Functional anatomy of T cell activation and synapse formation.

T cell activation and function require a structured engagement of antigen-presenting cells. These cell contacts are characterized by two distinct dynamics in vivo: transient contacts resulting from promigratory junctions called immunological kinapses or prolonged contacts from stable junctions called immunological synapses. Kinapses operate in the steady state to allow referencing to self-peptide-MHC (pMHC) and searching for pathogen-derived pMHC. Synapses are induced by T cell receptor (TCR) interactions with agonist pMHC under specific conditions and correlate with robust immune responses that generate effector and memory T cells. High-resolution imaging has revealed that the synapse is highly coordinated, integrating cell adhesion, TCR recognition of pMHC complexes, and an array of activating and inhibitory ligands to promote or prevent T cell signaling. In this review, we examine the molecular components, geometry, and timing underlying kinapses and synapses. We integrate recent molecular and physiological data to provide a synthesis and suggest ways forward.

Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter.

The germinal center (GC) reaction produces high-affinity antibodies by random mutation and selective clonal expansion of B cells with high-affinity receptors. The mechanism by which B cells are selected remains unclear, as does the role of the two anatomically defined areas of the GC, light zone (LZ) and dark zone (DZ). We combined a transgenic photoactivatable fluorescent protein tracer with multiphoton laser-scanning microscopy and flow cytometry to examine anatomically defined LZ and DZ B cells and GC selection. We find that B cell division is restricted to the DZ, with a net vector of B cell movement from the DZ to the LZ. The decision to return to the DZ and undergo clonal expansion is controlled by T helper cells in the GC LZ, which discern between LZ B cells based on the amount of antigen captured and presented. Thus, T cell help, and not direct competition for antigen, is the limiting factor in GC selection.

Author Correction: The bone marrow microenvironment at single-cell resolution.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The bone marrow microenvironment at single-cell resolution.

The bone marrow microenvironment has a key role in regulating haematopoiesis, but its molecular complexity and response to stress are incompletely understood. Here we map the transcriptional landscape of mouse bone marrow vascular, perivascular and osteoblast cell populations at single-cell resolution, both at homeostasis and under conditions of stress-induced haematopoiesis. This analysis revealed previously unappreciated levels of cellular heterogeneity within the bone marrow niche and resolved cellular sources of pro-haematopoietic growth factors, chemokines and membrane-bound ligands. Our studies demonstrate a considerable transcriptional remodelling of niche elements under stress conditions, including an adipocytic skewing of perivascular cells. Among the stress-induced changes, we observed that vascular Notch delta-like ligands (encoded by Dll1 and Dll4) were downregulated. In the absence of vascular Dll4, haematopoietic stem cells prematurely induced a myeloid transcriptional program. These findings refine our understanding of the cellular architecture of the bone marrow niche, reveal a dynamic and heterogeneous molecular landscape that is highly sensitive to stress and illustrate the utility of single-cell transcriptomic data in evaluating the regulation of haematopoiesis by discrete niche populations.

CD138 mediates selection of mature plasma cells by regulating their survival.

Antibody secreting cells (ASCs) are critical effector cells and long-lived sentinels for immune memory. ASCs are highly dependent on exogenous soluble factors such as interleukin-6 (IL-6) and APRIL, to prevent their cell death. We have found that the canonical surface marker of ASCs, CD138 (syndecan-1), which is upregulated during ASC maturation, is required in a cell-intrinsic manner to mount an effective long-term humoral immune response following immunization. Surface expression of CD138 increased heparan sulfate levels on ASCs, which are known to bind pro-survival cytokines, leading to increased survival in a cell-intrinsic manner in vivo. In IL-6 and APRIL-deficient hosts, ASCs underwent extensive apoptosis independently of CD138 expression. We propose a model in which CD138 expression on fully mature ASCs provides a selective survival advantage over less mature, newly minted ASCs, by enhancing pro-survival cytokine signaling.

Development and migration of plasma cells in the mouse lymph node.

