Urushiol (poison ivy)-triggered suppressor T cell clone generated from peripheral blood.

Allergic contact dermatitis to Toxicodendron radicans (poison ivy) is mediated by the hapten urushiol. An urushiol-specific, interleukin 2 (IL-2)-dependent T cell clone (RLB9-7) was generated from the peripheral blood of a patient with a history of allergic contact dermatitis to T. radicans. This clone proliferated specifically to both leaf extract and pure urushiol. Although the clone had the phenotype CD3+CD4+CD8+, proliferation to antigen was blocked by anti-CD8 and anti-HLA-A, B, C, but not by anti-CD4, suggesting that CD4 was not functionally associated with the T cell receptor. Furthermore, studies with antigen-presenting cells from MHC-typed donors indicated that the clone was MHC class 1 restricted. RLB9-7 was WT31 positive, indicating it bears the alpha beta T cell receptor. The clone lacked significant natural killer cell activity and produced only low levels of IL-2 or gamma-interferon upon antigen stimulation. Addition of RLB9-7 to autologous peripheral blood mononuclear cells in the presence of urushiol inhibited the pokeweed mitogen-driven IgG synthesis. This suppression was resistant to irradiation (2,000 rad) and was not seen when RLB9-7 was added to allogeneic cells, even in the presence of irradiated autologous antigen-presenting cells, suggesting that suppression was MHC restricted and not mediated by nonspecific soluble factors. However, RLB9-7 cells in the presence of urushiol inhibited the synthesis of tetanus toxoid-specific IgG by autologous lymphocytes, indicating that the suppression, although triggered specifically by urushiol, was nonspecific.

Processing of urushiol (poison ivy) hapten by both endogenous and exogenous pathways for presentation to T cells in vitro.

The antigen processing requirements for urushiol, the immunogen of poison ivy (Toxicodendron radicans), were tested by presentation of urushiol to cultured human urushiol-responsive T cells. Urushiol was added to antigen-presenting cells (APC) either before or after fixation with paraformaldehyde. Three distinct routes of antigen processing were detected. CD8+ and CD4+ T cells, which were dependent upon processing, proliferated if urushiol was added to APC before fixation, but did not proliferate when urushiol was added to APC after fixation. Processing of urushiol for presentation to CD8+ T cells was inhibited by azide, monensin, and brefeldin A. This suggests that urushiol was processed by the endogenous pathway. In contrast, presentation of urushiol to CD4+ T cells was inhibited by monensin but not by brefeldin A. This was compatible with antigen processing by the endosomal (exogenous) pathway. Finally, certain CD8+ T cells recognized urushiol in the absence of processing. These cells proliferated in response to APC incubated with urushiol after fixation. Classification of contact allergens by antigen processing pathway may predict the relative roles of CD4+ and CD8+ cells in the immunopathogensis of allergic contact dermatitis.

Autoimmune hair loss (alopecia areata) transferred by T lymphocytes to human scalp explants on SCID mice.

Alopecia areata is a tissue-restricted autoimmune disease of the hair follicle, which results in hair loss and baldness. It is often psychologically devastating. The role of T lymphocytes in this disorder was investigated with cell transfer experiments. Scalp explants from patients were transplanted to severe combined immunodeficiency (SCID) mice and injected with autologous T lymphocytes isolated from involved scalp. T lymphocytes which had been cultured with hair follicle homogenate along with antigen-presenting cells were capable of inducing the changes of alopecia areata, including hair loss and perifollicular infiltrates of T cells, along with HLA-DR and ICAM-1 expression of the follicular epithelium. Similar changes were not noted in grafts injected with scalp-derived T cells that had not been cultured with follicular homogenate. These data indicate that alopecia areata is mediated by T cells which recognize a follicular autoantigen.

Requirements for the construction of antibody heterodimers for the direction of lysis of tumors by human T cells.

We constructed a series of MAb heterodimers consisting of the J5 (anti-common acute lymphoblastic leukemia antigen [CALLA]) antibody and antibodies to a variety of structures present on the surface of activated human T cells, including CD3 antigen (T cell receptor-associated glycoproteins), CD2 antigen (T11/E-rosette receptor), CD25 antigen (IL-2 receptor), and the transferrin receptor. We tested the ability of these heterodimers to direct a CD2 + CD3 + CD8 + CD4 - CD25 + transferrin receptor + MHC-restricted human cytolytic T lymphocyte (CTL) clone to lyse a CALLA + human tumor in vitro. Only heterodimers containing an anti-CD3 antibody or activating antibodies to CD2 could direct the clone to lyse these human tumor targets, even when the clone was additionally activated with anti-CD3 or anti-CD2 antibodies. Our findings may have implications in the design of strategies for the use of such reagents in the treatment of human neoplasia.

