To the Editor: Atopic dermatitis (AD) is an inflammatory, relapsing skin disorder that affects 15% to 25% of children worldwide and persists in adulthood in around 25% of these cases.1 About 3% to 8% ...of patients with AD seem to have a disturbance in viral clearance, manifesting severe forms of molluscum contagiosum, papilloma virus, and most prominently, the generalized cutaneous infection with herpes simplex virus 1 (HSV1), called eczema herpeticum (EH),2,E1,E2 which can lead to life-threatening complications.E3 The occurrence of EH is associated with a more severe AD disease according to disease scoring systems, total serum IgE, polyallergen sensitization, and other measures.3,4 In addition, not only skin-affecting viral diseases but also respiratory tract infections, such as influenza, are more prevalent in patients with AD.5,E4 Several studies point to a defect in the antiviral IFN-γ response and the skin barrier in patients with AD with a history of EH (ADEH+).6-8 In this study, we aimed to characterize virus-specific T-cell responses in patients suffering from AD with or without history of EH applying state-of-the-art detection systems, namely, measurements of IL-4 and IFN-γ cytokine expression in stimulated T-cell lines (TCLs), direct ex vivo enrichment and characterization of virus-specific CD4+ and CD8+ T cells via CD154 (CD40L) or CD137 (4-1BB), respectively, as well as specific detection of virus-specific CD8+ T cells with MHC class I tetramers in combination with intracellular cytokine staining. The significantly reduced IFN-γ production by TCLs from patients with AD grown in the presence of HSV1 protein glycoprotein D (gD), HSV1 immunodominant peptides, or the immunodominant peptides from influenza hemagglutinin was more pronounced in ADEH+ subjects than in patients with AD without a history of EH. The observed type 2-skewed phenotype leads to presumably inappropriate cytokine responses and eventually ineffective expansion.E7 Noteworthy, these observed effects were not directly related to a more severe AD disease burden, an increased atopic predisposition, or higher age because the 3 groups were matched for age, total IgE, severity of AD (SCORing Atopic Dermatitis), and Sx1 positivity (see Fig E3 in this article's Online Repository at www.jacionline.org). 1 T. Werfel, J.P. Allam, T. Biedermann, K. Eyerich, S. Gilles, E. Guttman-Yassky, Cellular and molecular immunologic mechanisms in patients with atopic dermatitis, J Allergy Clin Immunol, Vol. 138, 2016, 336-349 2 L. Bin, M.G. Edwards, R. Heiser, J.E. Streib, B. Richers, C.F. Hall, Identification of novel gene signatures in patients with atopic dermatitis complicated by eczema herpeticum, J Allergy Clin Immunol, Vol. 134, 2014, 848-855 3 A. Wollenberg, S. Wetzel, W.H. Burgdorf, J. Haas, Viral infections in atopic dermatitis: pathogenic aspects and clinical management, J Allergy Clin Immunol, Vol. 112, 2003, 667-674 4 L.A. Beck, M. Boguniewicz, T. Hata, L.C. Schneider, J. Hanifin, R. Gallo, Phenotype of atopic dermatitis subjects with a history of eczema herpeticum, J Allergy Clin Immunol, Vol. 124, 2009, 260-269.e7 5 J.I. Silverberg, N.B. Silverberg, Childhood atopic dermatitis and warts are associated with increased risk of infection: a US population-based study, J Allergy Clin Immunol, Vol. 133, 2014, 1041-1047 6 R.A. Mathias, A. Weinberg, M. Boguniewicz, D.J. Zaccaro, B. Armstrong, L.C. Schneider, Br J Dermatol, Vol. 169, 2013, 700-703 7 P.S. Gao, N.M. Rafaels, T. Hand, T. Murray, M. Boguniewicz, T. Hata, Filaggrin mutations that confer risk of atopic dermatitis confer greater risk for eczema herpeticum, J Allergy Clin Immunol, Vol. 124, 2009, 507-513, 513.e1-7 8 A. De Benedetto, M.K. Slifka, N.M. Rafaels, I.H. Kuo, S.N. Georas, M. Boguniewicz, Reductions in claudin-1 may enhance susceptibility to herpes simplex virus 1 infections in atopic dermatitis, J Allergy Clin Immunol, Vol. 128, 2011, 242-246.e5 9 Y.D. Mahnke, M.H. Beddall, M. Roederer, Cytometry A, Vol. 83, 2013, 439-440
T cells recognize mycobacterial glycolipid (mycolipid) antigens presented by CD1b molecules, but the role of CD4 and CD8 co-receptors in mycolipid recognition is unknown. Here we show CD1b-mycolipid ...tetramers reveal a hierarchy in which circulating T cells expressing CD4 or CD8 co-receptor stain with a higher tetramer mean fluorescence intensity than CD4-CD8- T cells. CD4+ primary T cells transduced with mycolipid-specific T cell receptors bind CD1b-mycolipid tetramer with a higher fluorescence intensity than CD8+ primary T cells. The presence of either CD4 or CD8 also decreases the threshold for interferon-γ secretion. Co-receptor expression increases surface expression of CD3ε, suggesting a mechanism for increased tetramer binding and activation. Targeted transcriptional profiling of mycolipid-specific T cells from individuals with active tuberculosis reveals canonical markers associated with cytotoxicity among CD8+ compared to CD4+ T cells. Thus, expression of co-receptors modulates T cell receptor avidity for mycobacterial lipids, leading to in vivo functional diversity during tuberculosis disease.
