The Journal of Immunology, 2006, 177: 1376-1377.
Copyright © 2006 by The American Association of Immunologists
Response to Comment on "The Vast Majority of CLA+ T Cells Are Resident in Normal Skin"
Rachael A. Clark and
Thomas S. Kupper
Department of Dermatology Brigham and Women’s Hospital and Harvard Skin Disease Research Center Boston, MA 02115
We and others have access only to commercially available CCR8 Abs, one of which we used in our recently published studies (1). The letter authors apparently did not see reactivity using this Ab and have generated a polyclonal Ab that they believe is superior. Certainly, we agree that Ab staining is only as good as the Ab used. Polyclonal Abs can have the disadvantage of decreased specificity; for example, polyclonal Abs to CCR8 may also react with CCR5 or other chemokine receptors. Additional specificity controls, beyond the peptide immunogen blocking control reported, should be performed before we can verify that the authors’ polyclonal Ab is superior. However, it should be noted that both the letter authors and we agree that 
50% of skin resident T cells express CCR8 (2). In their original article, the authors found CCR8 expression on 46% of CD4 T cells and 61% of CD8 T cells. In the current letter, the percentage of positive cells is significantly higher, a variance from the previously published data that is not explained. Although the intensity of staining we report is lower, we believe that it is real. Our negative control was an isotype-matched Ab used at the same concentration as the CCR8 Ab. Gates for positive staining were based on this control, with 99% of isotype control-stained cells gated in the negative. Thus, we are comfortable that our CCR8 staining is not an artifact. Indeed, the fact that both our groups found similar expression of CCR8 on skin resident T cells should be considered independent confirmation of this observation, rather than a bone of contention. Of course, if we were provided with this group’s CCR8 polyclonal Ab, we could directly replicate their findings.
In contrast, we do take serious issue with the contention that skin T cells resident in normal skin do not express CCR4. We have clear, convincing, and abundant data that the vast majority of skin T cells isolated freshly from skin—whether using EDTA treatment, isolated freshly from skin using collagenase treatment, or isolated from skin using explant cultures—express high levels of CCR4 (Fig. 1a in Ref. 1). Moreover, we find high expression of CCR4 on virtually all T cells isolated from both sun-exposed (face) and sun-protected skin (breast and abdomen). The authors provide some data above in histogram form showing that collagenase treatment does not affect CCR4 levels. However, dot plots showing CCR8 staining vs CCR4 staining would be much more convincing. We have now tested upward of 30 normal skin donors and have found high expression of CCR4 on T cells from all donors. To settle this issue, we stained frozen sections of normal skin for CD3 and CCR4. Virtually all CD3+ T cells found in normal skin costained for CCR4 (Fig. 1), confirming that T cells in normal skin do express this homing receptor. Using frozen sections, we have likewise confirmed our finding regarding the percentage of CD4 vs CD8 T cells in normal skin and the expression of CLA and CCR6 by T cells resident in normal skin. These data are not shown but can be provided upon request.
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FIGURE 1. T cells in normal human skin express CCR4. Sections of normal human skin were stained with directly conjugated CD3 and CCR4 (1G1) Abs and analyzed by fluorescence microscopy. In the sections shown, CD3+ T cells are present within the epidermis and the dermis of normal skin (two epidermal T cells are specifically indicated by arrows). Virtually all CD3+ T cells (left panels) present in normal skin costained for CCR4 expression (right panels). Control sections stained with identical concentrations of isotype-matched directly conjugated Abs showed no staining aside from nonspecific staining of the stratum corneum.
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As for the "reduced chemotactic migration" of T cells that the authors describe, with due respect, we do not find the chemotaxis data they describe particularly compelling. The letter authors claim that CCR8 is present on at least 50% of skin T cells, but migration to CCL1 is only 12% of input T cells (compared with a random migration of 6%). This low percentage of migration is concerning, and calls into question the results using CCR4 ligands.
Lastly, we would stress the point that a homing receptor present on only 50% of skin resident T cells, according to the authors’ own data, is unlikely to be the major addressin guiding T cells into skin under conditions of normal immunosurveillance. In contrast, CLA and CCR4 are present on the vast majority of T cells from normal skin regardless of the method of T cell isolation, a result we have confirmed by staining frozen sections of normal skin. Moreover, the ligands for CLA and CCR4, E-selectin and thymus and activation-regulated chemokine, are expressed at low but detectable levels in resting cutaneous endothelium (3). This suggests that homing to normal skin uses the same receptors as trafficking to inflamed skin, and thus, it is our belief that homeostatic trafficking to skin relies on quantitative, not qualitative, differences in vascular homing receptor expression. While we do not mean to minimize a potential contributory role for CCR8 in skin homing, we think that the evidence is overwhelming that CLA and CCR4 are both better candidates for homing receptors that support migration of T cells into normal skin. We thank the letter authors for their comments but are confident that our results will stand the test of time.
References
- Clark, R. A., B. Chong, N. Mirchandani, N. K. Brinster, K. Yamanaka, R. K. Dowgiert, T. S. Kupper. 2006. The vast majority of CLA+ T cells are resident in normal skin. J. Immunol. 176: 4431-4439. [Abstract/Free Full Text]
- Schaerli, P., L. Ebert, K. Willimann, A. Blaser, R. S. Roos, P. Loetscher, B. Moser. 2004. A skin-selective homing mechanism for human immune surveillance T cells. J. Exp. Med. 199: 1265-1275. [Abstract/Free Full Text]
- Chong, B. F., J.-E. Murphy, T. S. Kupper, R. C. Fuhlbrigge. 2004. E-selectin, thymus- and activation-regulated chemokine/CCL17, and intercellular adhesion molecule-1 are constitutively coexpressed in dermal microvessels: a foundation for a cutaneous immunosurveillance system. J. Immunol. 172: 1575-1581. [Abstract/Free Full Text]
