The predominant drug-specific T-cell population may switch from cytotoxic T cells to regulatory T cells during the course of anticonvulsant-induced hypersensitivity

Background

Delayed hypersensitivity is responsible for severe cutaneous adverse drug reactions (cADRs), especially in Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis, and drug-induced hypersensitivity syndrome (DIHS) (also known as drug rash with eosinophilia and systemic symptoms [DRESS] syndrome). The drug-induced lymphocyte stimulation test (DLST), or lymphocyte transformation test (LTT), is used to identify the culprit drug in severe cADR cases.

Objective

The aim of this study was to examine the immune reactions in cADR patients through the identification of the drug-specific proliferating cells by flow cytometric DLST (FCM-DLST).

Methods

The peripheral blood mononuclear cells of 16 anticonvulsant-induced cADR patients were investigated by conventional DLST and a FCM-DLST protocol in which CFSE dilution and BrdU incorporation were combined. FCM-DLST allowed for the identification of the drug-specific proliferating cells in six cases. Three of these cases were DIHS cases, whereas there was one case of SJS, one case of maculopapular rash (MP), and one case of erythema multiforme (EM) among the six cases.

Results

In FCM-DLST, drug-specific proliferating T cells were detected as CFSE^low BrdU^high cells. These cells corresponded to the cells incorporating ³H-thymidine in conventional DLST. Although CD4⁺ T-cell proliferation dominated the observed proliferation in most of the cases (in the recovery stage of the three DIHS cases, the MP case, and the EM case), drug-specific CD8⁺ cytotoxic T lymphocytes (CTLs) were detected, especially in the acute stages of the SJS case and one of the DIHS cases. There was a dramatic switch in the predominant drug-specific proliferating T-cell population in the course of one of the cases of DIHS in which CD8⁺ CTLs were predominant initially, whereas CD4⁺ T cells were predominant later. Moreover, drug-specific CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells (Tregs) proliferated during the recovery stage in one DIHS case.

Conclusions

FCM-DLST revealed that the cell proliferation detected by conventional DLST is a heterogeneous proliferation of both CD8⁺ CTLs and CD4⁺ T cells that likely includes Tregs. However, the number of cADR cases in this study was limited, which limits the conclusions that can be drawn from it.     

Bullöse Arzneimittelexantheme

Bullous drug exanthems are clinically characteristic, usually severe cutaneous and mucosal drug hypersensitivity reactions. Commonly, they appear 5-14 days after onset of drug treatment. Therapy of choice is to avoid the culprit drug and systemic administration of glucocorticoids. A key element in the immune pathogenesis of bullous drug exanthems is presumably the activation of cytotoxic CD8(+) T lymphocytes which recognize drug metabolites as nominal antigens. These compounds form spontaneously (e.g. penicillins) or are metabolized by cytochrome P450-dependent enzymes (sulfonamides). The diagnosis of bullous drug exanthems is primarily based on skin tests and in vitro-techniques. Among the skin tests, prick as well as patch tests are important. Patch tests can be also applied at the former skin lesion in fixed drug eruption. In vitro techniques include analysis of drug-specific IgE (only available for anti-penicillin, anti-sulfamethoxazole) and cellular tests with the patients' lymphocytes (lymphocyte transformation test-LTT).     

