1 Journal of Investigative Dermatology 2006 Vol: 126(5):1059-1070. DOI: 10.1038/sj.jid.5700199

A Novel Method for the Isolation of Skin Resident T Cells from Normal and Diseased Human Skin

T cells resident in normal skin likely conduct immunosurveillance and are implicated in the development of inflammatory disorders such as psoriasis. This population of cells is difficult to study because existing techniques allow isolation of only few cells. We report here a novel method of isolating T cells from both normal and diseased human skin. Explants of skin cultured on three-dimensional matrices led to the outgrowth of dermal fibroblasts that elaborated T cell chemoattractant factors. These factors led to the migration of skin resident T cells out of skin explants where they could be collected and studied. Skin resident T cells isolated from explant cultures were CD45RO+ memory T cells and expressed high levels of cutaneous lymphocyte antigen (CLA) and chemokine receptor (CCR)4. Inclusion of IL-2 and IL-15 in explant cultures produced up to a 10-fold expansion of skin-resident T cells, while maintaining the CLA+CCR4+ skin-homing phenotype as well as a diverse T cell repertoire. This method also allowed efficient isolation of malignant T cells from the skin lesions of cutaneous T cell lymphoma and the isolation of tumor-infiltrating lymphocytes from primary squamous cell carcinomas and melanoma metastases.

