Phenotype and suppressor activity of T-lymphocyte clones extracted from lesions of oral lichen planus

Lymphocytes were extracted from six biopsy specimens of oral lichen planus. T-lymphocyte lines were expanded in culture with phytohaemagglutinin and interleukin 2, and cloned by limiting dilution. Fifteen T-cell clones were isolated with a probability of clonality of 96.3%. The majority of clones (n=13) expressed the αβ T-cell receptor, and of these, 11 were CD8⁺ and two were CD4⁺. Two clones were CD4^? and CD8^?, and expressed the γδ T-cell receptor. The ability of these clones (effectors) to suppress concanavalin-A-stimulated proliferation of autologous lesional T-cell lines (responders) was assessed. Maximum suppressor activity ranged from 17 to 100%. The majority of clones (n=12), including a CD3⁺ CD4⁺CD8^?αβ⁺ clone, displayed suppressor activity which was proportional to the effector to responder ratio. A CD3⁺CD4⁺CD8^?αβ⁺ clone and a CD3⁺CD4^?CD8^?γδ⁺ clone displayed substantial helper activity at higher effector to responder ratios. These results demonstrate differential helper and suppressor activity of T-lymphocyte clones extracted from oral lichen planus lesions. The balance between help and suppression may be a fundamental determinant of immunological activity within the lymphocytic infiltrate of oral lichen planus, and hence may dictate the clinical behaviour of the disease.     

Treatment of severe psoriasis with low dose cyclosporin A and the effect on the helper-suppressor T cell ratio in peripheral blood

Oral administration of cyclosporin A (CsA) 14 mg/kg/day has been reported to improve psoriasis in a double-blind study. The purpose of this study was to evaluate the efficacy of a lower dose of CsA in severe psoriasis and to monitor lymphocyte subpopulations in peripheral blood in order to detect its mechanism of action. Eleven patients with severe, active psoriasis were treated only with oral administration of 5 mg/kg/day CsA twice a day for 3 months, during which they were closely examined, including single- and two-color analysis of lymphocyte surface markers by flow cytometry using monoclonal antibodies. Seven patients showed improvement within a week, and the others within 2-3 weeks. Five patients had total remission, 2 showed marked improvement, and 4 showed moderate improvement; no clinically important side effects, except hypertrichosis in 2 females were seen. In the peripheral blood of patients treated with CsA there was a decrease in the percentages of helper inducer (CD4⁺4B4⁺)T(Thi) cells, while no significant decrease was found in those of suppressor inducer (CD4⁺2H4⁺)T(Tsi), suppressor effector (CD8⁺CD11⁺)T(Tse), or cytotoxic (CD8⁺CD11^-)T(Tc) cells, resulting in a decrease in the ratio of Thi to Tsi, Tse and Tc. The activated helper (CD4⁺HLA-DR⁺)—suppressor (CD8⁺HLA-DR⁺)T cell ratio was also decreased. These immunological findings obtained from the patients were consistent with in vitro studies of CsA reported earlier and may well explain the effectiveness of CsA in psoriasis as observed in this study.     

Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR γ-chain gene sequencing

Abstract Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30⁺ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30⁺ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR á-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells. Received: 8 September 2000 / Revised: 11 November 2000 / Accepted: 10 December 2000     

Active psoriasis and profound CD4+ lymphocytopenia

We report the case of a patient with a long-standing history of widespread chronic plaque psoriasis, who was recently found to have a profound CD4⁺ lymphocytopenia. He is human immunodeficiency virus (HIV) negative. His psoriasis remains active and widespread, and he has had 60 cutaneous malignancies, including many squamous cell carcinomas, excised over the last 10 years. In the past he has had numerous cutaneous viral warts. Despite a low peripheral blood CD4⁺ T-cell count, similar numbers of activated T cells, identified by double labelling for CD4 and HLA-DR antigens, were found in the epidermis of our patient as other individuals with psoriasis. Thus, there appear to be sufficient activated CD4⁺ T cells in our patient's psoriatic plaques to maintain the psoriatic process.     

