Fig 1.
Screening monoclonal antibody clones against the amino terminus of CCL21.
A) Screening of CCL21 monoclonal antibody clones by western blotting for binding to the human CCL21 protein as a single band. Clone #8 (C8) results are shown for all assays in this figure. B) Cross reactivity of monoclonal anti-CCL21 antibody to human and mouse CCL21 protein. We then screened the clones for lack of binding to murine CCL21 by slot blot hybridization. C) Screening of a peptide from the CCR7-interacting amino terminal region of CCL21 could compete off the monoclonal from binding CCL21 using slot blot hybridization. D) Screening of monoclonal antibody clones for lack of binding to the related human chemokine CCL19. Thirty-three monoclonal antibodies passed all of these screens.
Fig 2.
Screening of monoclonal antibody clones against CCL21-mediated T-cell chemotaxis.
A) Inhibition of migration in transwell chemotaxis assays of human helper T-cells (CD3+/CD4+) towards human CCL21 by the 33 murine monoclonal antibody clones that passed the screening process outlined in Fig 1. Clone 8 (C8) completely abrogated T-cell migration towards CCL21, while clone 9 partially inhibited migration. Several clones promoted migration of T-cells, suggesting they enhanced CCL21 presentation to its receptor, CCR7. B) C8 also blocked migration of three distinct normal donor human Th-cells towards CCL21, indicating this is a general phenomenon. For all migration assays, data are the mean ± SD of triplicate wells, performed twice. Student T tests were performed for statistical analysis for this figure and Fig 3. * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001 when compared to control.
Fig 3.
Inhibition of T-cell subset migration towards CCL21 by C8.
(A) Flow cytometric gating strategy for isolation of human Th-cell subpopulations for testing the effects of C8 on migration towards CCL21. Migration of defined populations of T cell subsets was determined using flow cytometry of the lower versus upper chambers in transwell chemotaxis assays with background migration (cells that migrated toward media with no chemokine) subtracted from total cells. (B) Surface biomarker identification of Th-cell subsets tested in these experiments. (C-F) Fractional migration towards CCL21 inhibited by C8 in these T-cell subsets. Naïve Th-cells had the greatest migration towards CCL21 and were the most inhibited. Effect of C8 on migration towards CCL21 decreases as Th-cells become more mature.
Fig 4.
Screening of C8 for immunohistologic recognition of CCL21 in a tonsilar lymphoid biopsy specimen.
Screening for binding of CCL21 in human lymphoid tissue. C8 was positive for binding to appropriate CCL21-expressing cells in human tonsil lymph node, while another monoclonal antibody in this series that failed screening in Fig 1 did not bind to any cells, indicating the utility of C8 as an immunohistologic tool (left panels). CCL21 is expressed in the venule endothelium of T-cell-mediated auto-immune skin diseases [15]. C8 specifically recognizes this expression in immunohistologic assays while it did not recognize synovium from rheumatoid arthritis serves as a negative control, as it is mediated by a distinct immunologic pathway (right panels).
Fig 5.
Immunohistology of venule endothelial expression of CCL21 in intestinal mucosal autoimmune diseases.
A) 40x magnification of 1 of 3 Crohn’s disease intestinal mucosa biopsy immunohistologically stained with the C8 (anti-CCL21 monoclonal antibody). Two of 3 had similar positive results as shown here. B) 100 x magnification of (A). C) 40x magnification of 1 of 3 ulcerative colitis colonic mucosa biopsy immunohistologically stained with C8. All 3 biopsies had results nearly identical to that show here. D) 100 x magnification of (C). E) 40x magnification of 1 of 6 colonic mucosal biopsies of celiac sprue disease immunohistologically stained with C8. Four of the 6 samples had nearly identical results with that shown here. F) 100 x magnification of (E). (G) Normal duodenal endothelium does not express CCL21. One of 3 biopsy samples shown here that is representative of all 3. H) Normal colonic endothelium does not express CCL21. One representative sample of 3 biopsies shown here.
Fig 6.
Screening of fully humanized anti-CCL21 monoclonal antibody clones derived from clone C8.
(A) Inhibition of migration in transwell chemotaxis assays of helper T cells (CD3+/CD4+) towards 1200 ng/ml rhCCL21 by the 16 humanized clones, referred to as V1 to V16, used at 100 μg/ml. All versions of humanized clones tested inhibited T cell migration towards CCL21 to some degree. However, V6 was the most potent at inhibiting T cell migration towards CCL21 (arrow). Data are the mean ± SD of triplicate wells. One-way ANOVA with post-hoc Tukey’s multiple comparison tests were performed for statistical analysis. All groups had a p<0.0001 when compared to control. (B-D) Confirmation of V6 inhibition of CD3+/CD4+/CD8-/CCR7+/CD45RA+/CD27+ Naïve Th-cell chemotaxis towards 100, 500, 800, 1200, and 1600 ng/ml rhCCL21 using CD3+ peripheral blood naïve Th-cells from 3 normal human donors. Data are the mean ± SD of triplicate wells.