In this study, we imaged the differentiation and migratory behavior of nascent plasma cells (PCs) in mouse lymph nodes by intravital microscopy. Pre-PCs exhibited a unique migration pattern characterized by long, linear paths that were randomly oriented. Although chemotaxis via Galphai coupled-receptors has been implicated in PC migration, treatment with Pertussis toxin (Ptx), which ablates these signals, did not prevent movement of pre-PCs while it arrested other lymphocytes. In vitro, pre-PCs displayed processive amoeboid locomotion on surfaces coated with integrin ligand, whereas fully differentiated PCs moved slowly or were arrested. Both PC arrest and differentiation occurred in the medullary cords. Ptx treatment before PC differentiation blocked their accumulation in the medullary cords but pre-PCs still differentiated in other lymph node regions. Taken together, we suggest pre-PCs undergo a persistent random walk to find the medullary cords, where localized chemokines help retain these cells until they undergo differentiation and arrest in situ.

Myeloid cells limit production of antibody-secreting cells after immunization in the lymph node.

Ab-secreting cell (ASC) expansion and survival are important processes in optimizing vaccines and controlling autoimmunity. The microenvironment of the medullary cords is positioned to control these key processes. Previously, we imaged and characterized ASC differentiation and migration by intravital microscopy in the lymph node (LN) by transferring and activating B cells expressing yellow fluorescent protein only in the ASC compartment. In this study, we observed that yellow fluorescent protein(+) ASCs in the medullary cords migrated along myelomonocytic cells and arrested in contact with them. Acute ablation of myeloid cells using the human diphtheria receptor system (diphtheria toxin receptor [DTR]) expressed in Lysmd1-cre-positive cells increased ASC and Ab production by 2-fold. Increases in ASC numbers were associated with cell proliferation based on Ki-67 staining, rather than reduced apoptosis, or changes in egress from the LN. Using DTR-mediated ablation targeted to Ccr2-expressing myeloid cells also generated increases in ASCs. In contrast, neither the depletion of Gr-1-positive cells with an Ab nor the ablation of cells using a cd11c-DTR resulted in any change in ASCs. IL-6 cytokine signaling can enhance ASC production and has been implicated in dampening ASCs in lupus mouse models through myeloid cells. Using mixed bone marrow chimeras, we observed that IL-6 enhances ASC production, but IL-6 production was not required by myeloid cells to dampen ASCs in the LN. Inhibition of ASCs by these myeloid cells in the LN provides a new regulatory mechanism with implications for tuning Ab responses.

New insights into the ontogeny, diversity, maturation and survival of long-lived plasma cells.

Plasma cells are unique immune effectors, capable of producing large amounts of high-affinity antibodies that protect against pathogenic infections. Although most plasma cells have short lifespans, certain conditions or vaccinations can give rise to long-lived plasma cells (LLPCs) that provide individuals with lifelong protection against pathogen exposure. The nature of these LLPCs is poorly understood; however, recent studies have shed new light on the ontogeny, diversity, maturation and survival of these unique cells. Whereas LLPCs had been thought to arise preferentially from germinal centres, novel genetic tools have revealed that they can originate from various stages throughout the humoral response. Furthermore, new single-cell analyses have shown that mouse and human plasma cells are heterogeneous and may undergo further maturation in situ in the bone marrow niche. Finally, plasma cells were previously considered to be sessile cells maintained in fixed survival niches, but new data show that plasma cell subsets can differentially migrate and organize into clusters that may be associated with survival niches. These descriptive findings provide new insights into how cell-intrinsic programmes and extrinsic factors may regulate the longevity of plasma cells in various contexts, which suggest new research avenues for their functional validation.

Dynamic organization of the bone marrow plasma cell niche.

Prophylactic, serological memory relies on maintaining stable reservoirs of plasma cells, capable of constitutively-secreting high-affinity, anti-pathogen antibody for a lifetime. Although antibody titers generated by some vaccines (e.g. measles) can last a lifetime, other vaccinations (e.g. tetanus) need repeated boosting because long-lived plasma cells are not produced or maintained. Moreover, in old age, the ability to generate long-lived humoral responses diminishes. Despite their importance to health, it is unknown how long-lived plasma cells survive over years, whereas most antibody secreting cells die off within weeks after vaccination. In this review, we focus on how known factors regulate the longevity of plasma cell fitness and survival, and how that landscape is shaped by environmental influences, such as inflammation, infection and aging. In addition, we highlight newly discovered cellular dynamics in the bone marrow that may reframe the mechanisms supporting long-lived plasma cell survival and function.

Germinal center expansion but not plasmablast differentiation is proportional to peptide-MHCII density via CD40-CD40L signaling strength.