Effective induction of melanoma‐antigen‐specific CD8+ T cells via Vγ9γδT cell expansion by CD56high+ Interferon‐α‐induced dendritic cells

Dendritic cells (DCs) can be differentiated from CD14⁺ monocytes in the presence of interferon‐α (IFNα) and granulocyte/macrophage‐colony stimulating factor (GM‐CSF) in vitro and are known as IFN‐DCs. Circulating blood CD56⁺ cells expressing high levels of CD14, HLA‐DR and CD86 have been shown to spontaneously differentiate into DC‐like cells in vitro after their isolation from blood. We show here that IFN‐DCs expressing high levels of CD56 (hereafter, CD56^high+ IFN‐DCs) can be differentiated in vitro from monocytes obtained as adherent cells from healthy donors and patients with metastatic melanoma. These cells expressed high levels of CD14, HLA‐DR and CD86 and possessed many pseudopodia. These CD56^high+ IFN‐DCs may be an in vitro counterpart of the circulating CD56⁺ CD14⁺ CD86⁺ HLA‐DR⁺ cells in blood. Conventional mature DCs differentiated from monocytes as adherent cells in the presence of GM‐CSF, IL‐4 and TNF‐α (hereafter, mIL‐4DCs) did not express CD56 or CD14. In contrast to mIL‐4DCs, the CD56^high+ IFN‐DCs exhibited a stronger capacity to stimulate autologous CD56⁺ Vγ9γδT cells highly producing IFNγ in the presence of zoledronate and IL‐2. The CD56^high+ IFN‐DCs possessing HLA‐A*0201 effectively induced Mart‐1‐modified melanoma peptide (A27L)‐specific CD8⁺ T cells through preferential expansion of CD56⁺ Vγ9γδT cells in the presence of A27L, zoledronate and IL‐2. Vaccination with CD56^high+ IFN‐DCs copulsed with tumor antigens and zoledronate may orchestrate the induction of various CD56⁺ immune cells possessing high effector functions, resulting in strong immunological responses against tumor cells. This study may be relevant to the design of future clinical trials of CD56^high+ IFN‐DCs‐based immunotherapies for patients with melanoma.     

Reduction of skin-homing cytotoxic T cells (CD8+ -CLA+) in patients with vitiligo

Background: Vitiligo is a frequently acquired, hereditary disease, characterized by achromic macules due to the absence of melanocytes. In contrast with earlier studies, in which the main pathogenic role was attributed to anti‐melanocyte antibodies, recent papers have emphasized a role for CD8⁺ cytotoxic T lymphocytes in melanocyte destruction. Fifteen percent of peripheral T cell express cutaneous lymphocyte‐associated antigen (CLA), responsible for skin‐homing T cell. Phototherapy is used to treat patients with generalized vitiligo and it has been shown to interfere with CLA⁺ T cells in other skin diseases. Objective: To describe peripheral blood T cell subpopulations' frequency and ability to express the skin‐homing molecule (CLA) in patients with non‐segmental vitiligo, before and after photochemotherapy (PUVA). Patients and Methods: Twenty‐two patients with generalized and active spreading vitiligo were submitted to 30 PUVA‐8MOP sessions. Lymphocyte immunophenotyping was performed by flow cytometry using anti‐CD3, anti‐CD8 and anti‐CLA monoclonal antibodies. Fifteen healthy volunteers, sex‐ and age‐matched, were included as a control group. Results: CD8⁺–CLA⁺ T cells were significantly reduced in number in untreated vitiligo patients (P=0.008) when compared with control individuals, albeit with a more intense CLA expression (P=0.028). These findings were not altered after PUVA. No significant difference was noticed in CD4/CD8 ratios nor in CD4–CLA⁺ T cell numbers between vitiligo patients and controls, both before and after PUVA. Conclusions: CD8–CLA⁺ T cells are reduced in peripheral blood of patients with non‐segmental vitiligo. This finding may be related to the previously reported increase of CD8⁺ cells in both lesions and perilesional skin of these patients.     

