A model of preferential pairing between epithelial and dendritic cells in thymic antigen transfer.

Medullary thymic epithelial cells (mTECs), which produce and present self-antigens, are essential for the establishment of central tolerance. Since mTEC numbers are limited, their function is complemented by thymic dendritic cells (DCs), which transfer mTEC-produced self-antigens via cooperative antigen transfer (CAT). While CAT is required for effective T cell selection, many aspects remain enigmatic. Given the recently described heterogeneity of mTECs and DCs, it is unclear whether the antigen acquisition from a particular TEC subset is mediated by preferential pairing with a specific subset of DCs. Using several relevant Cre-based mouse models that control for the expression of fluorescent proteins, we have found that, in regards to CAT, each subset of thymic DCs preferentially targets a distinct mTEC subset(s). Importantly, XCR1+-activated DC subset represented the most potent subset in CAT. Interestingly, thymic DCs can also acquire antigens from more than one mTEC, and of these, monocyte-derived dendritic cells (moDCs) were determined to be the most efficient. moDCs also represented the most potent DC subset in the acquisition of antigen from other DCs. These findings suggest a preferential pairing model for the distribution of mTEC-derived antigens among distinct populations of thymic DCs.

Francisella tularensis Outer Membrane Vesicles Participate in the Early Phase of Interaction With Macrophages.

Francisella tularensis is known to release unusually shaped tubular outer membrane vesicles (OMV) containing a number of previously identified virulence factors and immunomodulatory proteins. In this study, we present that OMV isolated from the F. tularensis subsp. holarctica strain FSC200 enter readily into primary bone marrow-derived macrophages (BMDM) and seem to reside in structures resembling late endosomes in the later intervals. The isolated OMV enter BMDM generally via macropinocytosis and clathrin-dependent endocytosis, with a minor role played by lipid raft-dependent endocytosis. OMVs proved to be non-toxic and had no negative impact on the viability of BMDM. Unlike the parent bacterium itself, isolated OMV induced massive and dose-dependent proinflammatory responses in BMDM. Using transmission electron microscopy, we also evaluated OMV release from the bacterial surface during several stages of the interaction of Francisella with BMDM. During adherence and the early phase of the uptake of bacteria, we observed numerous tubular OMV-like protrusions bulging from the bacteria in close proximity to the macrophage plasma membrane. This suggests a possible role of OMV in the entry of bacteria into host cells. On the contrary, the OMV release from the bacterial surface during its cytosolic phase was negligible. We propose that OMV play some role in the extracellular phase of the interaction of Francisella with the host and that they are involved in the entry mechanism of the bacteria into macrophages.

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid Lo phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.

TCR Triggering Induces the Formation of Lck-RACK1-Actinin-1 Multiprotein Network Affecting Lck Redistribution.

The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations. An adaptor protein, receptor for activated C kinase 1 (RACK1), was chosen as a viable option, and its capacity to bind Lck and aid the process of activation-induced redistribution of Lck was assessed. Our microscopic observation showed that T-cell activation induces a rapid, concomitant, and transient co-redistribution of Lck and RACK1 into the forming immunological synapse. Consistent with this observation, the formation of transient RACK1-Lck complexes were detectable in primary CD4+ T-cells with their maximum levels peaking 10 s after TCR-CD4 co-aggregation. Moreover, RACK1 preferentially binds to a pool of kinase active pY394Lck, which co-purifies with high molecular weight cellular fractions. The formation of RACK1-Lck complexes depends on functional SH2 and SH3 domains of Lck and includes several other signaling and cytoskeletal elements that transiently bind the complex. Notably, the F-actin-crosslinking protein, α-actinin-1, binds to RACK1 only in the presence of kinase active Lck suggesting that the formation of RACK1-pY394Lck-α-actinin-1 complex serves as a signal module coupling actin cytoskeleton bundling with productive TCR/CD4 triggering. In addition, the treatment of CD4+ T-cells with nocodazole, which disrupts the microtubular network, also blocked the formation of RACK1-Lck complexes. Importantly, activation-induced Lck redistribution was diminished in primary CD4+ T-cells by an adenoviral-mediated knockdown of RACK1. These results demonstrate that in T cells, RACK1, as an essential component of the multiprotein complex which upon TCR engagement, links the binding of kinase active Lck to elements of the cytoskeletal network and affects the subcellular redistribution of Lck.

Gastrointestinal Autoimmunity Associated With Loss of Central Tolerance to Enteric α-Defensins.