The use of human T-lymphocyte clones to study T-cell function in allergic contact dermatitis to urushiol.

Allergic contact dermatitis to poison ivy (Toxicodendron radicans) is believed to be mediated by T lymphocytes specific for the hapten urushiol. Activated T lymphocytes may produce pathology by a variety of mechanisms including direct cytotoxicity, production of lymphokines, recruitment of non-specific effector cells, non-specific cytotoxicity, and possibly autologous DR reactivity. The regulation and pathogenesis of this condition was studied by cloning and characterizing urushiol-specific T cells from the peripheral blood of patients with poison ivy dermatitis. Multiple CD8+ (T8+) urushiol-specific clones were derived. All clones that proliferated in response to a crude extract of T. radicans also proliferated in response to purified urushiol. Thus, urushiol appears to be the single immunogenic component of T. radicans resin. Pentadecylcatechol (PDC), which differs from urushiol only in the lack of unsaturated bonds in its lipophilic tail, stimulated only one of seven clones tested. This suggests that the double bonds in the C15 lipophilic tail of urushiol are required for antigenicity. Several of the CD8+ urushiol-specific clone suppressed pokeweed mitogen-induced IgG production in the presence of urushiol. Suppression was triggered specifically by urushiol and required MHC compatibility both for the antigen-presenting cells and the responding B cells. These suppressor clones were isolated from convalescent blood and may represent a mechanism for the termination of an allergic contact dermatitis.

Induction of hapten-specific tolerance of human CD8+ urushiol (poison ivy)-reactive T lymphocytes.

The interaction of CD28 with B7 molecules (CD80 or CD86) is an essential second signal for both the activation of CD4+ T cells through the T-cell receptor and the prevention of anergy. We studied the requirement of hapten-specific human CD8+ cells for CD28 co-stimulation in recognition of hapten, and anergy induction. Urushiol, the immunogenic hapten of poison ivy (Toxicodendron radicans), elicits a predominantly CD8+ T-cell response. Autologous PBMC were pre-incubated with urushiol prior to fixation by paraformaldehyde. Fixed antigen-presenting cells were unable to present urushiol to human CD8+ urushiol-specific T cells. Addition of anti-CD28, however, overcame this antigen-presenting defect, enabling CD8+ cells to proliferate. Fixation of antigen-presenting cells prevents upregulation of B7, and addition of anti-CD28 substitutes for this signal. Proliferation of CD8+ T cells in response to urushiol was blocked by CTLA4Ig, a recombinant fusion protein that blocks CD28/B7 interactions. Preincubation of urushiol-specific CD8+ cells with fixed PBMC + urushiol for 7 d induced anergy. Anergic CD8+ cells were viable and able to proliferate in response to IL-2, but not in response to urushiol. Induction of anergy required the presence of urushiol, and pre-incubation with irradiated PBMC + urushiol did not have this effect. It is proposed that anergy was induced by presentation of urushiol by fixed PBMC, in the absence of adequate co-stimulation signals. Induction of anergy by blocking of co-stimulation could potentially induce clinical hyposensitization to haptens.

Quantitation and cloning of human urushiol specific peripheral blood T-cells: isolation of urushiol triggered suppressor T-cells.

A limiting dilution assay was developed to quantitate urushiol (the antigen of poison ivy; Toxicodendron radicans) specific T cells from peripheral blood of a patient with a history of rhus (poison ivy) dermatitis. It was found that maximal sensitivity with minimal nonspecific proliferation could be produced with the use of 5 U/ml of recombinant IL2 added to the assay on day 6. This donor was found to have a frequency of urushiol specific peripheral blood T cells of (1/2935). Five interleukin 2 (IL2) dependent urushiol specific T-cell clones were generated from the peripheral blood of this patient. These T-cell clones had a CD8+ (T8+) phenotype and proliferated specifically to both extracts of Toxicodendron radicans (poison ivy) leaves and pure urushiol. Pentadecylcatechol was an inferior antigen, only stimulating proliferation of one clone. The ability of all clones to proliferate to pure urushiol, despite their having been induced with leaf extract, suggests that urushiol, or closely related catechols, represent the only allergenic constituents of Toxicodendron radicans. Lymphokine production in response to antigen varied between (0.6-5.0) units/ml of interleukin 2 (IL2) and (1.0-120) units/ml of gamma interferon. Although none of the clones showed significant cytotoxicity against NK targets, three of five lines showed considerable cytotoxicity against concanavalin A treated (lectin approximated) targets. However, cytotoxicity for rhus conjugated autologous targets was not detected. It was found that several of these CD8+ clones could suppress IgG production in the presence of rhus antigen. The isolation of these T-cells from peripheral blood several months after rhus dermatitis suggests that these clones may have a role in down regulating delayed hypersensitivity to urushiol.