The ICAM-3/LFA-1 interaction is critical for epidermal Langerhans cell alloantigen presentation to CD4+ T cells

Intercellular adhesion molecule (ICAM)-3 is a recently described member of the immunoglobulin superfamily and, as such, is closely related to ICAM-1 and ICAM-2. All three ICAMS are cognate for the counter-receptor lymphocyte function associated antigen-1 (LFA-L CD11a/CD18). Unlike ICAM-1 and ICAM-2. ICAM-3 is constitutively expressed at high levels on resting leucocytes. We investigated the expression and function of ICAM-3 in normal skin (n= 5), as well as its expression in psoriasis (n= 4). atopic eczema (n= 4), allergic (rhus) contact dermatitis (n=3). and cutaneous T-cell lymphoma (CTCL. n=2). Five-micrometre cryostat sections of skin were stained using monoclonal antibodies to ICAM-3 and A well characterized immunoperoxidase technique. In normal skin. ICAM-3 was expressed by all cutaneous leucocytes hut most striking was the strong expression of ICAM-3 by Langerhans cells within both epidermis and dermis. This observation was confirmed by double-labelling with CD1a and negative staining with an IgG1 isotype control. In psoriasis, atopic eczema, allergic contact dermatitis, and CTCL. ICAM-3 was co-expressed on all CD1a⁺ cells, although, in psoriasis, the intensity of ICAM-3 expression was reduced. Functional blocking experiments were performed to determine whether the observed ICAM-3 expression on Langerhans cells was functionally important in antigen presentation. CD4⁺ T cells were prepared from peripheral blood and 10⁵ CD4⁺ T cells combined with 10⁵ epidermal cells harvested from keratome biopsies of normal skin of an individual allogeneic to the T-cell donor. Addition of 50 μg anti-ICAM-3 to the co-culture resulted in a consistent (50%) reduction in degree of alloantigen presentation by Langerhans cells to T cells. Inhibition was 77% of that produced by the addition of anti-LFA-1. These data indicate that ICAM-3 is constitutively expressed by Langerhans cells and is a major ligand for LFA-1 on CD4⁺ T cells during their response to Langerhans cells. Because fresh Langerhans ceils constitutively express little ICAM-1. whereas ICAM-3 is constitutively expressed at high levels, it would appear that 1CAM-3 is the dominant functional ICAM on in situ Langerhans cells in the normal epidermis.     

Contact sensitization induces proliferation of heterogeneous populations of hapten-specific T cells1

Abstract To characterize the T cells that are activated during the induction of contact hypersensitivity (CH), two sets of studies were conducted: 1) dinitrophenol (DNP)-specific proliferative responses of T cells in draining lymph nodes of BALB/c mice sensitized epicutaneously to dinitro-fluorobenzene (DNFB) were examined, and 2) from these lymph node cells, DNP-specific T cells were cloned by limiting dilution microculture and analyzed by FACS for surface markers, by RT-PCR, HT2 bioassay and ELISA for cytokine expression at mRNA and protein levels respectively, and by proliferation assay for cytokine and antigen-presenting cell (APC) requirements. Our results show that αβ TCR-bearing T cells of both the CD4⁺ and CD8⁺ subtypes from lymph nodes of DNFB-skin-painted mice proliferate specifically to dinitrobenzene sulfonate (DNBS) in vitro. Four DNP-specific, CD4⁺ T-cell clones were characterized: clone 5S4 secreted IL-4 and required 11-4 for optimal growth; clone 5S10 secreted IL-2 and required 1L-2 for optimal growth; clone 5S2 secreted IL-4 but required IL-2 for optimal growth; and clone 5S8 secreted IL-2 predominantly at 5 months, but switched to production of IL-4 at 7 months. All four clones secreted IL-10, and proliferated to DNBS when Langerhans cell (LC)-enriched epidermal cells were used as APC. These findings indicate that heterogeneous populations of DNP-specific T cells are activated in draining lymph nodes during the induction of CH to DNFB in BALB/c mice.     

