Role of regulatory T cells in psoriasis pathogenesis and treatment.

Psoriasis is a chronic inflammatory disease with a strong genetic component that can be triggered by environmental factors. Disease pathogenesis is mainly driven by type 1 and type 17 cytokine-producing cells which, in healthy individuals, are modulated by regulatory T cells (Tregs). Tregs play a fundamental role in immune homeostasis and contribute to the prevention of autoimmune disease by suppressing immune responses. In psoriasis, Tregs are impaired in their suppressive function leading to an altered T-helper 17/Treg balance. Although Treg dysfunction in patients with psoriasis is associated with disease exacerbation, it is unknown how they are functionally regulated. In this review, we discuss recent insights into Tregs in the setting of psoriasis with an emphasis on the effect of current treatments on Tregs and how already available therapeutics that modulate Treg frequency or functionality could be exploited for treatment of psoriasis.

Emerging roles of innate lymphoid cells in inflammatory diseases: Clinical implications.

Innate lymphoid cells (ILC) represent a group of lymphocytes that lack specific antigen receptors and are relatively rare as compared to adaptive lymphocytes. ILCs play important roles in allergic and nonallergic inflammatory diseases due to their location at barrier surfaces within the airways, gut, and skin, and they respond to cytokines produced by activated cells in their local environment. Innate lymphoid cells contribute to the immune response by the release of cytokines and other mediators, forming a link between innate and adaptive immunity. In recent years, these cells have been extensively characterized and their role in animal models of disease has been investigated. Data to translate the relevance of ILCs in human pathology, and the potential role of ILCs in diagnosis, as biomarkers and/or as future treatment targets are also emerging. This review, produced by a task force of the Immunology Section of the European Academy of Allergy and Clinical Immunology (EAACI), encompassing clinicians and researchers, highlights the role of ILCs in human allergic and nonallergic diseases in the airways, gastrointestinal tract, and skin, with a focus on new insights into clinical implications, therapeutic options, and future research opportunities.

Dengue NS1 antigen contributes to disease severity by inducing interleukin (IL)-10 by monocytes.

Both dengue NS1 antigen and serum interleukin (IL)-10 levels have been shown to associate with severe clinical disease in acute dengue infection, and IL-10 has also been shown to suppress dengue-specific T cell responses. Therefore, we proceeded to investigate the mechanisms by which dengue NS1 contributes to disease pathogenesis and if it is associated with altered IL-10 production. Serum IL-10 and dengue NS1 antigen levels were assessed serially in 36 adult Sri Lankan individuals with acute dengue infection. We found that the serum IL-10 levels correlated positively with dengue NS1 antigen levels (Spearman's r = 0·47, P < 0·0001), and NS1 also correlated with annexin V expression by T cells in acute dengue (Spearman's r = 0·63, P = 0·001). However, NS1 levels did not associate with the functionality of T cell responses or with expression of co-stimulatory molecules. Therefore, we further assessed the effect of dengue NS1 on monocytes and T cells by co-culturing primary monocytes and peripheral blood mononuclear cells (PBMC), with varying concentrations of NS1 for up to 96 h. Monocytes co-cultured with NS1 produced high levels of IL-10, with the highest levels seen at 24 h, and then declined gradually. Therefore, our data show that dengue NS1 appears to contribute to pathogenesis of dengue infection by inducing IL-10 production by monocytes.

Expansion of highly activated invariant natural killer T cells with altered phenotype in acute dengue infection.

Invariant natural killer T (iNKT) cells are capable of rapid activation and production of cytokines upon recognition of antigenic lipids presented by CD1d molecules. They have been shown to play a significant role in many viral infections and were observed to be highly activated in patients with acute dengue infection. In order to characterize further their role in dengue infection, we investigated the proportion of iNKT cells and their phenotype in adult patients with acute dengue infection. The functionality of iNKT cells in patients was investigated by both interferon (IFN)-γ and interleukin (IL)-4 ex-vivo enzyme-linked immunospot (ELISPOT) assays following stimulation with alpha-galactosyl-ceramide (αGalCer). We found that circulating iNKT cell proportions were significantly higher (P = 0·03) in patients with acute dengue when compared to healthy individuals and were predominantly of the CD4(+) subset. iNKT cells of patients with acute dengue had reduced proportions expressing CD8α and CD161 when compared to healthy individuals. The iNKT cells of patients were highly activated and iNKT activation correlated significantly with dengue virus-specific immunoglobulin (Ig)G antibody levels. iNKT cells expressing Bcl-6 (P = 0·0003) and both Bcl-6 and inducible T cell co-stimulator (ICOS) (P = 0·006) were increased significantly in patients when compared to healthy individuals. Therefore, our data suggest that in acute dengue infection there is an expansion of highly activated CD4(+) iNKT cells, with reduced expression of CD161 markers.

