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

Abstract

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.

Presence of CD4+CD8+ double‐positive T cells with very high interleukin‐4 production potential in lesional skin of patients with systemic sclerosis

“…Although the culture conditions may have favored the outgrowth of certain clones, we do not believe this influenced the results considerably, because oligoclonality in T cells infiltrating SSc skin has also been observed previously in studies using techniques not involving T cell culture (43), and T cells cultured from normal skin have been shown to be polyclonal (44,45). Thus, our results are consistent with the hypothesis that T cells are recruited in SSc skin in Figure 5.…”

Whole-genome sequencing reveals oncogenic mutations in mycosis fungoides

“…For 1 patient, an additional biopsy was obtained from a separate tumor, and the tumor cells cultured (without a matrix) as previously described. 21 Additionally, frozen, banked skin biopsies from 25 MF patients were retrospectively identified. All samples were obtained under an institutional review board-approved protocol (IRB# 111143).…”

The Majority of Human Peripheral Blood CD4+CD25highFoxp3+ Regulatory T Cells Bear Functional Skin-Homing Receptors

“…We cultured explants of skin on specialized matrices that have previously been found to support the survival of skin-resident T cells without altering their phenotype (18,19). Skin T cells prepared by this method were 78% positive for CLA and 89% positive for CCR4 (18). In the present study, skin T cells were stained with anti-CD4, anti-CD25, and anti-CD69 Abs.…”

“…Skin T cells were prepared by three-dimensional skin explant cultures as previously described (18,19). Skin obtained from cosmetic surgery procedures was minced into explants ϳ2 mm ϫ 2 mm ϫ 2 mm in size.…”