T cells migrate to tumour sites after extracorporeal interleukin 2 stimulation and reinfusion in a patient with metastatic melanoma

Peripheral blood mononuclear cells (PBMC) were taken by leukapheresis from a patient with melanoma skin metastases and stimulated in vitro using 1000 IU recombinant interleukin 2 (IL-2)/ml to generate lymphokine-activated killer cells (LAK cells). Two-colour immunofluorescence analysis demonstrated an IL-2-induced up-regulation of CD25 on natural killer cells (CD56⁺) as well as on T lymphocytes (CD3⁺). After radiolabelling with indium-111, the cells were reinfuse. Gamma-camera imaging revealed an enrichment at the tumour sites. Immunostaining of tumour tissue taken before and after scintigraphy demonstrated CD25⁺ Tlymphocytes (CD2⁺, CD3⁺), but no natural killer cells (CD16⁺, CD56⁺) infiltrating the metastases. LAK cell enrichment at melanoma metastases in vivo did not involve natural killer cells, but was characterized by increased numbers of activated T lymphocytes in this patient.     

Detection of CD4+ CD25+ FOXP3+ regulatory T cells in peripheral blood of patients with chronic autoimmune urticaria

Background/Objectives: Compelling evidence indicates a significant role for a population of CD4⁺ T regulatory cells in suppressing immune responses and in maintaining immunological homeostasis. This study aims to investigate the potential role of CD4⁺CD25^HIGHFOXP3⁺ T regulatory cells in patients with chronic autoimmune urticaria and to define the characteristics of CD4⁺CD25^HIGHFOXP3⁺ cells in chronic urticaria. Methods: We used flow cytometry to assess the expression of CD4⁺CD25^HIGHFOXP3⁺ cells in the peripheral blood mononuclear cells of patients with chronic autoimmune urticaria. Results: In this study, we found that patients with chronic autoimmune urticaria have a significantly reduced frequency of CD4⁺CD25^HIGHFOXP3⁺ cells (1.39 ± 0.27% vs 2.09 ± 0.34%; P = 0.001) in their peripheral blood, accompanied by a decreased intensity of FOXP3 expression (50.13 ± 9.79 vs 68.19 ± 6.40; P < 0.001). Notably, although patients with chronic idiopathic urticaria had a reduced frequency of CD4⁺CD25^HIGHFOXP3⁺ cells (1.85 ± 0.46% vs 3.64 ± 0.48%; P < 0.001), their FOXP3 expression levels did not differ from those in healthy controls. Conclusions: Patients with chronic autoimmune urticaria displayed a reduced percentage of CD4⁺CD25⁺FOXP3⁺ regulatory T cells. The results imply CD4⁺CD25⁺FOXP3⁺ regulatory T cells may contribute to the autoimmune pathological process of chronic autoimmune urticaria.     

Lesional alopecia areata T lymphocytes downregulate epithelial cell proliferation

Alopecia areata is characterized by peribulbar infiltration by activated T cells. The function of these T cells in the pathogenesis is unknown. To elucidate the potential role of lesional T cells in the regulation of hair growth, T-cell clones from the margin of involved alopecia areata lesions from three patients were obtained by cloning, using the limiting dilution method. Of these T-cell clones, 31 were CD4 ⁺ CD8 ^– , 15 were CD8 ⁺ CD4 ^– and 2 were CD4 ^– CD8 ^– . The T-cell clones were activated and the supernatant harvested 24 h later and tested for its capacity to regulate proliferation of neonatal keratinocytes. The majority of the T-cell clone supernatants inhibited epithelial cell proliferation in a dose-dependent fashion. When the cytokine profiles of conditioned T-cell medium were compared with the growth-regulatory capacity, it was found that T-cell clones that released high amounts of interferon gamma and/or tumour necrosis factor alpha inhibited keratinocyte growth. In conclusion, T cells derived from the margin of active alopecia areata lesions are able to downregulate epithelial cell proliferation. This points to an important role of the immune system, especially the T cells, in this disease. Received: 28 May 1996     

