Dendritic cell-expressed common gamma-chain recruits IL-15 for trans-presentation at the murine immunological synapse.

Background: Mutations of the common cytokine receptor gamma chain (γc) cause Severe Combined Immunodeficiency characterized by absent T and NK cell development. Although stem cell therapy restores these lineages, residual immune defects are observed that may result from selective persistence of γc-deficiency in myeloid lineages. However, little is known about the contribution of myeloid-expressed γc to protective immune responses.  Here we examine the importance of γc for myeloid dendritic cell (DC) function. Methods: We utilize a combination of in vitro DC/T-cell co-culture assays and a novel lipid bilayer system mimicking the T cell surface to delineate the role of DC-expressed γc during DC/T-cell interaction. Results: We observed that γc in DC was recruited to the contact interface following MHCII ligation, and promoted IL-15Rα colocalization with engaged MHCII. Unexpectedly, trans-presentation of IL-15 was required for optimal CD4+T cell activation by DC and depended on DC γc expression. Neither recruitment of IL-15Rα nor IL-15 trans-signaling at the DC immune synapse (IS), required γc signaling in DC, suggesting that γc facilitates IL-15 transpresentation through induced intermolecular cis associations or cytoskeletal reorganization following MHCII ligation. Conclusions: These findings show that DC-expressed γc is required for effective antigen-induced CD4+ T cell activation. We reveal a novel mechanism for recruitment of DC IL-15/IL-15Rα complexes to the IS, leading to CD4+ T cell costimulation through localized IL-15 transpresentation that is coordinated with antigen-recognition.

Ultrastructure of Immune Synapses.

The immunological synapse is a critical event for immune response development. The use of planar supported bilayers as surrogate antigen-presenting cells is a useful tool to study this phenomenon. Here we describe electron microscopy methods and approaches to expand our knowledge of the events taking place during the initial phases of T cell activation after antigen recognition at the nanometer scale.

Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse.

The recognition events that mediate adaptive cellular immunity and regulate antibody responses depend on intercellular contacts between T cells and antigen-presenting cells (APCs). T-cell signalling is initiated at these contacts when surface-expressed T-cell receptors (TCRs) recognize peptide fragments (antigens) of pathogens bound to major histocompatibility complex molecules (pMHC) on APCs. This, along with engagement of adhesion receptors, leads to the formation of a specialized junction between T cells and APCs, known as the immunological synapse, which mediates efficient delivery of effector molecules and intercellular signals across the synaptic cleft. T-cell recognition of pMHC and the adhesion ligand intercellular adhesion molecule-1 (ICAM-1) on supported planar bilayers recapitulates the domain organization of the immunological synapse, which is characterized by central accumulation of TCRs, adjacent to a secretory domain, both surrounded by an adhesive ring. Although accumulation of TCRs at the immunological synapse centre correlates with T-cell function, this domain is itself largely devoid of TCR signalling activity, and is characterized by an unexplained immobilization of TCR-pMHC complexes relative to the highly dynamic immunological synapse periphery. Here we show that centrally accumulated TCRs are located on the surface of extracellular microvesicles that bud at the immunological synapse centre. Tumour susceptibility gene 101 (TSG101) sorts TCRs for inclusion in microvesicles, whereas vacuolar protein sorting 4 (VPS4) mediates scission of microvesicles from the T-cell plasma membrane. The human immunodeficiency virus polyprotein Gag co-opts this process for budding of virus-like particles. B cells bearing cognate pMHC receive TCRs from T cells and initiate intracellular signals in response to isolated synaptic microvesicles. We conclude that the immunological synapse orchestrates TCR sorting and release in extracellular microvesicles. These microvesicles deliver transcellular signals across antigen-dependent synapses by engaging cognate pMHC on APCs.

Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse.

The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity.

Monocyte subsets differentially employ CCR2, CCR5, and CX3CR1 to accumulate within atherosclerotic plaques.

Monocytes participate critically in atherosclerosis. There are 2 major subsets expressing different chemokine receptor patterns: CCR2(+)CX3CR1(+)Ly-6C(hi) and CCR2(-)CX3CR1(++)Ly-6C(lo) monocytes. Both C-C motif chemokine receptor 2 (CCR2) and C-X(3)-C motif chemokine receptor 1 (CX3CR1) are linked to progression of atherosclerotic plaques. Here, we analyzed mouse monocyte subsets in apoE-deficient mice and traced their differentiation and chemokine receptor usage as they accumulated within atherosclerotic plaques. Blood monocyte counts were elevated in apoE(-/-) mice and skewed toward an increased frequency of CCR2(+)Ly-6C(hi) monocytes in apoE(-/-) mice fed a high-fat diet. CCR2(+)Ly-6C(hi) monocytes efficiently accumulated in plaques, whereas CCR2(-)Ly-6C(lo) monocytes entered less frequently but were more prone to developing into plaque cells expressing the dendritic cell-associated marker CD11c, indicating that phagocyte heterogeneity in plaques is linked to distinct types of entering monocytes. CCR2(-) monocytes did not rely on CX3CR1 to enter plaques. Instead, they were partially dependent upon CCR5, which they selectively upregulated in apoE(-/-) mice. By comparison, CCR2(+)Ly-6C(hi) monocytes unexpectedly required CX3CR1 in addition to CCR2 and CCR5 to accumulate within plaques. In many other inflammatory settings, these monocytes utilize CCR2, but not CX3CR1, for trafficking. Thus, antagonizing CX3CR1 may be effective therapeutically in ameliorating CCR2(+) monocyte recruitment to plaques without impairing their CCR2-dependent responses to inflammation overall.

