Sandra L. King

Direct real-time observation of E- and P-selectin-mediated rolling on cutaneous lymphocyte-associated antigen immobilized on Western blots.

Human memory T cells associated with cutaneous inflammatory responses are characterized by their expression of cutaneous lymphocyte-associated Ag (CLA), a carbohydrate determinant differentially expressed on P-selectin glycoprotein ligand-1 (PSGL-1). Although expression of the CLA epitope on PSGL-1 (CLA+ PSGL-1) by memory T cells is associated with acquisition of E-selectin ligand activity, it is not known whether CLA+ PSGL-1, itself, is a ligand for E-selectin on human T cells or whether other glycoproteins, with or without CLA modification, support E-selectin-dependent rolling in shear flow. To address this issue, we developed a method for real-time analysis of functional adhesive interactions between selectin-bearing cells in shear flow with leukocyte ligands resolved by SDS-PAGE and immobilized on standard Western blots. The results of these studies provide direct evidence that CLA+ PSGL-1 is a functional ligand for both E- and P-selectin, confirm that the P-selectin ligand activity of PSGL-1 is independent of CLA modification, and identify a distinct, non-PSGL-1 E-selectin ligand on CLA-positive human memory T cells.

Ly6G ligation blocks recruitment of neutrophils via a β2-integrin–dependent mechanism

Ly6G is a glycosylphosphatidylinositol (GPI)-anchored protein of unknown function that is commonly targeted to induce experimental neutrophil depletion in mice. In the present study, we found that doses of anti-Ly6G Abs too low to produce sustained neutropenia remained capable of inhibiting experimental arthritis, leaving joint tissues free of infiltrating neutrophils. Thioglycollate-stimulated peritonitis was also attenuated. No alteration in neutrophil apoptosis was observed, implicating impaired recruitment. Indeed, Ly6G ligation abrogated neutrophil migration toward LTB(4) and other chemoattractants in a transwell system. Exploring the basis for this blockade, we identified colocalization of Ly6G and β2-integrins by confocal microscopy and confirmed close association by both coimmunoprecipitation and fluorescence lifetime imaging microscopy. Anti-Ly6G Ab impaired surface expression of β2-integrins in LTB(4)-stimulated neutrophils and mimicked CD11a blockade in inhibiting both ICAM-1 binding and firm adhesion to activated endothelium under flow conditions. Correspondingly, migration of β2-integrin-deficient neutrophils was no longer inhibited by anti-Ly6G. These results demonstrate that experimental targeting of Ly6G has functional effects on the neutrophil population and identify a previously unappreciated role for Ly6G as a modulator of neutrophil migration to sites of inflammation via a β2-integrin-dependent mechanism.

Cdc42 interacting protein 4 (CIP4) is essential for integrin-dependent T-cell trafficking

The F-BAR domain containing protein CIP4 (Cdc42 interacting protein 4) interacts with Cdc42 and WASP/N-WASP and is thought to participate in the assembly of filamentous actin. CIP4−/− mice had normal T- and B-lymphocyte development but impaired T cell-dependent antibody production, IgG antibody affinity maturation, and germinal center (GC) formation, despite an intact CD40L–CD40 axis. CIP4−/− mice also had impaired contact hypersensitivity (CHS) to haptens, and their T cells failed to adoptively transfer CHS. Ovalbumin-activated CD4+ effector T cells from CIP4−/−/OT-II mice migrated poorly to antigen-challenged skin. Activated CIP4−/− T cells exhibited impaired adhesion and polarization on immobilized VCAM-1 and ICAM-1 and defective arrest and transmigration across murine endothelial cell monolayers under shear flow conditions. These results demonstrate an important role for CIP4 in integrin-dependent T cell-dependent antibody responses and GC formation and in integrin-mediated recruitment of effector T cells to cutaneous sites of antigen-driven immune reactions.