Increased HLA-DR+ T-lymphocyte population in peripheral blood of alopecia areata

The populations of activated T-cell subsets [HLA-DR⁺ -Leu 4⁺ cells, interleukin 2 receptor positive (IL-2R ⁺)-Leu 4⁺ cells] in the peripheral blood of patients with alopecia areata (AA) were investigated using double direct immunofluorescence staining. Fifty-eight patients with AA were classified into one of three types: those with inactive single AA (type 1) lesions, active multiple alopecia areata (MAA) lesions and active alopecia totalis (AT) (type 2) and chronic alopecia universalis (AU) (type 3). Compared to normal controls, high percentages of HLA-DR⁺-Leu 4⁺ cells were detected in types 2 and 3 AA patients, but not in type 1 AA patients. These findings suggest that T cells are activated in the peripheral blood of active MAA, AT and chronic AU.     

Papuloerythroderma‐like cutaneous involvement of a CD62L− subclone of T‐cell prolymphocytic leukemia

We report the case of an 88‐year‐old Japanese man with erythrodermic involvement of T‐cell prolymphocytic leukemia (T‐PLL). He had a history of pharyngeal diffuse large B‐cell lymphoma successfully treated with polychemotherapy including cyclophosphamide and epirubicin, 6 years before the current illness. He presented with numerous reddish, coalescing, flat‐topped papules on the trunk and extremities, sparing the skin folds of the abdomen, the features of which mimicked those of papuloerythroderma. Immunohistochemistry showed perivascular and epidermotropic infiltration of CD3⁺ CD4⁺ T cells in the cutaneous lesion. However, flow cytometric analysis revealed that the skin infiltrating T cells were negative for surface CD4, and that CD3⁺ CD4^? CD8^? cells made up 92% of the T‐cell fraction of peripheral blood. The circulating atypical T cells had a round or oval nucleus and prominent nucleoli, and the deletion of chromosomes 6q, 13 and 17. These cytological profiles were consistent with those of T‐PLL and distinct from those of Sézary cells. The same T‐cell clone was detected in the cutaneous lesion and peripheral blood, but the expression of CD62L was absent in the skin infiltrates and present in the circulating cells. No specific mutation was detected in STAT3 or STAT5B. Although low‐dose oral etoposide had a beneficial effect on the skin rash, a fatal crisis of marked leukocytosis (169 × 10³/μL) occurred 19 months after the illness onset. CD62L‐leukemic cells of T‐PLL may infiltrate the skin to form papuloerythroderma‐like cutaneous lesions.     

Possible role of superantigens in inducing autoimmunity in pemphigus patients

The diagnostic and pathological relevance of anti‐desmoglein autoantibodies in common forms of pemphigus has been well established, and T cells have been shown to play a role in the onset and progression of these diseases. The role of superantigens in provoking polyclonal activation of T cells with many different fine specificities, possibly including autoreactive T cells and T‐cell mediated autoantibody response, is unknown. Further, abnormal T‐cell function may lead to opportunistic infections particularly with Candida. The response of T cells of pemphigus patients to recall antigens of these opportunists is not clear. The aim of this study was to investigate the in vitro response of T lymphocytes from pemphigus patients to common bacterial superantigens such as streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B, and recall antigens such as Candida antigen. Changes in CD3⁺CD4⁺ and CD3⁺CD8⁺ T‐cell sub‐populations and expression of naive/memory markers (CD45RA⁺/RO⁺) on different T cells were analyzed by flow cytometry. Significant elevation in CD3⁺CD4⁺ and expression of the memory (CD45RO⁺) markers on these cells was observed both in pemphigus vulgaris and pemphigus foliaceus patients, as compared to healthy controls, upon stimulation with streptococcal pyrogenic exotoxin A and staphylococcal enterotoxin B. However, only memory T cells (CD45RO⁺) were significantly increased upon Candida antigen stimulation. Our study suggests that CD4⁺ memory T lymphocytes may modulate the pathogenic autoantibody response in pemphigus patients, and also emphasizes the possibility that the superantigen‐reactive T cells participate in the triggering of autoimmunity in pemphigus.     

