The vast majority of CLA+ T cells are resident in normal skin.

There are T cells within normal, noninflamed skin that most likely conduct immunosurveillance and are implicated in the development of psoriasis. We isolated T cells from normal human skin using both established and novel methods. Skin resident T cells expressed high levels of CLA, CCR4, and CCR6, and a subset expressed CCR8 and CXCR6. Skin T cells had a remarkably diverse TCR repertoire and were mostly Th1 memory effector cells with smaller subsets of central memory, Th2, and functional T regulatory cells. We isolated a surprising number of nonexpanded T cells from normal skin. To validate this finding, we counted T cells in sections of normal skin and determined that there are approximately 1 x 10(6) T cells/cm(2) normal skin and an estimated 2 x 10(10) T cells in the entire skin surface, nearly twice the number of T cells in the circulation. Moreover, we estimate that 98% of CLA(+) effector memory T cells are resident in normal skin under resting conditions. These findings demonstrate that there is a large pool of memory T cells in normal skin that can initiate and perpetuate immune reactions in the absence of T cell recruitment from the blood.

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.

Expression of interleukin-18 and caspase-1 in cutaneous T-cell lymphoma.

PURPOSE: Cutaneous T-cell lymphoma (CTCL) is a malignancy of skin-homing Th2 T cells. Clonal T cells and CTCL skin lesions typically express Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-10, but fail to produce Th1 cytokines. However, the reason for Th2 bias is unknown. IL-18 is a pleiotropic proinflammatory cytokine produced by monocytes/macrophages lineage as well as epithelial cells, such as human keratinocytes. In the absence of IL-12, IL-18 leads to increased immunoglobulin E production from B cells and enhanced production of IL-4 and IL-13 by basophils, mast cells, and CD4(+) T cells. We have analyzed cytokines in CTCL patients, which may bias the immune response around the Th1/Th2 axis. EXPERIMENTAL DESIGN: We examined plasma of 95 CTCL patients and skin of 20 CTCL patients for IL-18, caspase-1, IL-12, and other cytokines. To identify the presence or absence of these cytokine proteins in CTCL and normal skin, we cultured explants from skin biopsies on three-dimensional matrices. RESULTS: Plasma levels of IL-18 and its converting enzyme, caspase-1, were significantly elevated in CTCL. mRNA levels for these factors were also elevated in CTCL skin lesions. Matrices populated with CTCL lesional skin produced significant amounts of IL-18 and caspase-1; however, production of IL-12 protein was barely detectable. CONCLUSIONS: We propose that the high levels of IL-18 expression in lesional CTCL skin contribute to increased plasma levels of IL-18 and that this, in the face of significantly lower levels of IL-12, may contribute to the Th2 bias seen in this disease.

Skin-derived interleukin-7 contributes to the proliferation of lymphocytes in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphomas (CTCLs) are malignancies of T cells that have a special affinity for the skin. We have previously reported that much of the T-cell receptor repertoire is altered in CTCL, and both malignant and nonmalignant clones are numerically expanded, presumably in response to T-cell trophic cytokines. We therefore examined levels of the T-cell trophic cytokines IL-2, IL-4, IL-7, IL-12, IL-13, and IL-15 in plasma in 93 CTCL patients and healthy controls. Only IL-7 levels were elevated in CTCL. We next looked at lesional skin from patients with CTCL and found elevated levels of IL-7 mRNA. Explant cultures of normal and lesional CTCL skin biopsies revealed significantly more IL-7 protein production in CTCL skin. Additionally, cultures of CTCL skin released greater numbers of T cells than normal skin; this was blocked by the addition of an IL-7 neutralizing antibody. Finally, these cultures induced proliferation of normal peripheral skin-homing T cells that were added to the cultures. These observations led us to postulate that IL-7 produced by skin cells contributes to the survival and proliferation of T cells within skin lesions and is likely the source of elevated circulating IL-7 in CTCL.

Human skin cells support thymus-independent T cell development.

Thymic tissue has previously been considered a requirement for the generation of a functional and diverse population of human T cells. We report that fibroblasts and keratinocytes from human skin arrayed on a synthetic 3-dimensional matrix support the development of functional human T cells from hematopoietic precursor cells in the absence of thymic tissue. Newly generated T cells contained T cell receptor excision circles, possessed a diverse T cell repertoire, and were functionally mature and tolerant to self MHC, indicating successful completion of positive and negative selection. Skin cell cultures expressed the AIRE, Foxn1, and Hoxa3 transcription factors and a panel of autoantigens. Skin and bone marrow biopsies can thus be used to generate de novo functional and diverse T cell populations for potential therapeutic use in immunosuppressed patients.