Varicella zoster virus (VZV) is a lymphotropic alpha-herpesvirinae subfamily member that produces varicella on primary infection and causes zoster, vascular disease and vision loss upon reactivation ...from latency. VZV-infected peripheral blood mononuclear cells (PBMCs) disseminate virus to distal organs to produce clinical disease. To assess immune evasion strategies elicited by VZV that may contribute to dissemination of infection, human PBMCs and VZV-specific CD8+ T cells (V-CD8+) were mock- or VZV-infected and analyzed for immunoinhibitory protein PD-1, PD-L1, PD-L2, CTLA-4, LAG-3 and TIM-3 expression using flow cytometry. All VZV-infected PBMCs (monocytes, NK, NKT, B cells, CD4+ and CD8+ T cells) and V-CD8+ showed significant elevations in PD-L1 expression compared to uninfected cells. VZV induced PD-L2 expression in B cells and V-CD8+. Only VZV-infected CD8+ T cells, NKT cells and V-CD8+ upregulated PD-1 expression, the immunoinhibitory receptor for PD-L1/PD-L2. VZV induced CTLA-4 expression only in V-CD8+ and no significant changes in LAG-3 or TIM-3 expression were observed in V-CD8+ or PBMC T cells. To test whether PD-L1, PD-L2 or CTLA-4 regulates V-CD8+ effector function, autologous PBMCs were VZV-infected and co-cultured with V-CD8+ cells in the presence of blocking antibodies against PD-L1, PD-L2 or CTLA-4; ELISAs revealed significant elevations in IFNγ only upon blocking of PD-L1. Together, these results identified additional immune cells that are permissive to VZV infection (monocytes, B cells and NKT cells); along with a novel mechanism for inhibiting CD8+ T cell effector function through induction of PD-L1 expression.
Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to ...characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy.
Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402-restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects.
We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402-restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide.
Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients.
Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading ...to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.
Orolabial herpes simplex virus type 1 (HSV-1) infection has a wide spectrum of severity in immunocompetent persons. To study the role of viral genotype and host immunity, we characterized oral HSV-1 ...shedding rates and host cellular response, and genotyped viral strains, in monozygotic (MZ) and dizygotic (DZ) twins.
A total of 29 MZ and 22 DZ HSV-1-seropositive twin pairs were evaluated for oral HSV-1 shedding for 60 days. HSV-1 strains from twins were genotyped as identical or different. CD4+ T-cell responses to HSV-1 proteins were studied.
The median per person oral HSV shedding rate was 9% of days that a swab was obtained (mean, 10.2% of days). A positive correlation between shedding rates was observed within all twin pairs, and in the MZ and DZ twins. In twin subsets with sufficient HSV-1 DNA to genotype, 15 had the same strain and 14 had different strains. Viral shedding rates were correlated for those with the same but not different strains. The median number of HSV-1 open reading frames recognized per person was 16. The agreement in the CD4+ T-cell response to specific HSV-1 open reading frames was greater between MZ twins than between unrelated persons (P = .002).
Viral strain characteristics likely contribute to oral HSV-1 shedding rates.
Antigen-specific T
persist and protect against skin or female reproductive tract (FRT) HSV infection. As the pathogenesis of HSV differs between humans and model organisms, we focus on humans with ...well-characterized recurrent genital HSV-2 infection. Human CD8+ T
persisting at sites of healed human HSV-2 lesions have an activated phenotype but it is unclear if T
can be cultivated
. We recovered HSV-specific T
from genital skin and ectocervix biopsies, obtained after recovery from recurrent genital HSV-2, using
activation by viral antigen. Up to several percent of local T cells were HSV-reactive
CD4 and CD8 T cell lines were up to 50% HSV-2-specific after sorting-based enrichment. CD8 T
displayed HLA-restricted reactivity to specific HSV-2 peptides with high functional avidities. Reactivity to defined peptides persisted locally over several month and was quite subject-specific. CD4 T
derived from biopsies, and from an extended set of cervical cytobrush specimens, also recognized diverse HSV-2 antigens and peptides. Overall we found that HSV-2-specific T
are abundant in the FRT between episodes of recurrent genital herpes and maintain competency for expansion. Mucosal sites are accessible for clinical monitoring during immune interventions such as therapeutic vaccination.
Tissue-resident-memory T cells (TRM) populate the body's barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve ...effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation
T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-
signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-
pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.
Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) ...is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.
Invariant NKT (iNKT) cells in both humans and non-human primates are activated by the glycolipid antigen, α-galactosylceramide (α-GalCer). However, the extent to which the molecular mechanisms of ...antigen recognition and in vivo phenotypes of iNKT cells are conserved among primate species has not been determined. Using an evolutionary genetic approach, we found a lack of diversifying selection in CD1 genes over 45 million years of evolution, which stands in stark contrast to the history of the MHC system for presenting peptide antigens to T cells. The invariant T cell receptor (TCR)-α chain was strictly conserved across all seven primate clades. Invariant NKT cells from rhesus macaques (
Macaca mulatta
) bind human CD1D-α-GalCer tetramer and are activated by α-GalCer-loaded human CD1D transfectants. The dominant TCR-β chain cloned from a rhesus-derived iNKT cell line is nearly identical to that found in the human iNKT TCR, and transduction of the rhesus iNKT TCR into human Jurkat cells show that it is sufficient for binding human CD1D-α-GalCer tetramer. Finally, we used a 20-color flow cytometry panel to probe tissue phenotypes of iNKT cells in a cohort of rhesus macaques. We discovered several tissue-resident iNKT populations that have not been previously described in non-human primates but are known in humans, such as TCR-γδ iNKTs. These data reveal a diversity of iNKT cell phenotypes despite convergent evolution of the genes required for lipid antigen presentation and recognition in humans and non-human primates.
PDF