The histamine-synthesizing enzyme histidine decarboxylase is upregulated by keratinocytes in atopic skin.

BACKGROUND: Histamine is an abundant mediator accumulating in the skin of atopic patients, where it is thought to be derived from immune cells. While keratinocytes express histidine decarboxylase (HDC), levels of the enzyme in normal or diseased epidermis and factors that influence its expression in human keratinocytes are not known. OBJECTIVES: To assess levels of HDC in inflammatory skin diseases and factors influencing its expression. METHODS: Normal and filaggrin-insufficient human keratinocytes, organotypic epidermal models and skin samples were investigated for the expression of HDC. The effect of cytokines, bacterial and allergen stimuli exposure and functional changes in differentiation were evaluated in vitro. RESULTS: We detected abundant expression of the HDC protein in all models studied; expression was increased in atopic skin samples. Filaggrin-insufficient keratinocytes maintained HDC levels, but exposure of keratinocytes to thymic stromal lymphopoietin, tumour necrosis factor-α, lipopolysaccharide (LPS) and house dust mite (HDM) extract increased HDC expression in vitro. Furthermore, filaggrin expression in cultured keratinocytes increased following histamine depletion. CONCLUSIONS: Keratinocytes express abundant HDC protein, and the levels increase in atopic skin. LPS, HDM and cytokines, which are implicated in allergic inflammation, promote the expression of the enzyme and upregulate histamine levels in keratinocytes. Actively produced histamine influences keratinocyte differentiation, suggesting functional relevance of the axis to atopic dermatitis. The findings therefore identify a new point of therapeutic intervention.

Histamine exerts multiple effects on expression of genes associated with epidermal barrier function.

BACKGROUND: The role of epidermal barrier genes in the pathogenesis of atopic skin inflammation has recently been highlighted. Cytokines that are abundant in the skin during inflammation have been shown to exert various effects on the expression of barrier genes, although the role of histamine in this area of skin biology is not yet fully understood. OBJECTIVE: To assess the effect of stimulation with histamine on keratinocytes by analysis of the pathways involved in epidermal barrier integrity. MATERIAL AND METHODS: We performed a gene expression analysis of histamine-stimulated keratinocytes. Functional changes were tested using the dye penetration assay. Differential changes in filaggrin and the filaggrin-processing enzyme bleomycin hydrolase (BLMH) were validated at the protein level, and expression was also assessed in filaggrin knock-down keratinocytes. RESULTS: Histamine altered expression of multiple barrier genes. Expression of filaggrin was downregulated, as was that of other markers, thus suggesting the presence of delayed/aberrant keratinocyte differentiation. Expression of genes involved in cellular adhesiveness and genes of protease expression was dysregulated, but expression of protease inhibitors was increased. BLMH was upregulated in keratinocytes subjected to histamine and filaggrin knockdown. CONCLUSIONS: Histamine exerts a dual effect on epidermal barrier genes; it suppresses keratinocyte differentiation and dysregulates genes of cellular adhesiveness, although it induces genes contributing to stratum corneum function. Upregulation of BLMH and protease inhibitors could support maintenance of the permeability barrier by enhanced generation of moisturizing compounds and suppressed desquamation. In contrast, in the case of stratum corneum damage, histamine could enhance transcutaneous sensitization.

Histamine enhances keratinocyte-mediated resolution of inflammation by promoting wound healing and response to infection.

BACKGROUND: The role of the epidermis in the immune response is well known. While multiple cytokines are implicated in keratinocyte-mediated infection clearance and wound healing, little is known about the involvement of keratinocytes in promoting resolution of inflammation. AIM: To assess effects of histamine stimulation on keratinocyte function. METHODS: We performed a combined microarray/Gene Ontology analysis of histamine-stimulated keratinocytes. Functional changes were tested by apoptosis assessment and scratch assays. Histamine receptor involvement was also assessed by blocking wound closure with specific antagonists. RESULTS: Histamine treatment had extensive effects on keratinocytes, including effects on proinflammatory responses and cellular functions promoting wound healing. At the functional level, there was reduced apoptosis and enhancement of wound healing in vitro. At the receptor level, we identified involvement of all keratinocyte-expressed histamine receptors (HRHs), with HRH1 blockage resulting in the most prominent effect. CONCLUSIONS: Histamine activates wound healing and infection clearance-related functions of keratinocytes. While enhancement of histamine-mediated wound healing is mediated predominantly via the HRH1 receptor, other keratinocyte-expressed receptors are also involved. These effects could promote resolution of skin inflammation caused by infection or superficial injury.