Non-M protein(s) on the cell wall and membrane of group A streptococci induce(s) IFN-γ production by dermal CD4+ T cells in psoriasis

Abstract Recently we have demonstrated that a disease-specific subpopulation of CD4⁺ T cells isolated from skin lesions of chronic plaque psoriasis produces interferon-γ in response to group A streptococcal (GAS) antigens. To determine if these T cells recognize M or non-M protein, extracts from cell wall of type M6 GAS (M6W) and its isogenic M gene deletion mutant (M-W), M6 membrane extract (M6M) and recombinant M6 protein (rM6) were used to stimulate GAS-reactive T-cell lines from nine patients with chronic plaque psoriasis. T-cell lines were incubated with or without streptococcal extracts for 18 h in the presence of a transport inhibitor, stained for surface CD4 and intracellular cytokine expression, and analysed by flow cytometry. Variable numbers (0.2–34%) of CD4⁺ T cells produced interferon-γ, in all but one of the T-cell lines tested, in response to M6W, M-W and M6M extracts. No significant difference between the response to M6W and M-W extracts was detected. In addition, rM6 protein failed to increase CD4⁺/interferon-γ⁺ T-cell numbers in seven of nine T-cell lines compared to medium alone. For the group, there was a highly significant correlation between the responses to the three extracts (M6W vs M-W, P = 0.0005; M6W vs M6M, P = 0.0003; M-W vs M6M, P = 0.0001). Low or minimal numbers of interleukin-4- and interleukin-10-producing CD4⁺ T cells were occasionally induced. These findings suggest that a subpopulation of CD4⁺ T cells isolated from skin lesions of chronic plaque psoriasis patients produces interferon-γ in response to non-M protein(s) present on the cell wall and membrane of GAS. Received: 11 September 2000 / Revised: 20 December 2000 / Accepted: 3 February 2001     

Evolutionary changes of immunohistological characteristics of secondary lesions in pityriasis rosea

Summary In 15 patients with pityriasis rosea, we studied the evolutionary changes of the immunohistological characteristics of the secondary lesions. Many CD1a⁺ cells were seen in the epidermis and dermis of early lesions. In the well-developed lesions, the number of CD1a⁺ cells greatly increased in the dermis. In the late lesions, CD1a⁺ cells in the dermis significantly decreased as compared with the well-developed lesions. Early lesions showed a moderate T-cell infiltrate. In the well-developed lesions, the dermal T-cell infiltrate was dense, and the CD4 CD8 ratio was 2.9. The late lesions had a moderate T-cell infiltrate, in which the CD4 CD8 ratio significantly decreased as compared with the well-developed lesions. Thus, the relative decrease in CD4⁺ helper inducer cells during lesion regression, concomitant with a decrease in number of CD1a⁺. Langerhans cells, is in accordance with a broader concept of increased suppressor mechanisms during healing.     

Increased frequencies of CD11b+CD33+CD14+HLA‐DRlow myeloid‐derived suppressor cells are an early event in melanoma patients

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous cell population characterized by immunosuppressive activity. Elevated levels of MDSC in peripheral blood are found in inflammatory diseases as well as in malignant tumors where they are supposed to be major contributors to mechanisms of tumor‐associated tolerance. We investigated the frequency and function of MDSC in peripheral blood of melanoma patients and observed an accumulation of CD11b⁺CD33⁺CD14⁺HLA‐DR^low MDSC in all stages of disease (I–IV), including early stage I patients. Disease progression and enhanced tumor burden did not result in a further increase in frequencies or change in phenotype of MDSC. By investigation of specific MDSC‐associated cytokines in patients’ sera, we found an accumulation of IL‐8 in all stages of disease. T‐cell proliferation assays revealed that MDSC critically contribute to suppressed antigen‐specific T‐cell reactivity and thus might explain the frequently observed transient effects of immunotherapeutic strategies in melanoma patients.     