Modulation of dendritic cell trafficking to and from the airways.

We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX(+) airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C(4) transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.

B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization.

Dendritic cell (DC) migration from the periphery to lymph nodes is regulated by the pattern of genes expressed by DCs themselves and by signals within the surrounding peripheral environment. Here, we report that DC mobilization can also be regulated by signals initiated within the downstream lymph nodes, particularly when lymph nodes enlarge as a consequence of immunization. Lymph node B lymphocytes orchestrate expansion of the lymphatic network within the immunized lymph node. This expanded network in turn supports increased DC migration from the periphery. These results reveal unique relationships between B cells, lymphatic vessels, and migratory DCs. Knowledge that DC migration from the periphery is augmented by B cell-dependent signals reveals new potential strategies to increase DC migration during vaccination.

Lymph node occupancy is required for the peripheral development of alloantigen-specific Foxp3+ regulatory T cells.

We previously demonstrated that L-selectin (CD62L)-dependent T cell homing to lymph nodes (LN) is required for tolerance induction to alloantigen. To explore the mechanisms of this observation, we analyzed the development and distribution of regulatory T cells (Treg), which play an important protective role against allograft rejection in transplantation tolerance. Alloantigen-specific tolerance was induced using either anti-CD2 plus anti-CD3 mAbs, or anti-CD40L mAbs plus donor-specific transfusion, in fully mismatched (BALB/c donor, C57BL/6 recipient) vascularized cardiac allografts. An expansion of CD4(+)CD25(+)CD62L(high) T cells was observed specifically within the LN of tolerant animals, but not in other anatomic sites or under nontolerizing conditions. These cells exhibited a substantial up-regulation of Foxp3 expression as measured by real-time PCR and by fluorescent immunohistochemistry, and possessed alloantigen-specific suppressor activity. Neither LN nor other lymphoid cells expressed the regulatory phenotype if recipients were treated with anti-CD62L mAbs, which both prevented LN homing and caused early allograft rejection. However, administration of FTY720, a sphingosine 1-phosphate receptor modulator that induces CD62L-independent T cell accumulation in the LNs, restored CD4(+)CD25(+) Treg in the LNs along with graft survival. These data suggest that alloantigen-specific Foxp3(+)CD4(+)CD25(+) Treg develop and are required within the LNs during tolerization, and provide compelling evidence that distinct lymphoid compartments play critical roles in transplantation tolerance.

Role of CCR8 and other chemokine pathways in the migration of monocyte-derived dendritic cells to lymph nodes.

Studying the influence of chemokine receptors (CCRs) on monocyte fate may reveal information about which subpopulations of monocytes convert to dendritic cells (DCs) and the migration pathways that they use. First, we examined whether prominent CCRs on different monocyte subsets, CCR2 or CX3CR1, mediated migration events upstream of the accumulation of monocyte-derived DCs in lymph nodes (LNs). Monocytes were labeled and traced by uptake of latex microspheres in skin. Unexpectedly, neither CCR2 nor CX3CR1 were required. However, absence of CCR2 led to an increased labeling of the minor Gr-1int monocyte population, and the number of latex+ DCs that emigrated to LNs was correspondingly increased. Characterization of Gr-1int monocytes revealed that they selectively expressed CCR7 and CCR8 mRNA in blood. CCR7 and CCR8 pathways were used by monocyte-derived DCs during mobilization from skin to LNs. The role of CCR8 in emigration from tissues also applied to human monocyte-derived cells in a model of transendothelial trafficking. Collectively, the data suggest that Gr-1int monocytes may be most disposed to become a lymphatic-migrating DCs. When these monocyte-derived DCs exit skin to emigrate to LNs, they use not only CCR7 but also CCR8, which was not previously recognized to participate in migration to LNs.

Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobilization.

High LDL and/or low HDL are risk factors for atherosclerosis and are also a common clinical feature in systemic lupus erythematosus, rheumatoid arthritis, and psoriasis. Here, we show that changes in lipid profiles that reflect atherosclerotic disease led to activation of skin murine dendritic cells (DCs) locally, promoted dermal inflammation, and induced lymph node hypertrophy. Paradoxically, DC migration to lymph nodes was impaired, suppressing immunologic priming. Impaired migration resulted from inhibitory signals generated by platelet-activating factor (PAF) or oxidized LDL that acts as a PAF mimetic. Normal DC migration and priming was restored by HDL or HDL-associated PAF acetylhydrolase (PAFAH), which mediates inactivation of PAF and oxidized LDL. Thus, atherosclerotic changes can sequester activated DCs in the periphery where they may aggravate local inflammation even as they poorly carry out functions that require their migration to lymph nodes. In this context, HDL and PAFAH maintain a normally functional DC compartment.

Emigration of monocyte-derived cells from atherosclerotic lesions characterizes regressive, but not progressive, plaques.

Some monocytes normally take up residence in tissues as sessile macrophages, but others differentiate into migratory cells resembling dendritic cells that emigrate to lymph nodes. In an in vitro model of a vessel wall, lipid mediators lysophosphatidic acid and platelet-activating factor, whose signals are implicated in promoting atherosclerosis, blocked conversion of monocytes into migratory cells and favored their retention in the subendothelium. In vivo studies revealed trafficking of monocyte-derived cells from atherosclerotic plaques during lesion regression, but little emigration was detected from progressive plaques. Thus, progression of atherosclerotic plaques may result not only from robust monocyte recruitment into arterial walls but also from reduced emigration of these cells from lesions.