SUMMARYSkin-infiltralitig lymphocytes (SIL) were isolated from skin biopsies of patients with hyperimmunoglobulin E (IgE) atopic dermatitis (AD) and expanded in vitro in the presence of IL-2 in combination with IL-4. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T helper/inducer phenotype in SIL populations. In ^H-thymidine incorporation assays, SIL showed proliferation in response to lL-2, IL-3, IL-4, ionomycin (Io)+ll-o-tetradecanoyl-phorbol-Hacetate (TPA) and OKT3 -\-TPA. OKT4 with and without TPA did not induce proliferation. Tumour necrosis factor alpha (TNF-a) did not block proliferative responses of SIL to IL-2 and IL-4. Cultured SIL showed no cytotoxic activity against K562 and Jurkat target cells. Expanded skin-derived T cells were tested for their capacity to secrete several cytokines in vitro. SIL secreted significant amounts of IL-4, GM-CSF and TNF-a upon stimulation with mitogens but failed to secrete IFN-y. Io in combination with phorbolester induced the secretion of larger amounts of IL-4, GM-CSF, TNF-a and low amounts of I FN-y. The data indicate that SIL derived from AD lesions were defective in their capacity to secrete IFN-y but were enriched in T cells capable of producing IL-4 upon stimulation. The results support the possibility of a predominant 'TH2-like' cell-mediated immune response in lesional skin of AD patients.

Functional studies of lymphocytes in atopic dermatitis (AD) have so far focused on peripheral blood mononuclear cells (PBMC), whereas cells at the involved site, the skin, have not been examined. Accordingly, we have developed methods to generate lymphocyte cultures from biopsies of inflammatory skin areas. Skin-infiltrating lymphocytes (SIL) were isolated from skin biopsies of 6 patients with severe AD and expanded in vitro in the presence of interleukin-2 (IL-2) without additional antigens. After 6-10 d in culture, outgrowth of mononuclear cells from biopsy tissue was observed in all cases. Phenotypic analysis of skin-derived cells revealed the predominance of CD4+ T-helper/inducer phenotype in SIL populations. Parallel cultures of SIL and PBMC showed an increase and expansion of CD8+ T cells in cultured PBMC, whereas the CD4+ phenotype was predominant in SIL cultures. As indicated by their expression of HLA-DR and CD25 antigens, most of the SIL were activated and the cells mainly expressed T-cell receptors (TCR) composed of alpha and beta chains. Different strategies for expansion of SIL in vitro were examined. High levels of IL-4 (1,000 U/ml) in combination with IL-2 (50 U/ml or 1,000 U/ml) preferentially promoted growth of SIL derived from AD and were much more effective than IL-2 alone. No cells expanded in cultures with IL-4 alone. SIL grown with high concentrations of IL-4 contained a significant proportion of double-positive CD4+8+ cells. No other marked differences were observed in the distribution of T cell subsets in cultures propagated under different conditions for 21 d. Our results demonstrate the feasibility of growing infiltrating T lymphocytes from inflammatory skin of AD patients. The use of high concentrations of IL-2 in combination with high levels of IL-4 allows a large expansion of these cells and thus represents a useful strategy to expand cells for further functional and molecular biologic studies.

Tumor‐infiltrating lymphocytes (TIL) were obtained from a biopsy of a patient with a Ki‐1–positive large cell lymphoma of the skin. Immunohistologic studies of the large anaplastic tumor cells showed an “aberrant” T “helper/inducer” phenotype (CD30+CD3–CD4+CD8–IL–2R+HLA–DR+). Using a cDNA probe for the constant region of the T‐cell receptor (TCR) beta gene, the cells were identified by their distinct monoclonal rearrangement of T‐cell receptor (TCR)‐beta DNA. Tumor cells isolated from biopsies were cultured in the presence of interleukin‐2 (IL‐2). Outgrowing lymphocytes were cloned, expanded in vitro, and 11 clones were subjected to phenotypic analysis: ten clones showed a predominantly CD4‐positive T “helper/inducer” phenotype whereas one clone expressed CD8 T “cytotoxic/suppressor” antigens. In contrast to the tumor cells, cells of all clones grown in vitro expressed the TCR‐associated CD3 complex. Furthermore, cells from all clones analyzed expressed CD5, CD7, CD45RO (UCHL1), CD11a (LFA‐1), CD25, and HLA‐DR antigens. Cells of two of ten CD4‐positive clones expressed CD45RA (2H4) in addition to UCHL1. T‐cell clones isolated from the tumor and grown in vitro exhibited individual DNA restriction band patterns different from that of a DNA tumor biopsy specimen. Therefore, the authors conclude that these T‐cell clones represent presumably nonmalignant TIL. All clones tested secreted interferon (IFN)‐gamma and tumor necrosis factor (TNF)‐alpha in vitro. Nine of 11 clones were found to secrete additionally IL‐2 and IL‐4 upon stimulation with phytohemagglutinin (PHA) whereas two clones did not secrete detectable amounts of IL‐4. Selective growth of TIL in the presence of IL‐2 opens the possibility to use these cells in adoptive immunotherapy of cutaneous T‐cell lymphoma (CTCL). Cytokines secreted by TIL cells in vitro (IL‐2, IL‐4, IFN‐gamma, TNF‐alpha) may be involved in their antitumorgenic activity. Moreover, these data implicate that CD4‐positive TIL derived from CTCL cannot be grouped into different subsets based on the production of IL‐2, IL‐4, IFN‐gamma, and TNF‐alpha. 68:2155‐2160, 1991.