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

Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL 60611

It is indeed important to dissect the mechanisms by which injection of anti-CD25 mAb may influence both CD4⁺CD25⁺ regulatory T cell (T_R) function and responder T cell functionality in vivo. This important need was the motivation of the current studies. While our current study does focus on the use of the 7D4 clone, we have observed similar findings using the PC61 clone (see Fig. 3 of Ref. 1). However, differences do exist.

Basic immunology tells us that an Ab of the isotype of IgM should be more efficient in fixing complement, and hence, serve to more efficiently deplete a target cell population. Interestingly, a previous study reported that neither PC61 nor 7D4 "induced a strong complement-mediated cytotoxicity" (2). We have tested this ourselves and have found that 7D4, but not PC61, fixes complement in vitro. Thus, we are left with the question that if PC61 indeed cannot fix complement, then by what mechanism does it deplete CD4⁺CD25⁺ T_R? Based on the capacity of fixing complement, it would seem that if either Ab were to be successful in depleting a target cell population, it would be the clone capable of fixing complement, i.e., 7D4. In vitro, as determined by triple staining of target T cells to measure the expression of CD4, CD25, and intracellular Foxp3, we have observed that CD25 expression is quickly down-regulated following exposure to either 7D4 or PC61 in the absence of cell depletion. In vivo, a fundamental difference between these two clones appears as revealed by the kinetics of CD25 expression, which is quickly down-regulated following in vivo treatment with 7D4. In comparison, the kinetics of PC61-induced CD25 down-regulation are slightly delayed; however, the effects appear to be longer-lasting than that of 7D4. Regardless of which isotype is used, we often see that CD25 expression returns to control levels within 7–10 days of the last treatment. It was this finding that originally prompted us to investigate the alternative actions of anti-CD25 mAb treatment, as it was hard to envision how the CD4⁺CD25⁺ T_R population could return to control levels within that time frame if the cells were physically depleted.

We are still investigating whether injection of PC61 results in a partial depletion of CD4⁺CD25⁺ T_R and the potential contribution that partial depletion may make to the differences in CD25 expression kinetics between the two Ab clones. While we cannot comment on the purported depletion of CD4⁺CD25⁺ T_R referenced by our colleagues (3), because Stephens and Anderton used a single dose of anti-CD25 mAb at a concentration double than we used in our studies, this difference is an obvious source for potential differences in the outcome of anti-CD25 mAb treatment. Furthermore, Zelenay and Demengeot (4) only observed partial deletion of CD4⁺CD25⁺ T_R using PC61. Our studies have revealed that decreasing the dose of anti-CD25 mAb treatment does decrease the duration of CD25 down-regulation.

In regards to the findings of McGeachy et al. (5) concerning the effects of PC61 on experimental autoimmune encephalomyelitis progression in disease induced under "optimal" priming conditions, it would be inappropriate for us to compare our findings to their report in light of the fact that the two model systems use distinct strains of mice, priming Ags, and priming protocols and that significant strain differences exist in regards to the functionality of CD4⁺CD25⁺ T_R. In sum, our data using clone 7D4 clearly demonstrate that CD25 down-regulation, in the absence of overt CD25 T_R depletion (as confirmed by Zelenay and Demengeot, Ref. 4), is capable of inactivating CD4⁺CD25⁺ T_R function allowing exacerbation autoreactive Th1 cell function and clinical experimental autoimmune encephalomyelitis progression.

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]
2. Dantal, J., Y. Jacques, J. P. Soulillou. 1991. Cluster-function relationship of rat-anti-mouse P55 IL-2 receptor monoclonal antibodies: in vitro studies of the CTL-L2 mouse cell line and in vivo studies in a delayed-type hypersensitivity model in mice. Transplantation 52: 110-115. [Medline]
3. Stephens, L. A., S. M. Anderton. 2006. Comment on "cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4⁺CD25⁺ T regulatory cells.". J. Immunol. 177: 2036[Free Full Text]
4. Zelenay, S., and J. Demengeot. 2006. Comment on "cutting edge: anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4⁺CD25⁺ T regulatory cells." J. Immunol. 177: 2036–2037.
5. McGeachy, M. J., L. A. Stephens, L. A., S. M. Anderton. 2005. Natural recovery and protection from autoimmune encephalomyelitis: contribution of CD4⁺CD25⁺ regulatory cells within the central nervous system. J. Immunol. 175: 3025-3032. [Abstract/Free Full Text]

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