BACKGROUND & AIMS: Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is an autoimmune disorder characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency, but patients also develop intestinal disorders. APECED is an autosomal recessive disorder caused by mutations in the autoimmune regulator (AIRE, which regulates immune tolerance) that allow self-reactive T cells to enter the periphery. Enteric α-defensins are antimicrobial peptides secreted by Paneth cells. Patients with APECED frequently have gastrointestinal symptoms and seroreactivity against secretory granules of Paneth cells. We investigated whether enteric α-defensins are autoantigens in humans and mice with AIRE deficiency. METHODS: We analyzed clinical data, along with serum and stool samples and available duodenal biopsies from 50 patients with APECED collected from multiple centers in Europe. Samples were assessed for expression of defensins and other molecules by quantitative reverse transcription polymerase chain reaction and flow cytometry; levels of antibodies and other proteins were measured by immunohistochemical and immunoblot analyses. Histologic analyses were performed on biopsy samples. We used Aire(-/-) mice as a model of APECED, and studied the effects of transferring immune cells from these mice to athymic mice. RESULTS: Enteric defensins were detected in extraintestinal tissues of patients with APECED, especially in medullary thymic epithelial cells. Some patients with APECED lacked Paneth cells and were seropositive for defensin-specific autoantibodies; the presence of autoantibodies correlated with frequent diarrhea. Aire(-/-) mice developed defensin-specific T cells. Adoptive transfer of these T cells to athymic mice resulted in T-cell infiltration of the gut, loss of Paneth cells, microbial dysbiosis, and the induction of T-helper 17 cell-mediated autoimmune responses resembling those observed in patients with APECED. CONCLUSIONS: In patients with APECED, loss of AIRE appears to cause an autoimmune response against enteric defensins and loss of Paneth cells. Aire(-/-) mice developed defensin-specific T cells that cause intestinal defects similar to those observed in patients with APECED. These findings provide a mechanism by which loss of AIRE-mediated immune tolerance leads to intestinal disorders in patients with APECED.

The pool of preactivated Lck in the initiation of T-cell signaling: a critical re-evaluation of the Lck standby model.

The initiation of T-cell receptor (TCR) signaling, based on the cobinding of TCR and CD4-Lck heterodimer to a peptide-major histocompatibility complex II on antigen presenting cells, represents a classical model of T-cell signaling. What is less clear however, is the mechanism which translates TCR engagement to the phosphorylation of immunoreceptor tyrosine-based activation motifs on CD3 chains and how this event is coupled to the delivery of Lck function. Recently proposed 'standby model of Lck' posits that resting T-cells contain an abundant pool of constitutively active Lck (pY394(Lck)) required for TCR triggering, and this amount, upon TCR engagement, remains constant. Here, we show that although maintenance of the limited pool of pY394(Lck) is necessary for the generation of TCR proximal signals in a time-restricted fashion, the total amount of this pool, ~2%, is much smaller than previously reported (~40%). We provide evidence that this dramatic discrepancy in the content of pY394(Lck)is likely the consequence of spontaneous phosphorylation of Lck that occurred after cell solubilization. Additional discrepancies can be accounted for by the sensitivity of different pY394(Lck)-specific antibodies and the type of detergents used. These data suggest that reagents and conditions used for the quantification of signaling parameters must be carefully validated and interpreted. Thus, the limited size of pY394(Lck) pool in primary T-cells invites a discussion regarding the adjustment of the quantitative parameters of the standby model of Lck and reevaluation of the mechanism by which this pool contributes to the generation of proximal TCR signaling.

Toll-like receptors expressed on embryonic macrophages couple inflammatory signals to iron metabolism during early ontogenesis.

Mammalian TLRs in adult animals serve indispensable functions in establishing innate and adaptive immunity and contributing to the homeostasis of surrounding tissues. However, the expression and function of TLRs during mammalian embryonic development has not been studied so far. Here, we show that CD45(+) CD11b(+) F4/80(+) macrophages from 10.5-day embryo (E10.5) co-express TLRs and CD14. These macrophages, which have the capability to engulf both apoptotic cells and bacteria, secrete a broad spectrum of proinflammatory cytokines and chemokines upon TLR stimulation, demonstrating that their TLRs are functional. Comparative microarray analysis revealed an additional set of genes that were significantly upregulated in E10.5 TLR2(+) CD11b(+) macrophages. This analysis, together with our genetic, microscopic, and biochemical evidence, showed that embryonic phagocytes express protein machinery that is essential for the recycling of cellular iron and that this expression can be regulated by TLR engagement in a MyD88-dependent manner, leading to typical inflammatory M1 responses. These results characterize the utility of TLRs as suitable markers for early embryonic phagocytes as well as molecular triggers of cellular responses, the latter being demonstrated by the involvement of TLRs in an inflammation-mediated regulation of embryonic homeostasis via iron metabolism.