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Figures
Figure 1: Skin explant cultures. (a) Three 2 mm 2 mm 2 mm explants of human skin were cultured on collagen-coated three-dimensional cell growth matrices. (b) Scanning electron micrograph of the Cellfoam three-dimensional matrix (provided by Cellsciences). (c) Immunofluorescence micrograph of dermal fibroblasts (stained green with vimentin antibody) populating the matrix after 3 weeks of explant culture. Figure 2: Efficient isolation of skin-resident memory T cells from explant cultures of normal human skin. (a) Maximal T cell production was observed at 3 weeks after initiation of culture in all donors. No exogenous cytokines were added. (b) Donor variability and mean cell yields from eight different skin donors. (c) T cells isolated from explant cultures were CD45RO+ memory T cells with (d) almost universal expression of CLA and CCR4. (e) T cells isolated from explants cultured without matrices showed loss of both CLA and CCR4 expression. (f–h) Functionality of T cells isolated from normal skin in explant cultures. T cells (f) proliferated in response to TCR crosslinking, (g) upregulated the activation CD69 and (h) produced TNF after treatment with the T cell mitogen concanavalin A. ***P<0.0001. Figure 3: Explant cultures elaborate T cell chemoattractants and T cell antiapoptotic factors but do not produce cytokines that induce T cell proliferation. (a) Conditioned medium from explant cultures contained significant levels of the T cell chemoattractants IL-8, (b) IP-10, MCP-1, MCP-3, MDC, MIG, and MIP-3. (c) Inclusion of PT blocked the migration of T cells from skin explants and neutralizing antibodies to the T cell chemokines IL-8 and MDC also partially inhibited migration. (d) Conditioned medium from explant cultures contained significant levels of IL-6 but lacked expression of the T cell proliferative cytokines IL-2, IL-7, and IL-15. Bars indicate the mean and standard deviation of multiple measurements. Figure 4: Peripheral blood T cells cultured in fibroblast-colonized matrices showed little change in CLA expression. CLA+-enriched and CLA- naïve and memory T cell subsets were isolated from peripheral blood and cultured in matrices colonized with dermal fibroblasts for 3 weeks without endogenous cytokines. (a) Fibroblast-colonized matrices preferentially supported the survival of CLA+ cutaneous T cells. (b) The presence of living fibroblasts on matrices greatly enhanced CLA+ T cell survival. Irradiation of fibroblasts (irrad) only slightly decreased T cell survival, but fixation of fibroblasts (fixed) largely destroyed their ability to maintain T cell viability. (c, d) CLA+ T cells remained CLA+ after culture in fibroblast-colonized matrices. CLA expression is shown (a) before and (b) after culture. Memory CD45RO+CLA+ remained CLA+ and naïve CD45RO-CLA- T cells and memory CD45RO+CLA- T cells failed to acquire CLA expression during culture. A duplicate experiment using different fibroblast and T cell donors produced similar results. Figure 5: Inclusion of IL-2 and IL-15 in explant cultures led to a 10-fold expansion of skin-resident T cells without loss of skin-homing receptors and with maintenance of significant diversity. (a) Total number of skin resident T cells isolated at 3 weeks when IL-2 (100 U/ml) and/or IL-15 (20 ng/ml) were included in explant cultures. Values represent the mean and standard deviation of three matrices per group; all groups shown used the same sample of normal skin. Comparable results were obtained from two additional donors. **P<0.001; ***P<0.0001. (b) Skin-resident T cells isolated from cytokine-treated explants retained expression of CLA and CCR4. (c) Flow cytometry analysis of TCR V usage showed that, whereas shifts occurred, all V present in unexpanded populations were also present in expanded populations. Similar results were obtained from a second skin donor. (e) T cells produced from explants treated with both IL-2 and IL-15 retained significant diversity as assessed by T cell spectratyping analysis. Multiple peaks represent diversity within each V subfamily. Results from a second donor showed similar results. Figure 6: Skin explant cultures allowed efficient isolation and expansion of skin-infiltrating T cells from CTCL lesions and TILs from SCC and melanoma metastases. (a) T cell production from CTCL lesional skin peaked between days 7 and 14 of explant cultures in the absence of exogenous cytokines. (b) Inclusion if IL-2 and IL-15 in CTCL explant cultures led to an expansion of skin-infiltrating T cells (c) without loss of CLA and CCR4 expression. (d) Explant culture of CTCL skin allowed isolation of malignant T cells. In explant cultures of skin from a patient with a clonal population of V11+ malignant T cells in the blood, 64% of all CD3+ T cells isolated were V11+ malignant T cells and (e) a pure population of malignant cells were identifiable based on its higher forward and side scatter characteristics. (f) Explant cultures allowed efficient isolation and expansion of TILs from human primary SCC. Similar results were obtained from a second, independent sample. (g) Explant cultures containing IL-2 and IL-15 allowed isolation of many more T cells from melanoma metastases than conventional methods of mechanical dissociation. Duplicate samples have confirmed this observation.
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References
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    • . . . Culture of CLA+ cutaneous T cells in serum-containing medium has been previously shown to lead to loss of CLA expression (Fuhlbrigge et al., 1997; Armerding and Kupper, 1999) . . .
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    • . . . Despite the key role these cells play in cutaneous immunity, the study of skin-resident T cells has been hampered by the difficulty of extracting them from the skin and the fact that these cells tend to lose expression of cutaneous homing receptors when cultured in serum-containing medium (Armerding and Kupper, 1999) . . .
    • . . . Although T cells isolated freshly from normal skin express CLA and CCR4, expression of these key cutaneous homing receptors is lost when these cells are cultured in serum-containing medium (Armerding and Kupper, 1999) . . .
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    • . . . Study of these cells has been difficult because they can be isolated only in low numbers using existing methods of mechanical dissociation combined with chelating agents or collagenase (Campbell et al., 2001; Schaerli et al., 2004) . . .
    • . . . T cells freshly isolated from normal skin using mechanical dissociation methods express both the cutaneous lymphocyte antigen (CLA) and the chemokine receptor (CCR)4 (Campbell et al., 2001; Ferenczi et al., 2002) . . .
    • . . . We found that skin resident T cells isolated from explant cultures expressed high levels of CLA and CCR4, as did T cells freshly isolated from normal skin using mechanical methods (Campbell et al., 2001; Ferenczi et al., 2002) . . .
    • . . . Moreover, the T cells we isolate from normal skin have high expression of CLA and CCR4, identical to T cells freshly isolated from skin using mechanical methods (Campbell et al., 2001; Ferenczi et al., 2002) . . .
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