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Comment on "Cutting Edge: Anti-CD25 Monoclonal Antibody Injection Results in the Functional Inactivation, Not Depletion, of CD4⁺CD25⁺ T Regulatory Cells"

Instituto Gulbenkian de Ciência, Oeiras, Portugal

The injection of anti-CD25 mAb to deplete CD25⁺ regulatory T cells (T_R) in mice has been common practice to infer the function of T_R. In a recent report, Kohm et al. (1) claimed that injection of anti-CD25 mAb in mice results in the functional inactivation, not depletion, of CD4⁺CD25⁺ T_R. This conclusion was based on the analysis of mice that received the rat-IgM 7D4 mAb or the rat IgG PC61. CD25 and FOXP3 expression was determined by FACS analysis after 7D4 treatment but solely by RT-PCR after PC61 administration (1). Using FACS analysis in both cases, we enumerated CD4⁺CD25⁺ and CD4⁺FOXP3⁺ cells in blood, spleen, and lymph nodes isolated from mice treated with either 7D4 or PC61 (Fig. 1). Similarly to Kohm et al., we found that despite the disappearance of surface CD25 expression, FOXP3⁺ cell frequency is not affected by 7D4 administration, irrespective of the tissue analyzed and independently of the dose used (up to 3 mg/mouse). In contrast, injection of as little as 500 µg/mouse of PC61 resulted in both the disappearance of CD25⁺ cells and the loss of CD4⁺FOXP3⁺ cells. In PBL, the frequency of CD4 cells expressing FOXP3 is reduced from an average of 8 to 3%, indicating that >65% of the total FOXP3⁺ cells are depleted. The disappearing cells are the bulk of the FOXP3⁺CD25⁺ T_R, while the pre-existing FOXP3⁺CD25^– subset appeared maintained. In spleen, 45% of the FOXP3⁺ cells were depleted. T_R originally CD25⁺ that lost CD25 expression and pre-existing CD25^– T_R appeared to contribute equally to the remaining FOXP3⁺ cell pool. In lymph nodes, depletion was less marked and corresponded to the disappearance of 33% of the FOXP3⁺CD25⁺. Increasing the amount of PC61 injected (up to 3 mg/mouse) did not dramatically increase the depletion efficiency (data not shown). We conclude that injection of the rat-IgG PC61 mAb, which has been the most frequently used to interfere with T_R function in vivo, does result in depletion of a large fraction of T_R.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. C57BL/6 mice (8 wk old) raised in specific pathogen-free conditions were injected i.p. once with anti-CD25 mAb clone 7D4 (3 mg) or PC61 (500 µg). Splenocytes, lymph node cells, and PBL were isolated 3 days after from either control or treated mice and analyzed by flow cytometry for CD4, FOXP3, and CD25 expression (for CD25 visualization, clone 7D4 or PC61 was used when PC61 or 7D4 was injected, respectively). A, Representative staining of splenocytes (gated on CD4+ cells) analyzed for CD25 and FOXP3 expression. Number inside dot plots represents average ± SD of the percentage of FOXP3+ among CD4+ cells. B, Percentage of FOXP3+CD25+ () and FOXP3+CD25– () among CD4+ cells. **, p < 0.01 for CD4+FOXP3+ in PC61-injected mice vs control or 7D4-injected mice. Data represent the average ± SD for each group (n = 3). One of three independent experiments is shown.

References

1. Kohm, A. P., J. S. McMahon, J. R. Podojil, W. S. Begolka, M. DeGutes, D. J. Kasprowicz, S. F. Ziegler, S. D. Miller. 2006. Cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4⁺CD25⁺ T regulatory cells. J. Immunol. 176: 3301-3305. [Abstract/Free Full Text]

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