CD8+ cytolytic T lymphocytes and the skin

Abstract: T lymphocytes show a special affinity for the skin. Although the roles played by the CD4⁺ population of T lymphocytes in immunodermatology were so far actively investigated, much less is known about the roles played in the skin by CD8⁺ cytolytic T lymphocytes (CTL). The activity of CD8⁺ CTL in the immunodermatological context, however, is likely to be most important; the immuno-biology itself of CD8⁺ CTL, moreover, although far from being fully understood, shows intriguing characteristics. Immunophenotype, function and cytokine profile of CD8⁺ CTL are overviewed in the first section of this review. Phenotypically, not only CD8⁺ CTL can be subdivided into CD8⁺ CD28⁺ CD11b⁺ and CD8⁺ CD28^- CD11b⁺ subsets, but also an up-to-now undetected CD8⁺ CD28^- CD11b^- subset does exist. Functionally, not only “cytotoxic” but even “suppressor” subpopulations have been shown to exert cytolytic capabilities indeed, and “suppression” itself may be due to such a lytic capacity. According to cytokine synthesis, CD8⁺ CTL can be split into Tc1 and Tc2 subsets, each able to influence specific patterns of immune responses. The impact of CD8⁺ CTL in immunodermatology, overviewed in the second section of the current review, is crucial. The pathophysiology of inflammatory dermatoses is deeply influenced by the activity of CD8⁺ CTL: e.g., CD8⁺ CTL within psoriateic epidermis are possibly associated to the persistence of psoriatic lesions not undergoing resolution; on the other hand, in late lesions of lichen planus CD8⁺ CTL predominate, thus explaining presumably both the cytolytic attack against keratinocytes and the modulation of the inflammatory reaction up to the final resolution of the lesions; Tc1 cells are decreased in atopic dermatitis, and such a decrease can account both for IgE overproduction and for development of infections. Finally, CD8⁺ CTL can sustain against cutaneous viruses/tumors cytolytic immune responses not only of secondary but even of primary type, i.e. induced by Langerhans cells/dendritic cells either transfected or pulsed with skin virus/tumor-associated antigens, thus allowing the production of vaccines against cutaneous viral/neoplastic diseases.     

Where does the antigen of cutaneous sarcoidosis come from?

Background: The antigen pathway of cutaneous sarcoidosis remains obscure. We have investigated topographic involvement of inflammatory cells and lymphatic vessels. Methods: Eleven cutaneous biopsies from eight patients were studied, along with controls from other granulomatous disorders and various skin lesions. Markers for lymphocytes, dendritic cells (DCs), and lymphatic vessel endothelial cells were detected using immunohistochemistry. Results: S100⁺ and CD1a⁺ immature DCs (Langerhans cells) occurred more frequently within the epidermis, whereas S100⁺, fascin⁺, or CD83⁺ maturing DCs occurred more frequently beneath the epithelium in cutaneous sarcoidosis cases than in controls (e.g. CD83, cutaneous sarcoidosis vs. other granulomatous disorders: r = 0.557, p = 0.011). Fascin⁺ and CD83⁺ mature DCs were often closely attached to CD3⁺ T‐lymphocytes around dermal granulomas. D2‐40⁺ lymphatic vessels were often found surrounding dermal granulomas, especially those located in the deeper dermis, in contrast to fascin⁺ blood vessels. Conclusions: Antigen‐capturing by immature DCs seems to take place initially in the epidermis, followed by maturation of DCs. These mature DCs may present the processed antigen to T‐lymphocytes that cause dermal granulomas either in the interstitium of the upper dermis, or in or around lymphatic vessels of the lower dermis. Environmental antigen could be verified by skin test. Kurata A, Terado Y, Izumi M, Fujioka Y, and Franke FE. Where does the antigen of cutaneous sarcoidosis come from?     

Induction of tumor peptide-specific cytotoxic T cells under serum-free conditions by mature human dendritic cells

Abstract Tumor vaccination strategies using antigen-pulsed dendritic cells (DC) are currently under development. We established an in vitro system using cultured DC from HLA-typed volunteers for the induction of tumor peptide-specific CD8⁺ T cells. The strength and specificity of the resulting CTL responses were investigated. For stimulation of syngeneic CD8⁺ T cells two well-defined DC populations were generated: CD1a⁺ immature DC cultured in the presence of GM-CSF and IL-4 and mature CD83⁺ DC generated by additional stimulation with a cytokine cocktail. Stimulations were performed under serum-free conditions and in the absence of exogenous cytokines. Analysis of T cell responses showed that mature DC, but not immature DC, were able to induce the expansion of syngeneic tumor peptide-specific CD8⁺ T cells. Priming of CD8⁺ T cells with peptide-pulsed mature DC rapidly increased the frequency of antigen-specific T cells (ELISPOT technique). T cells induced by mature DC showed strong antigen-specific cytotoxicity in ⁵¹Cr-release assays whereas no antigen-specific cytotoxicity was detectable in CTL generated by immature DC. These data show that terminally differentiated mature DC are necessary for the induction of tumor antigen-specific CTL responses. Received: 13 January 2000 / Revised: 23 March 2000 / Accepted: 23 March 2000     

T-cell subsets in drug-induced toxic epidermal necrolysis. Possible pathogenic mechanism induced by CD8-positive T cells