A121: In Search of Infectious Triggers of Periodic Fever, Aphthous Stomatitis, Pharyngitis and Adenitis Syndrome

Periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome is the most prevalent pediatric autoinflammatory fever disorder. As there is no known genetic cause or confirmatory test, diagnosis of PFAPA is challenging. Clockwork interval between episodes is a characteristic feature, which aids in diagnosis. The true prevalence and the etiology of PFAPA are not known, but genetic factors leading to immune dysregulation as well as infectious agents have been suggested as causative factors. Oral corticosteroids abort the episodes in majority of patients, and in recent years, tonsillectomy has been shown to be effective in inducing remission. Our center has a significant success rate with tonsillectomy in PFAPA patients. Using unbiased next‐generation sequencing, we investigated the presence of potential infectious agents and gene expression signatures in tonsils from patients with PFAPA, chronic tonsillitis and obstructive sleep apnea (OSA).

Disruption of TNF-α/TNFR1 function in resident skin cells impairs host immune response against cutaneous vaccinia virus infection.

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1−/− mice developed larger primary lesions, numerous satellite lesions and higher skin virus levels after VV scarification. Following their recovery, these TNFR1−/− mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice developed an effective memory immune response. A functional systemic immune response of TNFR1−/− mice was further demonstrated by enhanced production of VV-specific IFNγ and VV-specific CD8+ T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM) reconstitution studies using WT BM in TNFR1−/− host mice, but not TNFR1−/− BM in WT host mice, reproduced the original results seen in TNFR1−/− mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency and that resident skin cells play a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNFα/TNFR1 signaling.

A161: Novel 3‐Dimensional Explant Method Facilitates the Study of Lymphocyte Populations in the Synovium and Reveals a Large Population of Resident Memory T cells in Rheumatoid Arthritis

Traditionally, immunologic memory was thought to be maintained by populations of central (TCM) and effector (TEM) memory lymphocytes that circulate in the blood and lymphatics, only temporarily extravagating into peripheral tissue to execute immunologic responses. Recently, this theory of adaptive memory has been challenged by the discovery of long‐lived and stable populations of tissue‐resident memory T cells (TRM). While TRM have been implicated in the pathogenesis of skin, intestinal, and lung inflammatory diseases, little information is available about TRM in synovium. The study of TRM in arthritis has been impaired by the scarcity of synovium samples coupled with the meager yield of lymphocytes from this tissue using conventional tissue digestion protocols. We have employed novel culturing techniques, previously used to examine TRM in skin, to further characterize TRM in inflammatory arthritis.

IL-1R Type 1–Deficient Mice Demonstrate an Impaired Host Immune Response against Cutaneous Vaccinia Virus Infection

The IL-1 superfamily of cytokines and receptors has been studied extensively. However, the specific roles of IL-1 elements in host immunity to cutaneous viral infection remain elusive. In this study, we applied vaccinia virus (VACV) by scarification to IL-1R1 knockout mice (IL-1R1−/−) and found that these mice developed markedly larger lesions with higher viral genome copies in skin than did wild-type mice. The phenotype of infected IL-1R1−/− mice was similar to eczema vaccinatum, a severe side effect of VACV vaccination that may develop in humans with atopic dermatitis. Interestingly, the impaired cutaneous response of IL-1R1−/− mice did not reflect a systemic immune deficiency, because immunized IL-1R1−/− mice survived subsequent lethal VACV intranasal challenge, or defects of T cell activation or T cell homing to the site of inoculation. Histologic evaluation revealed that VACV infection and replication after scarification were limited to the epidermal layer of wild-type mice, whereas lack of IL-1R1 permitted extension of VACV infection into dermal layers of the skin. We explored the etiology of this discrepancy and determined that IL-1R1−/− mice contained significantly more macrophages and monocyte-derived dendritic cells in the dermis after VACV scarification. These cells were vulnerable to VACV infection and may augment the transmission of virus to adjacent skin, thus leading to larger skin lesions and satellite lesions in IL-1R1−/− mice. These results suggest new therapeutic strategies for treatment of eczema vaccinatum and inform assessment of risks in patients receiving IL-1 blocking Abs for treatment of chronic inflammatory disorders.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.Aberrant glycosylation patterns on cancer cells promote several pro-tumorigenic functions, including enhancing tumor cell proliferation. Here the authors provide data that show melanoma cells downregulate GCNT2 with consequent loss of I-branched glycans; this leads to the formation of extended i-linear glycans and enhances melanoma growth via increases, in part, by IGF-1- and extracellular matrix-induced signaling.