Cytokine regulation of the epidermal barrier.

Studies published in recent years have highlighted the role of epidermal barrier defects in both atopic skin disease and the development of broader allergic manifestations. While genetic determinants of barrier function are important, it is clear that local acquired effects are also involved in disease pathogenesis. In this review, we aimed to summarize the known influences of cytokines abundantly expressed during atopic skin disease on components of epidermal barrier integrity and function.

Identification of an immunodominant region of the major house dust mite allergen Der p 2 presented by common human leucocyte antigen alleles.

BACKGROUND: Better understanding of the relevance of the immune response to common environmental allergens, such as the major house dust mite (HDM) allergen Der p 2, requires characterization of constituent T-cell epitopes. AIM: To identify CD4(+) T-cell epitopes within Der p 2 recognized by commonly expressed human leucocyte antigen (HLA) alleles. METHODS: HLA-blocking antibodies, peptide pools and truncations were used in ELISpot assays to establish restricted T-cell epitopes. RESULTS: People with and without atopic dermatitis have detectable Der p 2-specific T cells in the peripheral blood, which can proliferate in response to Der p 2 peptides. Interleukin-4-specific responses, both ex vivo and cultured to Der p 2 peptides, had a significant positive correlation with HDM-specific serum IgE. Within one pool of Der p 2 peptides, the 20mer D11 was found to induce multiple responses restricted through several alleles, including HLA-DPB1*0401 and HLA-DRB1*01. CONCLUSIONS: We have identified an immunogenic region of Der p 2 presented by common HLA class II alleles, including the most commonly expressed HLA allele DPB1*0401. Identification of such epitopes may be of future value in peptide immunotherapeutic approaches.

Human antimicrobial peptides LL-37 and human ß-defensin-2 reduce viral replication in keratinocytes infected with varicella zoster virus.

BACKGROUND: There is mounting evidence that antimicrobial peptides have an important role in cutaneous defence, but the expression of these antimicrobial peptides in atopic eczema (AE) is still unclear. There are several families of antimicrobial peptides, including cathelicidins and human ß-defensins. Patients with AE are more susceptible to severe cutaneous viral infections, including varicella zoster virus (VZV). AIM: To characterize the functional activity of the antimicrobial peptides LL-37 (human cathelicidin) and human ß-defensin (hBD)-2 keratinocytes were infected with VZV, in a skin-infection model. METHODS: Flow-cytometry analysis was used to investigate LL-37 expression in normal human keratinocytes, and quantitative PCR was used to determine viral loads in infected HaCaT keratinocytes and B cells, with and without exogenous LL-37 and hBD-2. RESULTS: LL-37 expression was present in keratinocytes, and both exogenous LL-37 and hBD-2 significantly reduced VZV load in infected keratinocytes and B cells. Specific antibodies blocked the antiviral action exhibited by these antimicrobial peptides. Pre-incubation of VZV with LL-37, but not hBD-2, further reduced VZV load. CONCLUSIONS: Both LL-37 and hBD-2 have an antiviral effect on VZV replication in the keratinocyte HaCaT cell line and in B cells, but their mechanism of action is different. Evidence of the relationship between antimicrobial peptide expression and higher susceptibility to infections in AE skin is still emerging. Developing novel antiviral therapies based on antimicrobial peptides may provide improved treatment options for patients with AE.

Phenotypic analysis of perennial airborne allergen-specific CD4+ T cells in atopic and non-atopic individuals.

BACKGROUND: Accumulating evidence suggests that T cells play an important role in the pathogenesis of atopic dermatitis (AD); yet, little is known of the differentiation status of CD4+ T cells specific for common environmental allergens, such as the major cat allergen, Fel d 1. OBJECTIVE: To determine the frequency, differentiation phenotype and function of circulating Fel d 1-specific CD4+ T cells in adult individuals with severe persistent AD in comparison with healthy controls. METHODS: Using HLA class II tetrameric complexes based on a HLA-DPB1*0401-restricted Fel d 1 epitope, ex vivo and cultured T cell frequency and phenotype were analysed in individuals with AD and healthy controls. Cytokine secretion was measured by ex vivo and cultured IL-4 and IFN-γ ELISpots. RESULTS: Ex vivo Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopics and non-atopics express high levels of CCR7, CD62L, CD27 and CD28, placing the cells largely within the central memory subgroup. However, the functional phenotype was distinct, with greater IL-4 production from the cells derived from atopics, which correlated with disease severity. CONCLUSIONS AND CLINICAL RELEVANCE: Circulating Fel d 1-specific DPB1*0401-restricted CD4+ T cells in both atopic and non-atopic donors maintain a central memory phenotype; however in atopics, the cells had greater Th2 effector function, compatible with a disease model of altered antigen delivery in atopic individuals.

