Cezary Ciszewski

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

Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon γ–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.

The role of HLA-DQ8 β57 polymorphism in the anti-gluten T-cell response in coeliac disease

Major histocompatibility complex (MHC) class II alleles HLA-DQ8 and the mouse homologue I-Ag7 lacking a canonical aspartic acid residue at position β57 are associated with coeliac disease and type I diabetes. However, the role of this single polymorphism in disease initiation and progression remains poorly understood. The lack of Asp 57 creates a positively charged P9 pocket, which confers a preference for negatively charged peptides. Gluten lacks such peptides, but tissue transglutaminase (TG2) introduces negatively charged residues at defined positions into gluten T-cell epitopes by deamidating specific glutamine residues on the basis of their spacing to proline residues. The commonly accepted model, proposing that HLA-DQ8 simply favours binding of negatively charged peptides, does not take into account the fact that TG2 requires inflammation for activation and that T-cell responses against native gluten peptides are found, particularly in children. Here we show that β57 polymorphism promotes the recruitment of T-cell receptors bearing a negative signature charge in the complementary determining region 3β (CDR3β) during the response against native gluten peptides presented by HLA-DQ8 in coeliac disease. These T cells showed a crossreactive and heteroclitic (stronger) response to deamidated gluten peptides. Furthermore, gluten peptide deamidation extended the T-cell-receptor repertoire by relieving the requirement for a charged residue in CDR3β. Thus, the lack of a negative charge at position β57 in MHC class II was met by negatively charged residues in the T-cell receptor or in the peptide, the combination of which might explain the role of HLA-DQ8 in amplifying the T-cell response against dietary gluten.

Cytosolic PLA2 is required for CTL-mediated immunopathology of celiac disease via NKG2D and IL-15

IL-15 and NKG2D promote autoimmunity and celiac disease by arming cytotoxic T lymphocytes (CTLs) to cause tissue destruction. However, the downstream signaling events underlying these functional properties remain unclear. Here, we identify cytosolic phospholipase A2 (cPLA2) as a central molecule in NKG2D-mediated cytolysis in CTLs. Furthermore, we report that NKG2D induces, upon recognition of MIC+ target cells, the release of arachidonic acid (AA) by CTLs to promote tissue inflammation in association with target killing. Interestingly, IL-15, which licenses NKG2D-mediated lymphokine killer activity in CTLs, cooperates with NKG2D to induce cPLA2 activation and AA release. Finally, cPLA2 activation in intraepithelial CTLs of celiac patients provides an in vivo pathophysiological dimension to cPLA2 activation in CTLs. These results reveal an unrecognized link between NKG2D and tissue inflammation, which may underlie the emerging role of NKG2D in various immunopathological conditions and define new therapeutic targets.

Distinct and Synergistic Contributions of Epithelial Stress and Adaptive Immunity to Functions of Intraepithelial Killer Cells and Active Celiac Disease

BACKGROUND & AIMS The mechanisms of tissue destruction during progression of celiac disease are poorly defined. It is not clear how tissue stress and adaptive immunity contribute to the activation of intraepithelial cytotoxic T cells and the development of villous atrophy. We analyzed epithelial cells and intraepithelial cytotoxic T cells in family members of patients with celiac disease, who were without any signs of adaptive antigluten immunity, and in potential celiac disease patients, who have antibodies against tissue transglutaminase 2 in the absence of villous atrophy. METHODS We collected blood and intestinal biopsy specimens from 268 patients at tertiary medical centers in the United States and Italy from 2004 to 2012. All subjects had normal small intestinal histology. Study groups included healthy individuals with no family history of celiac disease or antibodies against tissue transglutaminase 2 (controls), healthy family members of patients with celiac disease, and potential celiac disease patients. Intraepithelial cytotoxic T cells were isolated and levels of inhibitory and activating natural killer (NK) cells were measured by flow cytometry. Levels of heat shock protein (HSP) and interleukin 15 were measured by immunohistochemistry, and ultrastructural alterations in intestinal epithelial cells (IECs) were assessed by electron microscopy. RESULTS IECs from subjects with a family history of celiac disease, but not from subjects who already had immunity to gluten, expressed higher levels of HS27, HSP70, and interleukin-15 than controls; their IECs also had ultrastructural alterations. Intraepithelial cytotoxic T cells from relatives of patients with celiac disease expressed higher levels of activating NK receptors than cells from controls, although at lower levels than patients with active celiac disease, and without loss of inhibitory receptors for NK cells. Intraepithelial cytotoxic T cells from potential celiac disease patients failed to up-regulate activating NK receptors. CONCLUSIONS A significant subset of healthy family members of patients with celiac disease with normal intestinal architecture had epithelial alterations, detectable by immunohistochemistry and electron microscopy. The adaptive immune response to gluten appears to act in synergy with epithelial stress to allow intraepithelial cytotoxic T cells to kill epithelial cells and induce villous atrophy in patients with active celiac disease.