T-lymphocyte dependence of psoriatic pathology in human psoriatic skin grafted to SCID mice.

Considerable indirect evidence suggests that T lymphocytes have a role in the pathogenesis of psoriasis. The goal of this study was to directly test the ability of T cells to maintain psoriasis pathology. Psoriatic skin was transplanted onto SCID mice, which were then injected with autologous T cells. T cells were cultured from either psoriatic skin lesions or peripheral blood and injected intradermally or intravenously. Control SCID mice transplanted with psoriasis grafts were not injected with T cells. After 10 wk, control psoriatic skin grafts not injected with T cells lost many of the features of psoriasis. Injection of peripheral blood T cells was not able to maintain these psoriatic features. In contrast, the injection of T cells derived from psoriatic skin was able to maintain the psoriatic phenotype. Psoriatic features that were maintained included epidermal thickness and labeling index and expression of HLA-DR, involucrin, and ICAM-1, as well as loss of expression of filaggrin. Injection of skin infiltrating T cells into skin of normal donors on SCID mice did not induce changes of psoriasis. The ability of T cells from lesional skin, but not peripheral blood, to maintain psoriasis suggests that psoriasis is mediated by an autoantigen reactive T cell, which is present at a higher frequency in the psoriatic lesion.

Melanocyte-associated T cell epitopes can function as autoantigens for transfer of alopecia areata to human scalp explants on Prkdc(scid) mice.

Alopecia areata is a tissue restricted autoimmune condition affecting the hair follicle, resulting in hair loss. The goal of this study was to test the hypothesis that the autoantigen of alopecia areata is melanocyte associated. Potential autoantigens were tested in the human scalp explant/Prkd(scid) CB-17 mouse transfer system. Scalp T cells from lesional (bald) alopecia areata scalp were cultured with antigen-presenting cells, and antigen, along with interleukin-2. The T cells were then injected into autologous lesional scalp grafts on SCID mice, and hair regrowth was measured. Hair follicle homogenate was used as an autoantigen control. T cells cultured with melanoma homogenate induced statistically significant reduction in hair growth (p <0.01 by ANOVA). HLA-A2-restricted melanocyte peptide epitopes were then tested with lesional scalp T cells from HLA-A2-positive alopecia areata patients. Melanocyte-peptide-activated T cells significantly reduced the number of hairs regrowing in two experiments with six patients (p <0.001 by ANOVA). Injected scalp grafts showed histologic and immunochemical changes of alopecia areata. The most consistent peptide autoantigens were the Gp100-derived G9-209 and G9-280 peptides, as well as MART-1 (27-35). Melanocyte peptide epitopes can function as autoantigens for alopecia areata. Multiple peptides were recognized, suggesting epitope spreading.

Recent developments in the pathogenesis of allergic contact dermatitis.

Allergic contact dermatitis is both an important clinical problem and a model system for lymphocyte-mediated pathologic changes. Elicitation of allergic contact dermatitis requires interaction of antigen with epidermal Langerhans cells, followed by migration of the Langerhans cells to the lymph nodes to present antigen to T lymphocytes. These activated T lymphocytes must then home to the antigen-exposed skin. Adhesion molecules such as LFA-1 and ICAM-1 have a role in this homing. Only a small proportion of the T lymphocytes in the skin lesion are specific for the inducing antigen. Studies of poison ivy (urushiol dermatitis) have determined this fraction to be less than one per 100 infiltrating lymphocytes. By a variety of amplification mechanisms, it is possible for this small number of antigen-specific T lymphocytes to induce the pathologic changes of allergic contact dermatitis. Improved understanding of this condition should result in increased knowledge of the pathogenesis of a variety of T lymphocyte-mediated skin conditions.

Alopecia areata. Autoreactive T cells are variably enriched in scalp lesions relative to peripheral blood.