Common filaggrin null alleles are not associated with hymenoptera venom allergy in Europeans.

The association of filaggrin mutations with atopic eczema (atopic dermatitis, AD) is well established and it is thought that filaggrin dysfunction impairs the skin's barrier function allowing allergen penetration and subsequent cutaneous sensitisation and inflammation. However, as most forms of barrier dysfunction are not associated with allergic sensitisation to common allergens, the possibility that filaggrin itself is involved in Th1/Th2 polarisation remains. We tested the hypothesis that allergen delivered to the skin independently of the stratum corneum is not associated with filaggrin mutations. Wasp stings bypass the stratum corneum and deliver antigen to the dermis. We found that European individuals with AD (n = 32) have an increased frequency of the 2 commonest filaggrin null mutations (R501X and 2282del4) compared to those with vespid allergy (n = 56) and healthy controls (n = 30). Thus, filaggrin does not appear to have a downstream effect on the development of allergic disease, and it is indeed filaggrin's role in the epithelial function that is likely to determine the link between filaggrin mutations and allergic sensitisation.

Interleukin-22 downregulates filaggrin expression and affects expression of profilaggrin processing enzymes.

BACKGROUND: The identification of filaggrin mutations has contributed towards our understanding of hereditary factors associated with epidermal dysfunction observed in individuals with atopic eczema (AE). However, factors that predispose to acquired filaggrin modulation are not well understood. Interleukin (IL)-22 is upregulated in lesional AE tissue, but its effects on filaggrin expression and genes associated with epidermal function have not yet been comprehensively addressed. OBJECTIVES: To investigate the effects of IL-22 on expression of filaggrin and genes encoding proteins relevant to epidermal function. METHODS: Microarray analysis was performed on IL-22-stimulated HaCaT keratinocytes. Filaggrin protein level was assessed by an intracellular enzyme-linked immunosorbent assay (ELISA) and Western blot in HaCaT cells and the findings were validated in primary keratinocytes. RESULTS: Exposure to IL-22 cytokine resulted in a downregulation of profilaggrin mRNA expression in HaCaT keratinocytes. The expression of genes involved in enzymatic processing of profilaggrin as well as the generation of natural moisturizing factor was also altered. Furthermore, there was an upregulation of many transcripts encoding proteins of the S100 family. Profilaggrin/filaggrin downregulation was detected by intracellular ELISA and Western blot in HaCaT cells. The relevance to the primary setting was confirmed in primary keratinocytes by Western blot. CONCLUSIONS: IL-22 downregulates profilaggrin/filaggrin expression in keratinocytes at both mRNA and protein levels and affects genes relevant to epidermal function. This novel pathway may have relevance to the pathogenesis and treatment of atopic and other skin disease.

Characteristics of Staphylococcus aureus colonization in patients with atopic dermatitis in Sri Lanka.

BACKGROUND: Colonization of the skin of patients with atopic dermatitis (AD) by Staphylococcus aureus (SA) is associated with more severe disease. AIM: To determine the association of SA colonization patterns and densities in lesional and nonlesional skin in patients with varying severities of AD, and to determine the antibiotic sensitivity patterns of SA isolates from Sri Lanka. METHODS: Skin and nasal swabs collected from 100 patients with AD and 120 controls were used to investigate the presence of SA. Severity of AD was graded using the Nottingham Eczema Severity Score. Colony counts were obtained for skin samples, and antibiotic sensitivity testing was performed in cases positive for SA. RESULTS: Skin colonization was seen in 57 patients (57%) but in only 10 controls (8%). Lesional skin of most patients (52/57; 91%) had SA densities of > 300 colony-forming units/cm(2) . Colonization rates with SA significantly increased with increasing age (Spearman correlation coefficient R = 0.9, P < 0.05) and increasing duration of lesions in patients with AD (Spearman R = 0.87, P < 0.05). Isolates from eight patients (13.5%) were found to be methicillin-resistant S. aureus (MRSA). Only 6 isolates (10%) were susceptible to penicillin and 22 (37%) to erythromycin, while 28 (47%) isolates had erythromycin-induced resistance to clindamycin. CONCLUSIONS: SA colonization rates were significantly associated with increasing age and severity of AD, and particularly with duration of lesions. Patients with severe disease were also more likely to be colonized with SA strains resistant to conventional antibiotics.