NKG2D Initiates Caspase-Mediated CD3ζ Degradation and Lymphocyte Receptor Impairments Associated with Human Cancer and Autoimmune Disease

Deficiencies of the T cell and NK cell CD3ζ signaling adapter protein in patients with cancer and autoimmune diseases are well documented, but mechanistic explanations are fragmentary. The stimulatory NKG2D receptor on T and NK cells mediates tumor immunity but can also promote local and systemic immune suppression in conditions of persistent NKG2D ligand induction that include cancer and certain autoimmune diseases. In this paper, we provide evidence that establishes a causative link between CD3ζ impairment and chronic NKG2D stimulation due to pathological ligand expression. We describe a mechanism whereby NKG2D signaling in human T and NK cells initiates Fas ligand/Fas-mediated caspase-3/-7 activation and resultant CD3ζ degradation. As a consequence, the functional capacities of the TCR, the low-affinity Fc receptor for IgG, and the NKp30 and NKp46 natural cytotoxicity receptors, which all signal through CD3ζ, are impaired. These findings are extended to ex vivo phenotypes of T and NK cells among tumor-infiltrating lymphocytes and in peripheral blood from patients with juvenile-onset lupus. Collectively, these results indicate that pathological NKG2D ligand expression leads to simultaneous impairment of multiple CD3ζ-dependent receptor functions, thus offering an explanation that may be applicable to CD3ζ deficiencies associated with diverse disease conditions.

IL-15 in tumor microenvironment causes rejection of large established tumors by T cells in a noncognate T cell receptor-dependent manner

A major challenge of cancer immunotherapy is the persistence and outgrowth of subpopulations that lose expression of the target antigen. IL-15 is a potent cytokine that can promote organ-specific autoimmunity when up-regulated on tissue cells. Here we report that T cells eradicated 2-wk-old solid tumors that expressed IL-15, eliminating antigen-negative cells. In contrast, control tumors that lacked IL-15 expression consistently relapsed. Interestingly, even tumors lacking expression of cognate antigen were rejected when expressing IL-15, indicating that rejection after adoptive T-cell transfer was independent of cognate antigen expression. Nevertheless, the T-cell receptor of the transferred T cells influenced the outcome, consistent with the notion that T-cell receptor activation and effector status determine whether IL-15 can confer lymphokine killer activity-like properties to T cells. The effect was limited to the microenvironment of tumors expressing IL-15; there were no noticeable effects on contralateral tumors lacking IL-15. Taken together, these results indicate that expression of IL-15 in the tumor microenvironment may prevent the escape of antigen loss variants and subsequent tumor recurrence by enabling T cells to eliminate cancer cells lacking cognate antigen expression in a locally restricted manner.

Interleukin 15 Primes Natural Killer Cells to Kill via NKG2D and cPLA2 and This Pathway Is Active in Psoriatic Arthritis

NK cells are large granular lymphocytes that form a critical component of the innate immune system, whose functions include the killing of cells expressing stress-induced molecules. It is increasingly accepted that despite being considered prototypical effector cells, NK cells require signals to reach their full cytotoxic potential. We previously showed that IL-15 is capable of arming CD8 effector T cells to kill independently of their TCR via NKG2D in a cPLA2-dependent process. As NK cells also express NKG2D, we wanted to investigate whether this pathway functioned in an analogous manner and if resting NK cells could be primed to the effector phase by IL-15. Furthermore, to establish relevance to human disease we studied a possible role for this pathway in the pathogenesis of psoriatic arthritis, since there are aspects of this disease that suggest a potential effector role for the innate immune system. We found that PsA patients had upregulated IL-15 and MIC in their affected synovial tissues, and that this unique inflammatory environment enabled NK cell activation and killing via NKG2D and cPLA2. Moreover, we were able to reproduce the phenotype of joint NK cells from blood NK cells by incubating them with IL-15. Altogether, these findings suggest a destructive role for NK cells when activated by environmental stress signals during the pathogenesis of PsA and demonstrate that IL-15 is capable of priming resting NK cells in tissues to the effector phase.

Human NKG2E Is Expressed and Forms an Intracytoplasmic Complex with CD94 and DAP12

The NKG2 family of NK receptors includes activating and inhibitory members. With the exception of the homodimer-forming NKG2D, NKG2 receptors recognize the nonclassical MHC class I molecule HLA-E, and they can be subdivided into two groups: those that associate with and signal through DAP12 to activate cells, and those that contain an ITIM motif to promote inhibition. The function of NKG2 family member NKG2E is unclear in humans, and its surface expression has never been conclusively established, largely because there is no Ab that binds specifically to NKG2E. Seeking to determine a role for this molecule, we chose to investigate its expression and ability to form complexes with intracellular signaling molecules. We found that NKG2E was capable of associating with CD94 and DAP12 but that the complex was retained intracellularly at the endoplasmic reticulum instead of being expressed on cell surfaces, and that this localization was dependent on a sequence of hydrophobic amino acids in the extracellular domain of NKG2E. Because this particular sequence has emerged and been conserved selectively among higher order primates evolutionarily, this observation raises the intriguing possibility that NKG2E may function as an intracellular protein.

Cysteinyl leukotrienes mediate lymphokine killer activity induced by NKG2D and IL-15 in cytotoxic T cells during celiac disease

Tang et al. show that cytotoxic effector cells produce and respond to cysteinyl leukotrienes to allow target cell killing dependent on NKG2D and IL-15. They further demonstrate a role for cysteinyl leukotrienes in celiac disease pathogenesis.