A patient with bromisovalum-induced toxic epidermal necrolysis showed pronounced delayed hypersensitivity to bromisovalum by patch testing. Biopsy specimens from the cutaneous lesion and the site of the positive patch test reaction were analyzed and compared immunohistologically. The findings were similar: most of the mononuclear cells disposed along the dermoepidermal junction and migrating into the epidermis were CD8-positive lymphocytes, whereas the dermal inflammatory infiltrates were composed predominantly of CD4-positive lymphocytes. This case showed the potential usefulness of patch testing in evaluating cases of toxic epidermal necrolysis. We believe that delayed hypersensitivity plays a crucial role in the development of drug-induced toxic epidermal necrolysis. Furthermore, potential effector cells with phenotypic characteristics of CD8-positive lymphocytes (suppressor/cytotoxic T cells) seem to represent important mediators of the epidermal damage of the cutaneous lesion in our case.     

Selective generation of CD8+ T-cell clones from the peripheral blood of patients with cutaneous reactions to beta-lactam antibiotics

The presence, phenotype, and functional characteristics of peripheral blood penicillin-specific T lymphocytes in individuals with cutaneous allergic reactions to penicillin were investigated using in vitro long-term culture techniques. Peripheral blood mononuclear cells from two penicillin-allergic patients were stimulated in vitro with penicillin, and T-cell blasts were clonally expanded by limiting dilution. Seven T-cell clones were derived, all of which were CD3⁺ CD4^? CD8⁺ HLA-DR⁺, and produced IL-2 and IFN-γ upon stimulation. T-cell proliferation required the presence of antigen and autologous, but not allogeneic, antigen-presenting cells. In addition to the parent compound, the T-cell clones also developed a proliferative response to penicilloyl, the major metabolite of penicillin. The cloned T-cell lines were found to exhibit marked suppressor activity for Con A mitogenesis. The observed suppressor activity required cell-to-cell contact, as supernatants from these T-cell clones had no comparable inhibitory effect. These findings indicate that there is a predominance of penicillin-specific CD8⁺ T cells in the peripheral blood of individuals sensitized to beta-lactam antibiotics.     

Characterization of the T cell response in allergic contact dermatitis caused by corticosteroids

Background Delayed allergic hypersensitivity reactions have classically been described as type IV reactions, which are caused by T cells; however, the respective roles of CD4⁺ and CD8⁺ cells are yet to be defined. A central role for CD8⁺ cytotoxic T cells as effector cells has been suggested. Objectives To determine the type of T cell involved in corticosteroid allergy. Methods We analysed the kinetics of T cell recruitment and the cytokine production profile in positive patch tests of 27 corticosteroid‐sensitized patients, as compared with control sites and control subjects. Skin biopsies, collected at 8, 24 and 48 hr following drug application, were embedded in paraffin for histological and immunohistological staining, and, in some cases, also deep‐frozen for gene expression analyses. Results CD3⁺ T cells were rapidly recruited in concert with the positivity of the patch test sites. High levels of interleukin (IL)‐4, IL‐5 and, to a lesser extent, interferon‐γ suggested that both Th2 and Th1 cytokines were implicated. IL‐4 was also produced by γδ T cell receptor (TCR) lymphocytes. Conclusions This study showed that, in allergic contact dermatitis caused by corticosteroids, the inflammatory infiltrate is composed of CD3⁺ T cells with a predominant Th2 cytokine profile, among which IL‐4 is also produced by γδ TCR lymphocytes.     

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.     

Phenotypic determination of T-lymphocytes responding to chemotactic stimulation from fMLP,IL-8, human IL-10, and epidermal lymphocyte chemotactic factor

Summary Human T lymphocytes were collected after they had migrated towards N-formyl-methionyl-leukyl-phenylalanine (fMLP), rIL-8, human IL-10 (hIL-10), and epidermal lymphocyte chemotactic factor (ELCF). They were stained for determination of their phenotype by FACS analysis using anti-CD4, -CD8, -CD18, -CD45R0 and OPD4 antibodies. Human IL-10 increased the percentage of CD8⁺ T lymphocytes in the migrating cell population by 152% compared with cells migrating towards the medium and decreased the number of CD4⁺ T lymphocytes by 79%. ELCF increased the number of CD4⁺ T lymphocytes by 18%, and the number of CD45R0⁺ T lymphocytes by 52%, while the number of CD8⁺ T lymphocytes was decreased by 20%. rIL-8 increased the number of CD4⁺ T lymphocytes and decreased the CD8⁺ T lymphocytes. The distribution of the different subpopulations of T lymphocytes was not changed significantly by fMLP. The observed changes in the phenotypes did not occur when incubating T lymphocytes with the chemotaxins. Our observations demonstrate that individual chemotactic factors will attract specific subsets of T lymphocytes. They may help to explain the predominance of memory T lymphocytes (CD4R0⁺, CD4⁺) in allergic contact dermatitis and certain other skin diseases. They also confirm the results of a recent study, that showed hIL-10 to be selectively chemotactic for CD8⁺ T lymphocytes.     

