Mouse Anti-CCR4 Recombinant Antibody (clone 1G1)

Stable L1.2 transfectants expressing different chemokine receptors, CCR1–CCR8, were stained with anti-CCR4 mAb 1G1. Negative control staining for all the L1.2 transfectants resembled that of the CCR1/L1.2 cells.

L1.2/CCR4 transfectants (at 2×10⁶ cells per ml in RPMI, 0.5% bovine serum albumin, 10 mM Hepes) were pre-treated with various concentrations of purified anti-CCR4 monoclonal antibodies 10E4 (IgG1), 2B10 (IgG2a) and 1G1 (IgG1) for ten minutes on ice, and then aliquots of 200 μl of cells used in chemotaxis assays with Costar 3.0 μM Transwell Filters (Costar, Cambridge, Mass.) and allowed to chemotax to 50 ng/ml of MDC (FIG. A) or 100 ng/ml of TARC (FIG. B). Cells were then counted on a Becton Dickinson FACSCAN. 10E4 proved to be the best blocking anti-CCR4 monoclonal antibody, followed by 2B10 and then 1G1. An IgG1 control monoclonal antibody had no effect on migration of the L1.2/CCR4 transfectants to MDC (315±30) or TARC (382±11).

Twenty-four hour-old CD4 lymphocytes were pre-incubated with 50 μg/ml of a control mAb, anti-CCR4 mAb 1G1 or anti-CCR4 mAb 2B10. After 10 minutes on ice the CD4 lymphocytes were then allowed to migrate for two hours to 100 ng/ml of MDC, TARC, or RANTES. In one case, the MDC was pre-incubated with a rabbit polyclonal to MDC for 10 minutes before use (MDC/−MDC). To establish the background migration, no chemokine was used in the lower well (−). After this time, the cells accumulated in the lower well were counted using a FACSCAN.

Chronically activated Th1/Th2 were generated by two cycles of activation from umbilical CD4 lymphocytes and were pre-incubated with 50 μg/ml of a IgG1 control mAb, anti-CCR4 mAb 1G1 or anti-CCR4 mAb 2B10. After 10 minutes on ice the Th2 cells were then allowed to migrate for two hours to 100 ng/ml of MDC, TARC, or RANTES. In one case the MDC was pre-incubated with a rabbit polyclonal to MDC for 10 minutes before use (MDC/−MDC). To establish the background migration no chemokine was used in the lower well (−). After this time the cells accumulated in the lower well were counted using a FACSCAN.

Peer cells were allowed to chemotax to TARC or MDC in the presence or absence of mAb 1G1 in a transwell chemotaxis assay. A range of concentration of TARC or MDC was used, with 50 ug/ml of anti-CCR4 mAb 1G1 and mAb MOPC21 (isotype control). The number of migrated cells was counted by flow cytometry using forward and side scatter.

Peer cells were allowed to chemotax to TARC or MDC in the presence or absence of mAb 1G1 in a transwell chemotaxis assay. A range of concentration of TARC or MDC was used, with 50 ug/ml of anti-CCR4 mAb 1G1 and mAb MOPC21 (isotype control). The number of migrated cells was counted by flow cytometry using forward and side scatter.

CCR4/L1.2 cells were incubated with 0.1 nM ¹²⁵I-TARC in the absence (total binding) or presence (competitive binding) of a dose range of either mAb 1G1, mAb 2B10, or MOPC21, an IgG1 isotype control. After 60 mintes, excess antibody and chemokine were washed away and reactions were counted. Data shown is the % inhibition of total binding.