Coexpression of CCR6 and CD146 (MCAM) is a marker of effector memory T-helper 17 cells

Effector memory T (T_EM) cells are a subpopulation of memory T cells that express receptors mediating migration to inflamed tissues and produce various cytokines. Effector memory T‐helper (Th)17 (Th17_EM) cells are thought to be essential for inflammation in Th17‐mediated diseases, but have not been studied in detail. To identify superior surface markers to isolate a homogeneous population of Th17_EM cells from peripheral blood, CD4⁺ T cells were isolated from the peripheral blood of healthy donors based on the expression of CCR7, CCR6 and CD146 using six‐color flow cytometry. After 4 days of culture in the presence of anti‐CD3/28 beads, intracellular cytokines were determined by flow cytometric analysis. To investigate the relevance of Th17_EM cells in Th17‐mediated disease, the frequencies of T_EM‐cell subsets in psoriasis were quantified using six‐color flow cytometry. An enzyme‐linked immunosorbent assay was performed to confirm the interleukin (IL)‐17‐producing capacity of T_EM‐cell subsets from the peripheral blood of a patient with psoriasis. CCR6⁺CD146⁺ T_EM (CD4⁺CD45RA^?CCR7^?) cells had a greater capacity to produce IL‐17 than CCR6⁺CD146^? or CCR6^?CD146⁺ T_EM cells. Although the percentage of CCR6⁺CD146⁺ cells in T_EM cells was not significantly different between patients with psoriasis and controls, three of eight patients had a higher percentage of CCR6⁺CD146⁺ T_EM cells than the mean +5 standard deviations of the controls. Coexpression of CCR6 and CD146 is a useful marker for Th17_EM cells. Increasing the number of CCR6⁺CD146⁺ Th17_EM cells in peripheral blood may facilitate estimation of systemic Th17‐cell activity in Th17‐mediated diseases.     

CXCL10 produced from hair follicles induces Th1 and Tc1 cell infiltration in the acute phase of alopecia areata followed by sustained Tc1 accumulation in the chronic phase

BackgroundAlopecia areata (AA) is an organ-specific and cell-mediated autoimmune disease. T lymphocytes densely surround lesional hair bulbs, which is histologically referred to as “swarm of bees”. However, pathomechanisms of “swarm of bees” are still uncertain.ObjectiveWe investigated the pathological mechanisms of “swarm of bees”, focusing on T-cell chemotaxis so that inhibition of chemotaxis may be strong candidate of novel treatments for AA.MethodsWe investigate the expression of chemokine receptors on T cells obtained from peripheral blood mononuclear cells (PBMCs) and skin infiltrating cells in AA patients. In addition, real-time chemotaxis assay was also demonstrated.ResultsIn PBMCs, the frequency of CXCR3⁺CD4⁺ T cells (Th1) was significantly higher in acute-phase AA than in chronic-phase AA or healthy control, while CXCR3⁺CD8⁺ T cells (Tc1) were significantly increased in chronic-phase AA. In the skin lesions of acute-phase AA, CXCR3⁺CD4⁺ and CXCR3⁺CD8⁺ T cells infiltrated in the juxta-follicular area. In chronic-phase AA, CXCR3⁺CD8⁺ T cells dominated the infiltrate around hair bulbs, possibly contributing to the prolonged state of hair loss. Lymphocytes obtained from a lesional skin of acute-phase AA contained CXCR3⁺CD4⁺ and CXCR3⁺CD8⁺ T cells at higher percentages than those of PBMCs, suggesting preferential emigration from the blood. Immunohistochemical and real-time RT-PCR studies demonstrated that hair follicles of acute-phase AA expressed a high level of Th1-associated chemokine CXCL10. By chemotaxis assay, freshly isolated PBMCs from acute-phase AA patients had a strong velocity of chemotaxis toward CXCL10 with increased expression of F-actin.ConclusionsThese results suggest that the increased production of CXCL10 from hair follicles induces preferential infiltrates of highly chemoattracted Th1 and Tc1 cells in the acute phase of AA, and Tc1 infiltration remains prolonged in the chronic phase.