Lck, Membrane Microdomains, and TCR Triggering Machinery: Defining the New Rules of Engagement.

In spite of a comprehensive understanding of the schematics of T cell receptor (TCR) signaling, the mechanisms regulating compartmentalization of signaling molecules, their transient interactions, and rearrangement of membrane structures initiated upon TCR engagement remain an outstanding problem. These gaps in our knowledge are exemplified by recent data demonstrating that TCR triggering is largely dependent on a preactivated pool of Lck concentrated in T cells in a specific type of membrane microdomains. Our current model posits that in resting T cells all critical components of TCR triggering machinery including TCR/CD3, Lck, Fyn, CD45, PAG, and LAT are associated with distinct types of lipid-based microdomains which represent the smallest structural and functional units of membrane confinement able to negatively control enzymatic activities and substrate availability that is required for the initiation of TCR signaling. In addition, the microdomains based segregation spatially limits the interaction of components of TCR triggering machinery prior to the onset of TCR signaling and allows their rapid communication and signal amplification after TCR engagement, via the process of their coalescence. Microdomains mediated compartmentalization thus represents an essential membrane organizing principle in resting T cells. The integration of these structural and functional aspects of signaling into a unified model of TCR triggering will require a deeper understanding of membrane biology, novel interdisciplinary approaches and the generation of specific reagents. We believe that the fully integrated model of TCR signaling must be based on membrane structural network which provides a proper environment for regulatory processes controlling TCR triggering.

Eosinophils from patients with type 1 diabetes mellitus express high level of myeloid alpha-defensins and myeloperoxidase.

Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Recently, small cationic α-defensin molecules have been implicated in the pathogenesis of certain inflammatory and autoimmune diseases. The purpose of this study was to assess the α-defensin expression in patients with T1D and elucidate the cellular source of their production. Our results show that 30% of patients exhibit increased levels of α-defensin mRNAs in their capillary blood. Quantitative RT-PCR performed on FACS-sorted granulocytes identified CD15(dull)/CD14(weak) population as the cellular source of α-defensins. Surprisingly, this granulocyte subpopulation displayed augmentation of α-defensin expression in all T1D patients tested. The determination of cell surface markers, expression of cell-specific genes and confocal microscopy identified CD15(dull)/CD14(weak) cells as eosinophils. The presence of transcriptionally active eosinophils in diabetic patients suggests that eosinophils could be a part of an intricate innate immune cellular network involved in the development of diabetes.

A specific type of membrane microdomains is involved in the maintenance and translocation of kinase active Lck to lipid rafts.

Lck is the principal signal-generating tyrosine kinase of the T cell activation mechanism. We have previously demonstrated that induced Lck activation outside of lipid rafts (LR) results in the rapid translocation of a fraction of Lck to LR. While this translocation predicates the subsequent production of IL-2, the mechanism underpinning this process is unknown. Here, we describe the main attributes of this translocating pool of Lck. Using fractionation of Brij58 lysates, derived from primary naive non-activated CD4(+) T cells, we show that a significant portion of Lck is associated with high molecular weight complexes representing a special type of detergent-resistant membranes (DRMs) of relatively high density and sensitivity to laurylmaltoside, thus called heavy DRMs. TcR/CD4 coaggregation-mediated activation resulted in the redistribution of more than 50% of heavy DRM-associated Lck to LR in a microtubular network-dependent fashion. Remarkably, in non-activated CD4(+) T-cells, only heavy DRM-associated Lck is phosphorylated on its activatory tyrosine 394 and this pool of Lck is found to be membrane confined with CD45 phosphatase. These data are the first to illustrate a lipid microdomain-based mechanism concentrating the preactivated pool of cellular Lck and supporting its high stoichiometry of colocalization with CD45 in CD4(+) T cells. They also provide a new structural framework to assess the mechanism underpinning the compartmentalization of critical signaling elements and regulation of spatio-temporal delivery of Lck function during the T cell proximal signaling.

Lck-dependent Fyn activation requires C terminus-dependent targeting of kinase-active Lck to lipid rafts.

Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active (Y505F)Lck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of (Y505F)Lck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.