T follicular helper (TFH) cells promote expansion of germinal center (GC) B cells and plasma cell differentiation. Whether cognate peptide-MHCII (pMHCII) density instructs selection and cell fate decisions in a quantitative manner remains unclear. Using αDEC205-OVA to differentially deliver OVA peptides to GC B cells on the basis of DEC205 allelic copy number, we find DEC205+/+ B cells take up 2-fold more antigen than DEC205+/- cells, leading to proportional TFH cell help and B cell expansion. To validate these results, we establish a caged OVA peptide, which is readily detected by OVA-specific TFH cells after photo-uncaging. In situ uncaging of peptides leads to multiple serial B-T contacts and cell activation. Differential CD40 signaling, is both necessary and sufficient to mediate 2-fold differences in B cell expansion. While plasmablast numbers are increased, pMHCII density does not directly control the output or quality of plasma cells. Thus, we distinguish the roles TFH cells play in expansion versus differentiation.

Plasma cell dynamics in the bone marrow niche.

Using intravital imaging, we report that bone marrow (BM) plasma cells (PCs) are motile. BM PCs exhibit a unique migration pattern, characterized by intermittent periods of high motility and longer stretches of confined migration or arrest. BM PCs accumulate into clusters, which have reduced cell motility. APRIL promotes cluster formation and overall PC motility in the BM. Although CXCL12 and its receptor, CXCR4, promote PC motility in the BM, VLA4 activity promotes arrest. However, blocking either pathway promotes PC egress from the BM. Under steady-state conditions, BM PCs recirculate to other bones and spleen. In older mice, overall PC motility and recirculation increase, and this is correlated with increased CXCR4 expression, which depends on PC age or maturation rather than mouse age. Altogether, these results suggest that changes in PC motility and CXCR4 expression are linked with survival of long-lived PCs in the BM.

Intravital Imaging Reveals Motility of Adult Hematopoietic Stem Cells in the Bone Marrow Niche.

Adult mammalian hematopoietic stem cells (HSCs) reside in the bone marrow (BM) but can be mobilized into blood for use in transplantation. HSCs interact with BM niche cells that produce growth factor c-Kit ligand (Kitl/SCF) and chemokine CXCL12, and were thought to be static and sessile. We used two-photon laser scanning microscopy to visualize genetically labeled HSCs in the BM of live mice for several hours. The majority of HSCs showed a dynamic non-spherical morphology and significant motility, undergoing slow processive motion interrupted by short stretches of confined motion. HSCs moved in the perivascular space and showed intermittent close contacts with SCF-expressing perivascular stromal cells. In contrast, mobilization-inducing blockade of CXCL12 receptor CXCR4 and integrins rapidly abrogated HSC motility and shape dynamics in real time. Our results reveal an unexpectedly dynamic nature of HSC residence in the BM and interaction with the SCF+ stromal niche, which is disrupted during HSC mobilization.

Dynamic CD138 surface expression regulates switch between myeloma growth and dissemination.

The canonical plasma cell marker CD138 (syndecan-1) is highly expressed on the myeloma cell surface, but its functional role in vivo is unclear, as well as the ontogeny of CD138-high and CD138-negative (neg) myeloma cells. In this study we used an in vivo murine Vk*MYC myeloma model where CD138 is heterogeneously expressed depending on tumor size. We find that in comparison to CD138-neg myeloma cells, the CD138-high subset of myeloma cells is highly proliferative, less apoptotic, and enhanced IL-6R signaling, which is known to promote survival. In addition CD138-high myeloma engrafts better than its CD138-neg counterpart. In contrast, CD138-neg cells are more motile both in vitro and in vivo, and more readily disseminate and spread to other bones in vivo than CD138-high subset. Neutralizing CD138 rapidly triggers migration of myeloma cells in vivo and leads to intravasation, which results in increased dissemination to other bones. Both murine and human myeloma cells can rapidly recycle CD138 surface expression through endocytic trafficking, in response to serum levels. Blocking CD138 enhances myeloma sensitivity to bortezomib chemotherapy and significantly reduces tumor size compared to bortezomib treatment alone. Thus, our data show that CD138 surface expression dynamically regulates a switch between growth vs. dissemination for myeloma, in response to nutrient conditions.

Measuring rotational diffusion of MHC class I on live cells by polarized FPR.