High frequency of skin-homing melanocyte-specific cytotoxic T lymphocytes in autoimmune vitiligo.

Vitiligo is an autoimmune condition characterized by loss of epidermal melanocytes. Using tetrameric complexes of human histocompatibility leukocyte antigen (HLA) class I to identify antigen-specific T cells ex vivo, we observed high frequencies of circulating MelanA-specific, A*0201-restricted cytotoxic T lymphocytes (A2-MelanA tetramer+ CTLs) in seven of nine HLA-A*0201-positive individuals with vitiligo. Isolated A2-MelanA tetramer+ CTLs were able to lyse A*0201-matched melanoma cells in vitro and their frequency ex vivo correlated with extent of disease. In contrast, no A2-MelanA tetramer+ CTL could be identified ex vivo in all four A*0201-negative vitiligo patients or five of six A*0201-positive asymptomatic controls. Finally, we observed that the A2-MelanA tetramer+ CTLs isolated from vitiligo patients expressed high levels of the skin homing receptor, cutaneous lymphocyte-associated antigen, which was absent from the CTLs seen in the single A*0201-positive normal control. These data are consistent with a role of skin-homing autoreactive melanocyte-specific CTLs in causing the destruction of melanocytes seen in autoimmune vitiligo. Lack of homing receptors on the surface of autoreactive CTLs could be a mechanism to control peripheral tolerance in vivo.

Oligoclonal expansions of CD8(+) T cells in chronic HIV infection are antigen specific.

Acute HIV infection is associated with a vigorous immune response characterized by the proliferation of selected T cell receptor V beta (BV)-expressing CD8(+) T cells. These 'expansions', which are commonly detected in the peripheral blood, can persist during chronic HIV infection and may result in the dominance of particular clones. Such clonal populations are most consistent with antigen-driven expansions of CD8(+) T cells. However, due to the difficulties in studying antigen-specific T cells in vivo, it has been hard to prove that oligoclonal BV expansions are actually HIV specific. The use of tetrameric major histocompatibility complex-peptide complexes has recently enabled direct visualization of antigen-specific T cells ex vivo but has not provided information on their clonal composition. We have now made use of these tetrameric complexes in conjunction with anti-BV chain-specific monoclonal antibodies and analysis of cytotoxic T lymphocyte lines/clones to show that chronically clonally expanded CD8(+) T cells are HIV specific in vivo.

Ex vivo staining of metastatic lymph nodes by class I major histocompatibility complex tetramers reveals high numbers of antigen-experienced tumor-specific cytolytic T lymphocytes.

Characterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential.

Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus In vivo.

Primary infection with virus can stimulate a vigorous cytotoxic T cell response. The magnitude of the antigen-specific component versus the bystander component of a primary T cell response remains controversial. In this study, we have used tetrameric major histocompatibility complex-peptide complexes to directly visualize antigen-specific cluster of differentration (CD)8+ T cells during the primary immune response to Epstein-Barr virus (EBV) infection in humans. We show that massive expansion of activated, antigen-specific T cells occurs during the primary response to this virus. In one individual, T cells specific for a single EBV epitope comprised 44% of the total CD8+ T cells within peripheral blood. The majority of the antigen-specific cells had an activated/memory phenotype, with expression of human histocompatibility leukocyte antigen (HLA) DR, CD38, and CD45RO, downregulation of CD62 leukocyte (CD62L), and low levels of expression of CD45RA. After recovery from AIM, the frequency of antigen-specific T cells fell in most donors studied, although populations of antigen-specific cells continued to be easily detectable for at least 3 yr.

The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

HIV-specific CD8(+) T cells produce antiviral cytokines but are impaired in cytolytic function.

The use of peptide-human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8(+) T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8(+) T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, macrophage inflammatory protein (MIP)-1beta, and perforin is analyzed by FACS((R)) within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8(+) T cells compared with cytomegalovirus (CMV)-specific CD8(+) T cells in HIV chronic infection. We show that the majority of circulating CD8(+) T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-gamma and MIP-1beta but not TNF-alpha. However, a striking finding is that HIV-specific CD8(+) T cells express significantly lower levels of perforin than CMV-specific CD8(+) T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8(+) T cells are impaired in cytolytic activity.