γδ T lymphocytes in oriental cutaneous leishmaniasis: occurrence and variable δ gene expression

It has been suggested that T lymphocytes expressing γδ T-cell receptors could play an important role in defence against some intracellular infectious pathogens. The present study was undertaken to characterize the occurrence and variable δ gene expression of T lymphocytes expressing the γδ T-cell receptor in oriental cutaneous leishmaniasis. Eleven cases of oriental cutaneous leishmaniasis were investigated by immunohistological analysis using an alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. In three cases, we observed an increased percentage of γδ T cells (about 20% of CD3⁺ cells). In these cases γδ T cells generally expressed the Vδ2 segment, and only rarely the Vδ1 gene product. Vδ2⁺ cells were predominantly localized in the dermis, and were virtually absent in the epidermal compartment. The rare γδ T cells observed in the epidermis were almost exclusively Vδ1⁺. This study demonstrates that an increase of γδ T cells may be found in oriental cutaneous leishmaniasis, although it is not a constant feature of the disease. The finding of a preferential expansion of the Vδ2 subset suggests that this subpopulation of yδ T cells might be selectively involved in the recognition of Leishmania antigens. The distinct compartmentalization of yδ T-cell subpopulations indicates that these subsets may recognize distinct sets of antigens.     

Expression of IFNγ, coexpression of TNFα and matrix metalloproteinases and apoptosis of T lymphocytes and macrophages in granuloma annulare

Abstract Granuloma annulare, a prototype noninfectious granulomatous dermatitis, is morphologically characterized by a necrobiotic core surrounded by a cellular infiltrate. Because of many morphological similarities to tuberculosis, granuloma annulare has been suggested to represent a delayed-type hypersensitivity (Th1) reaction in the course of which inflammatory cells elicit matrix degradation. In the present study we (1) investigated the expression of interferon-á as the most important Th1-associated cytokine, (2) sought in situ evidence for the coexpression of the proinflammatory cytokine tumor necrosis factor-· and cytokine-regulated matrix metalloproteinases 2 (gelatinase A) and 9 (gelatinase B), and (3) sought to determine whether shrunken cells seen within necrobiotic areas of granuloma annulare are apoptotic cells. In situ hybridization combined with immunofluorescence showed that large numbers of infiltrating CD3⁺ lymphocytes express interferon-á. Application of catalyzed signal amplification in immunodetection revealed that the vast majority of CD3⁺ lymphocytes and CD68⁺ macrophages contained tumor necrosis factor-·. Immunohistochemistry demonstrated that macrophages producing tumor necrosis factor-· coexpress matrix metalloproteinases 2 and 9. In situ end-labeling combined with immunofluorescence detected few apoptotic T cells in perivascular regions and numerous apoptotic macrophages within necrobiotic areas. These results suggest that in granuloma annulare interferon-á⁺ Th-1 lymphocytes may cause a delayed-type hypersensitivity reaction whereby macrophages are differentiated to aggressive effector cells expressing tumor necrosis factor-α and matrix metalloproteinases. In parallel, activation-induced apoptosis in lymphocytes and macrophages may serve to restrict the destructive potential of the inflammatory cells. Received: 23 November 1999 / Received after revision: 28 March 2000 / Accepted: 14 April 2000     