BACKGROUND AND DESIGN: Alopecia areata is a condition characterized by hair loss in association with perifollicular infiltration of T cells and antigen-presenting cells. Autoreactive T cells are postulated to amplify this abnormality by interacting with DR+ follicular epithelium. These cells may recognize either autologous major histocompatibility complex class II antigen or an autoantigen restricted by major histocompatibility complex class II. Limiting dilution analysis was used to determine the frequency of autoreactive lymphocytes in scalp biopsy specimens and peripheral blood from seven adult patients with alopecia areata. Autoreactive T cells are defined for this study as those that proliferate in response to autologous irradiated peripheral blood mononuclear cells. RESULTS: Autoreactive lymphocytes were enriched in scalp biopsy specimens relative to peripheral blood in five of seven patients. This enrichment was statistically significant in four of five patients. Five autoreactive T-cell clones derived from lesional scalp were characterized. Four of these clones were CD3+CD4+CD8- and one clone was CD3+CD4-CD8+. CONCLUSIONS: Enrichment of autoreactive cells in lesions of alopecia areata supports a role for these cells in the pathogenesis of this condition. Enrichment of autoreactive lymphocytes is also found in allergic contact dermatitis. Thus, these autoreactive lymphocytes may have a general role in inflammation.

Favourable melanoma prognosis associated with the expression of the tumour necrosis factor receptor and the alpha1beta1 integrin: a preliminary report.

Recently we encountered a patient (p1) with a Clark's level IV melanoma associated with recurrent cutaneous metastasis who subsequently experienced a complete remission after a period of therapy with dichloronitrobenzene (DCNB) and interferon-alpha (IFNalpha). Another patient (p2) with a similar Clark's level of disease but with a fatal prognosis and melanoma cells from the Sk-Mel 28 and MeWo cell lines served as control groups. Since cytokines and members of the alpha1 integrin family have been implicated in the growth and metastatic behaviour of melanomas, we evaluated the cytokine effects and integrins expressed by melanoma cells in the patients' tumours. P1, but not p2 nor MeWo melanoma cells, expressed HLA-DR, alpha1beta1 and the tumour necrosis factor receptor (TNF-R). Culturing p1 melanoma cells with TNFalpha, but not MeWo or p2 melanoma cells, increased their expression of alpha1beta1 integrin and was cytotoxic for the cells. Significant in vivo growth of metastatic p1 and p2 melanoma explants as well as MeWo cells grafted subcutaneously onto nude mice was noted over 36 days. Intralesional injection of TNFalpha induced complete regression of p1 explants, but not of p2 or MeWo explants. Intralesional injection of IFNalpha or anti-alpha1beta1 integrin arrested p1 graft growth. These results suggest that the slow growth of the melanoma cells in nude mice, as well as the susceptibility to tumour killing by TNFalpha and the dependence of the tumour on signals mediated by the alpha1beta1 integrin are features that may have contributed to the beneficial therapeutic response in patient 1.

Non-specifically activated human peripheral blood mononuclear cells are cytotoxic for human keratinocytes in vitro.

Cultured human keratinocytes were lysed by activated PBMC in a 4-h 51Cr release assay. PBMC were activated by incubation with 50 U/ml of rIL-2 for 4 days. The cytotoxic precursors were found to be NKH1+ and included both CD2+ and CD2- phenotypes. This cytotoxicity was not genetically restricted, as cells killed both allogeneic and autologous keratinocytes without priming. Cytotoxicity was blocked by pre-incubation of effector cells with mAb against LFA-1 alpha-(TS1/22) and beta-chains (TS1/18), but not by antibodies directed against CD4, CD8, or leukocyte common Ag (T200) suggesting that LFA-1 is an important interactive molecule in this cytotoxicity. IFN-gamma is reported to upregulate ICAM-1, the ligand for LFA-1. Pre-treatment of target keratinocytes with IFN-gamma was also found to greatly increase the sensitivity of keratinocytes to lysis. This increased sensitivity to lysis was blocked by anti-LFA-1 and anti-ICAM-1, but not by anti-DR (L243), and thus was not the result of increased DR expression. Such treated targets were lysed at low levels (15 to 18%) by an Ag-specific CD8+ cytotoxic clone as well as a T cell line derived from a skin lesion of allergic contact dermatitis. In contrast, control keratinocytes were only sensitive to IL-2-activated PBMC as described above. The above findings may be relevant to a variety of conditions in which epidermal damage is associated with lymphocytic infiltrate. These conditions include graft-vs-host disease, erythema multiforme, and lupus erythematosus. DR+ keratinocytes, which may be a marker for IFN-gamma are also found in the above conditions. It is suggested that epidermal pathology may be mediated by non-specific cytotoxicity induced in the course of an immune response.