The Journal of Immunology, 2006, 177: 1375-1376.
Copyright © 2006 by The American Association of Immunologists
Comment on "The Vast Majority of CLA+ T Cells Are Resident in Normal Skin"
Patrick Schaerli*,
Lisa M. Ebert
and
Bernhard Moser
* Department of Pathology Harvard Medical School The CBR Institute for Biomedical Research Boston, MA 02115
Ludwig Institute for Cancer Research (Melbourne Branch) Heidelberg, Victoria, Australia
Institute of Cell Biology University of Bern Bern, Switzerland
In a recent article, Clark et al. (1) characterized T cells present in normal human skin. Some of the conclusions they have drawn challenge our own published (2, 3, 4) and unpublished findings.
We were unable to identify cell surface CCR8 with the rat anti-CCR8 Ab that they have used (Fig. 1A). This was precisely the reason why we have generated our own anti-CCR8 Ab reagent (Fig. 1A and supplemental Fig. 1 in Ref. 2). In our study, staining for CCR8 was carefully controlled by the use of peptide-blocked Ab as negative control, and moreover, the level of CCR8 cell surface staining we observed on cultured T cells fully matched their chemotactic responsiveness to the CCR8 ligand CCL1 (Fig. 2 in Ref. 2). In the absence of controls being shown, it is difficult to determine whether the CCR8 stainings documented by Clark et al. represent an artifact.
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FIGURE 1. Expression of chemokine receptors on T cells from healthy human skin. A, Difficulty in studying cell surface CCR8 using commercial Abs. Flow cytometric analysis of surface CCR8 expression on 300-19 pre-B cells stably transfected with human CCR8, freshly isolated CD3+ T cells from normal skin, or CD4+ skin T cells cultured for 7 days in 200 U/ml IL-2. No staining was observed with the PE-conjugated rat anti-CCR8 Ab from R&D Systems (191704) (filled histograms) compared with the IgG2b isotype control (141945) (bold lines), both at 6.25 µg/ml (upper panel). In contrast, clear CCR8 positivity was detected with our affinity-purified rabbit anti-CCR8 Ab (percent positive cells indicated by numbers) (lower panel). Anti-CCR8 Ab was used at 15 µg/ml, and negative control included 1 µg/ml blocking CCR8 peptide (2 ). B, Detection of CCR4 is unaffected by collagenase D treatment. CCR4 expression on CD3+ T cells isolated from minced, EDTA-treated (5 h/4°C) normal skin (dotted line) or recovered from normal skin after collagenase D treatment (1 mg/ml, 30 min/37°C) (filled histograms), using mouse anti-CCR4 (1G1) obtained from BD Biosciences. Also, the fraction of CCR4+ cells in peripheral blood CD4+ memory T cells was not reduced upon collagenase D treatment (filled histograms) as compared with untreated cells (dotted line). Normal lines show isotype control stainings.
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Also, using their reagent, Clark et al. concluded that CCR8+ T cells extracted from normal human skin uniformly expressed CCR4 and CCR6, two chemokine receptors with prominent expression on inflammatory T cells (references in Ref. 5). In contrast, we found that CCR4 is present at low levels (Fig. 1B), and CCR6 is expressed on a small cell fraction (Fig. 3 in Ref. 4), which is in agreement with the reduced chemotactic migration of freshly isolated skin T cells in response to the corresponding chemokines (2). Clark et al. explained this obvious discrepancy by the treatment we have selected for extracting lymphocytes out of fresh skin tissue, which involved a brief collagenase D digestion step (2, 3, 4). However, in control experiments with cutaneous and peripheral blood T cells, this treatment did not reduce the stainings for cell surface CCR4 (Fig. 1B) or CCR6 (data not shown). We only observed an effect in chemokine receptor stainings after prolonged (>90 min) protease digestion (data not shown). Finally, minor differences between the study of Clark et al. (1) and our own findings (2, 3, 4) relate to the ratio of CD4+ vs CD8+ T cells in healthy human skin, which in our hands is not biased toward CD4+ T cells, and their effector status, as measured by the percentage of T cells able to produce cytokines in response to in vitro stimulation. These differences may be due to the distinct T cell isolation procedures used (recovery of migratory cells after >7 days of in vitro culture of full thickness skin tissue, representing 20% of total skin T cells (1), vs rapid cell extraction out of dermatomed split skin (2, 3, 4)).
In our view, CCR8 marks a population of skin-resident lymphocytes, including 
T cells, 
T cells, and NK cells that appear to be largely underrepresented in inflamed skin tissue (2, 3, 4). CCL1, the ligand for CCR8, is present in normal human skin and, importantly, is not up-regulated under inflammatory conditions, suggesting to us that this chemokine system controls cutaneous T cell traffic under steady-state conditions. In contrast, numerous reports describe the presence of CCR4 and CCR6 on T cells from inflammatory sites (references in Ref. 5). The report by Clark et al. argues against a distinct role for CCR8 in the control of steady-state T cell traffic within human skin because these cells would also respond to the CCR4 and CCR6 ligands that are greatly up-regulated during inflammation in diverse peripheral tissues.
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]
- Schaerli, P., M. Britschgi, M. Keller, U. C. Steiner, L. S. Steinmann, B. Moser, W. J. Pichler. 2004. Characterization of human T cells that regulate neutrophilic skin inflammation. J. Immunol. 173: 2151-2158. [Abstract/Free Full Text]
- Ebert, L. M., S. Meuter, B. Moser. 2006. Homing and function of human skin T cells and NK cells: relevance for tumor surveillance. J. Immunol. 176: 4331-4336. [Abstract/Free Full Text]
- Schaerli, P., B. Moser. 2005. Chemokines: control of primary and memory T cell traffic. Immunol. Res. 31: 57-74. [Medline]
