (C) Levels of soluble CD318 are significantly higher in synovial fluids from patients with RA (n = 36) or JIA (n = 10) than in those from patients with OA (n = 28). Sr, serum; SF, synovial fluid. (D) Soluble CD318 is chemotactic to T cells. T-cell migration toward various concentrations of CD318 (50, 100, 200, 400, 800, and 1,600 pg/mL) was assessed in Boyden chemotaxis chambers. PBS was the negative control, and TARC was the positive control. Readings represent the number of cells migrating through the membrane (the sum of three high power 40x fields per well, averaged for each quadruplicate well). (E) T-cell adhesion to IFN-γ–stimulated synovial fibroblasts in the presence of mouse IgG (control), anti-CD318, anti-CD166, or both was measured by a Synergy plate reader.

Fig. S2.

Immunofluorescence microscopy of synovial tissue. To further define the cellular CD318 expression in RA and OA synovium, synovial tissue (ST) slides were fixed with cold acetone for 20 min and blocked with 20% FBS and 5% goat serum for 1 h at 37 °C. Rabbit anti-human cadherin-11 antibody (GeneTex) and mouse anti-human CD318 antibody (3A11 antibody made in house) were used at 10 µg/mL for 1-h incubation at 37 °C. Nonspecific rabbit (Invitrogen) and mouse IgG (BD Biosciences) were used as respective antibody controls. STs were then washed and incubated for an additional hour with 10 µg/mL fluorescently conjugated goat anti-mouse/rabbit secondary antibodies [Alexa Fluor 555 (PE) for cadherin-11 binding; and 488 (FITC) for CD318 binding (Invitrogen)]. For nuclear staining, DAPI was used at 1:5,000 diluted in PBS. Images were taken at 400× magnification. Anti-CD318 positive synoviocytes are shown by fluorescent green staining, and cadherin-11 staining is shown by fluorescent red. Yellow cells are a result of a merger of the green and red fields showing dual fluorescence of cadherin-11 and CD318 expression on the same cells within the ST.