Characterization of human skin-derived CD1a-positive lymph cells

Abstract The phenotype and function of CD1a⁺ lymph cells is of considerable interest. By means of microsurgical lymph cannulation human lymph derived from normal skin was sampled. Cells were isolated and processed for immunocytochemistry, electron microscopy, flow cytometry and functional assays. The majority of the cells, (62%), were T cells. The other cells comprised CD1a⁺ cells (7%), monocytes/macrophages (8%), and B cells (1%); the remainder were erythrocytes or uncharacterized cells. The CD1a⁺ cells reacted with antibodies against protein S-100, HLA-DR, the Lag antigen, CD4, CD11a, CD11b, CD18, CD25, CD40, CD54, CD80 and CD86. Interestingly, a small prolow portion the of CD1a⁺ cells (about 5%) reacted with an antibody to CD14. The CD1a⁺ cells did not react with an antibody against human follicular dendritic cells nor were they CD19-, CD23-, E-cadherin- or factor XIIIa-positive. Both allogenic and antigen-specific T cell proliferation stimulated by antigen-presenting lymph cells were strongly inhibited by adding anti-CD80 and anti-CD86 antibodies. By electron microscopy Birbeck granules were detected in only 22% of the CD1a⁺ lymph cells and these cells exhibited an extensive ruffling of the surface. These findings demonstrate that CD1a⁺ lymph cells, which do not express the dermal dendritic cell marker factor XIIIa, resemble dendritic cells formerly designated as ‘veiled’ as well as lymphoid dendritic cells, suggesting that after migration to the regional lymphoid organs, Langerhans cells form a more differentiated population of dendritic cells specialized in sensitizing T lymphocytes. Our results add further support to the view that resident Langerhans cells may be precursors of lymphoid dendritic cells acquiring the final phenotype in the microenvironment of the lymph node. Received: 30 July 1998 / Received after revision: 30 September 1998 / Accepted: 19 October 1998     

Multiple cutaneous reticulohistiocytosis with T‐cell large granular lymphocyte clonopathy

A 63‐year‐old Caucasian man presented with a 4‐month history of disseminated asymptomatic reddish‐brown papulonodular lesions. A skin biopsy showed dermal infiltration with CD68⁺ histiocytes, predominantly with eosinophilic cytoplasm, some with a ground‐glass cytoplasm, and a small number of giant cells. The diagnosis of multiple cutaneous reticulohistiocytosis was made. Bone marrow immunophenotyping due to peripheral blood lymphocytosis revealed the presence of a monoclonal population of CD3⁺, CD8⁺ CD57⁺ large granular lymphocytes. The present case suggests the coexistence of multiple cutaneous reticulohistiocytosis with an underlying disorder.     

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.     

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.     

Characterization of CD45RO+ memory T lymphocytes in keloid disease

Keloid is a type of scar which seems like an overgrown scar, and can even be larger or more visible than the original wound. They are most common in darker skin types. Although the exact causes of keloid remain unknown, we know that the immune system is involved. Memory T lymphocytes are cells that can provide a rapid and highly effective immune response against pathogens (harmful bacteria or viruses) and can recognize a wide variety of antigens (toxins or foreign substances in the body). In this study, the authors sought to investigate the abnormalities of a specific memory T cell called CD3⁺CD45RO⁺ in keloid scars by analyzing blood and tissue samples. The authors, from Shanghai, China, found that most of T lymphocytes in keloid scars are CD3⁺CD45RO⁺ memory T cells, and they describe the ways in which this type differs from another type of memory T cells called FOXP3^‐CD8^‐mT. The authors also identified a profound decrease in the number of cells, called CD4⁺CD25^highFOXP3⁺ regulatory T cells, in patients with multiple keloid scars, and a significant increase of CD8⁺CD103⁺ memory T cells in keloid scars. The authors concluded that there exist distinct abnormalities in CD45RO⁺ memory T‐cell subsets in keloid scars and it may imply that dysregulation of T cell responses may play a role in the development of keloid scarring. Further research is required.     

Linfoma cutáneo primario T CD8+ tipo acral inducido por hipersensibilidad retardada persistente a pendientes de oro

Primary cutaneous CD8⁺ T-cell lymphoma has been included as a provisional entity within the new revised classification of lymphoid neoplasms of the World Health Organization in 2016¹. It was initially described as indolent CD8⁺ lymphoid proliferation of the ear² and a total of 29 cases of such neoplasm have been published in the literature so far. None of them have been linked to delayed contact hypersensitivity reactions. We present a case of acral type primary cutaneous lymphoma T CD8⁺ involving both earlobes clearly related with the prolonged use of gold earrings, confirmed with epicutaneous tests, histopathology, immunohistochemical and molecular studies. Auricular skin lesions were induced again with a provocation test with identical histopathologycal and the same clonality, confirming both the diagnosis of lymphoma and its induction by the antigenic stimulus of gold.