Clustering of membrane proteins is a dynamic process which can regulate cellular function and signaling. The size of receptor and other membrane protein clusters can in principle be measured in terms of their rotational diffusion. However, in practice, measuring rotation of membrane proteins of live cells has been difficult, largely because of the difficulty of rigidly attaching reporter groups to the molecules of interest. Here we show that polarized photobleaching recovery can detect rotation of membrane proteins genetically tagged with yellow fluorescent protein, YFP. MHC class I molecules were engineered with a rigid, in-sequence, YFP tag followed at the C-terminus by a pair of crosslinkable domains. When crosslinker was added we could detect changes in rotational anisotropy decay consistent with clustering of the MHC molecules. This result points the way to use of engineered fluorescent fusion proteins to measure rotational diffusion in native cell membranes.

CXCL12-Producing Vascular Endothelial Niches Control Acute T Cell Leukemia Maintenance.

The role of the microenvironment in T cell acute lymphoblastic leukemia (T-ALL), or any acute leukemia, is poorly understood. Here we demonstrate that T-ALL cells are in direct, stable contact with CXCL12-producing bone marrow stroma. Cxcl12 deletion from vascular endothelial, but not perivascular, cells impeded tumor growth, suggesting a vascular niche for T-ALL. Moreover, genetic targeting of Cxcr4 in murine T-ALL after disease onset led to rapid, sustained disease remission, and CXCR4 antagonism suppressed human T-ALL in primary xenografts. Loss of CXCR4 targeted key T-ALL regulators, including the MYC pathway, and decreased leukemia initiating cell activity in vivo. Our data identify a T-ALL niche and suggest targeting CXCL12/CXCR4 signaling as a powerful therapeutic approach for T-ALL.

Aging-related anatomical and biochemical changes in lymphatic collectors impair lymph transport, fluid homeostasis, and pathogen clearance.

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time-lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging-related increase in the glycation and carboxylation of lymphatic's endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.

Organizing MHC Class II Presentation.

Major histocompatibility complex (MHC) class II molecules are ligands for CD4(+) T cells and are critical for initiating the adaptive immune response. This review is focused on what is currently known about MHC class II organization at the plasma membrane of antigen presenting cells and how this affects antigen presentation to T cells. The organization and diffusion of class II molecules have been measured by a variety of biochemical and microscopic techniques. Membrane lipids and other proteins have been implicated in MHC class II organization and function. However, when compared with the organization of MHC class I or TCR complexes, much less is known about MHC class II. Since clustering of T cell receptors occurs during activation, the organization of MHC molecules prior to recognition and during synapse formation may be critical for antigen presentation.

A dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into the germinal center.

The germinal center (GC) reaction is essential for the generation of the somatically hypermutated, high-affinity antibodies that mediate adaptive immunity. Entry into the GC is limited to a small number of B cell clones; however, the process by which this limited number of clones is selected is unclear. In this study, we demonstrate that low-affinity B cells intrinsically capable of seeding a GC reaction fail to expand and become activated in the presence of higher-affinity B cells even before GC coalescence. Live multiphoton imaging shows that selection is based on the amount of peptide-major histocompatibility complex (pMHC) presented to cognate T cells within clusters of responding B and T cells at the T-B border. We propose a model in which T cell help is restricted to the B cells with the highest amounts of pMHC, thus allowing for a dynamic affinity threshold to be imposed on antigen-binding B cells.

Affinity measured by microcluster.

Like T cell activation, B cell activation is driven by aggregation of B cell receptors (BCRs) into microclusters. New work suggests that the early dynamics of BCR mobility and microcluster formation "translate" BCR affinity for antigen into B cell responsiveness.

Clustering class I MHC modulates sensitivity of T cell recognition.

T cell recognition of peptide-MHC is highly specific and is sensitive to very low levels of agonist peptide; however, it is unclear how this effect is achieved or regulated. In this study we show that clustering class I MHC molecules on the cell surface of B lymphoblasts enhances their recognition by mouse and human T cells. We increased clustering of MHC I molecules by two methods, cholesterol depletion and direct cross-linking of a dimerizable MHC construct. Imaging showed that both treatments increased the size and intensity of MHC clusters on the cell surface. Enlarged clusters correlated with enhanced lysis and T cell effector function. Enhancements were peptide-specific and greatest at low concentrations of peptide. Clustering MHC class I enhanced recognition of both strong and weak agonists but not null peptide. Our results indicate that the lateral organization of MHC class I on the cell surface can modulate the sensitivity of T cell recognition of agonist peptide.