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Reprogramming of CTLs into natural killer-like cells in celiac disease

ABSTRACT: Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon g–producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases. Keywords: NKG2C; LAK; CTL; NK receptor; IEL; Mucosal Immunity; Celiac Disease

ORGANISM(S): Homo sapiens

PROVIDER: GSE4592 | GEO | 2006/05/15

SECONDARY ACCESSION(S): PRJNA94447

REPOSITORIES: GEO

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Project description:Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon gâproducing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases. Experiment Overall Design: Using a standard GeneChip preparation protocol (Affymetrix, Santa Clara CA), gene array hybridizations and data collection on 5 paired NKG2C+ and NKG2C- IE and PB âCTL samples was preformed at the Functional Genomics Facility, University of Chicago. Briefly, 10ug of total RNA, extracted using the RNeasy Mini kit (Qiagen, Valencia, CA), was used to generate double-stranded cDNA using a T7 linked oligo dT primer and the Superscript II RT system (Invitrogen Carlsbad CA). Purified cDNA was then used to generate biotin-labeled cRNA using the BioArray High Yield RNA Transcript Labeling Kit (Enzo Diagnostics, Farmingdale, NY) according to the manufacturers protocol. This biotinylated-cRNA was then fragmented and hybridized to an Affymetrix GeneChip HG-U133. The arrays were then washed and stained with phycoerythrin-conjugated streptavidin according to the Affymetrix GeneChip protocol and scanned using the Affymetrix Agilent GeneArray Scanner (Affymetrix, Santa Clara CA). Data analyses were preformed using DNA-Chip Analyzer 1.3 (dChip) with the *.CEL files obtained from GeneChip Operating System 1.3 (GCOS 1.3). A PM-only model was used to estimate gene expression level and the invariant set approach was used for normalization. For comparison analyses of the paired NKG2C+ and NKG2C- populations, thresholds for selecting significant genes were set at a relative difference > 1.5 fold, absolute difference > 100 and a significant paired t-test for the signal intensity difference at p < 0.05. Duplicate genes and genes of unknown function were removed. The remaining genes were then grouped based on function and cluster analysis was preformed using dChip.

Project description:Previous research on adaptive NK cells in rhesus macaques suffered from the lack of specific antibodies to differentiate between inhibitory CD94/NKG2A and stimulatory CD94/NKG2C heterodimeric receptors. Recently we reported an expansion of NKG2C receptor-encoding genes in rhesus macaques, but their expression and functional role on primary NK cells remained unknown due to this deficit. Thus, we established monoclonal antibodies 4A8 and 7B1 which show identical specificities and bind to both NKG2C-1 and NKG2C-2 but neither react with NKG2C-3 nor NKG2A on transfected cell lines. Using a combination of 4A8 and Z199 antibodies in multicolor flow cytometry we detected broad expression (4-73%) of NKG2C-1 and/or NKG2C-2 (NKG2C-1/2) on primary NK cells in rhesus macaques from our breeding colony. Stratifying our data to CMV-positive and CMV-negative animals, we noticed a higher proportion (23-73%) in primary NK cells expressing NKG2C-1/2 in CMV+ as compared to CMV- macaques (4-5%). These NKG2C-1/2-positive NK cells in CMV+ macaques are characterized by lower expression of IL12RB2, ZBTB16 and SH2D1B as well as high expression of IFN-gamma, indicating that antibody 4A8 detects CMV-associated adaptive NK cells. Single cell RNA seq data of 4A8-positive NK cells from a CMV-positive individual demonstrated that a high proportion of these adaptive NK cells transcribe in addition to NKG2C-1/2 also NKG2C-3, but interestingly NKG2A as well. Remarkably, NKG2C-1 and in particular NKG2C-2 have a higher affinity to Mamu-E as compared to NKG2A. Primary NK cells exposed to Mamu-E-expressing target cells displayed strong degranulation as well as IFN-gamma expression of 4A8+ adaptive NK cells of rhCMV+ animals that was not evident in rhCMV- animals. Thus, despite co-expression of inhibitory and stimulatory CD94/NKG2 receptors the higher number of different stimulatory NKG2C receptors and their higher binding avidity to Mamu-E outreach inhibitory signaling via NKG2A. These data demonstrate the evolutionary conservation of the CMV-driven development of NKG2C-positive adaptive NK cells with particular molecular signatures in primates and with changes in gene copy numbers and regulation and in ligand binding strength of NKG2C isotypes. Thus, rhesus macaques represent a suitable and valuable nonhuman primate animal model to study the CMV-NKG2C liaison in vivo.