Regulation of pulmonary plasma cell responses during secondary infection with influenza virus.

During secondary infection with influenza virus, plasma cells (PCs) develop within the lung, providing a local source of antibodies. However, the site and mechanisms that regulate this process are poorly defined. Here, we show that while circulating memory B cells entered the lung during rechallenge and were activated within inducible bronchus-associated lymphoid tissues (iBALTs), resident memory B (BRM) cells responded earlier, and their activation occurred in a different niche: directly near infected alveoli. This process required NK cells but was largely independent of CD4 and CD8 T cells. Innate stimuli induced by virus-like particles containing ssRNA triggered BRM cell differentiation in the absence of cognate antigen, suggesting a low threshold of activation. In contrast, expansion of PCs in iBALTs took longer to develop and was critically dependent on CD4 T cells. Our work demonstrates that spatially distinct mechanisms evolved to support pulmonary secondary PC responses, and it reveals a specialized function for BRM cells as guardians of the alveoli.

Macrophages in the synovial lining niche initiate neutrophil recruitment and articular inflammation.

The first immune-activating changes within joint resident cells that lead to pathogenic leukocyte recruitment during articular inflammation remain largely unknown. In this study, we employ state-of-the-art confocal microscopy and image analysis in a systemic, whole-organ, and quantitative way to present evidence that synovial inflammation begins with the activation of lining macrophages. We show that lining, but not sublining macrophages phagocytose immune complexes containing the model antigen. Using the antigen-induced arthritis (AIA) model, we demonstrate that on recognition of antigen-antibody complexes, lining macrophages undergo significant activation, which is dependent on interferon regulatory factor 5 (IRF5), and produce chemokines, most notably CXCL1. Consequently, at the onset of inflammation, neutrophils are preferentially recruited in the vicinity of antigen-laden macrophages in the synovial lining niche. As inflammation progresses, neutrophils disperse across the whole synovium and form swarms in synovial sublining during resolution. Our study alters the paradigm of lining macrophages as immunosuppressive cells to important instigators of synovial inflammation.

It takes a village to skew a lymph node.

Unconventional T cells (UTCs) play important roles in protecting barrier tissues. In this issue of Immunity, Ataide and colleagues show an additional function for these cells: populating downstream lymph nodes and skewing their cytokine profiles. This process allows tissues to adjust the function of draining lymph nodes through imprinting UTC cytokine production, subsequently influencing the immune responses that are triggered.

Secondary influenza challenge triggers resident memory B cell migration and rapid relocation to boost antibody secretion at infected sites.

Resident memory B (BRM) cells develop and persist in the lungs of influenza-infected mice and humans; however, their contribution to recall responses has not been defined. Here, we used two-photon microscopy to visualize BRM cells within the lungs of influenza -virus immune and reinfected mice. Prior to re-exposure, BRM cells were sparsely scattered throughout the tissue, displaying limited motility. Within 24 h of rechallenge, these cells increased their migratory capacity, localized to infected sites, and subsequently differentiated into plasma cells. Alveolar macrophages mediated this process, in part by inducing expression of chemokines CXCL9 and CXCL10 from infiltrating inflammatory cells. This led to the recruitment of chemokine receptor CXCR3-expressing BRM cells to infected regions and increased local antibody concentrations. Our study uncovers spatiotemporal mechanisms that regulate lung BRM cell reactivation and demonstrates their capacity to rapidly deliver antibodies in a highly localized manner to sites of viral replication.

The Zinc Finger Protein Zbtb18 Represses Expression of Class I Phosphatidylinositol 3-Kinase Subunits and Inhibits Plasma Cell Differentiation.

The PI3K pathway plays a key role in B cell activation and is important for the differentiation of Ab producing plasma cells (PCs). Although much is known about the molecular mechanisms that modulate PI3K signaling in B cells, the transcriptional regulation of PI3K expression is poorly understood. In this study, we identify the zinc finger protein Zbtb18 as a transcriptional repressor that directly binds enhancer/promoter regions of genes encoding class I PI3K regulatory subunits, subsequently limiting their expression, dampening PI3K signaling and suppressing PC responses. Following activation, dividing B cells progressively downregulated Zbtb18, allowing gradual amplification of PI3K signals and enhanced development of PCs. Human Zbtb18 displayed similar expression patterns and function in human B cells, acting to inhibit development of PCs. Furthermore, a number of Zbtb18 mutants identified in cancer patients showed loss of suppressor activity, which was also accompanied by impaired regulation of PI3K genes. Taken together, our study identifies Zbtb18 as a repressor of PC differentiation and reveals its previously unappreciated function as a transcription modulator of the PI3K signaling pathway.

Marginal zone SIGN-R1+ macrophages are essential for the maturation of germinal center B cells in the spleen.

The mechanisms that regulate germinal center (GC) B cell responses in the spleen are not fully understood. Here we use a combination of pharmacologic and genetic approaches to delete SIGN-R1+ marginal zone (MZ) macrophages and reveal their specific contribution to the regulation of humoral immunity in the spleen. We find that while SIGN-R1+ macrophages were not essential for initial activation of B cells, they were required for maturation of the response and development of GC B cells. These defects could be corrected when follicular helper T (Tfh) cells were induced before macrophage ablation or when Tfh responses were enhanced. Moreover, we show that in the absence of SIGN-R1+ macrophages, DCIR2+ dendritic cells (DCs), which play a key role in priming Tfh responses, were unable to cluster to the interfollicular regions of the spleen and were instead displaced to the MZ. Restoring SIGN-R1+ macrophages to the spleen corrected positioning of DCIR2+ DCs and rescued the GC B cell response. Our study reveals a previously unappreciated role for SIGN-R1+ macrophages in regulation of the GC reaction and highlights the functional specification of macrophage subsets in the MZ compartment.

Visualization of T Cell Migration in the Spleen Reveals a Network of Perivascular Pathways that Guide Entry into T Zones.

Lymphocyte homeostasis and immune surveillance require that T and B cells continuously recirculate between secondary lymphoid organs. Here, we used intravital microscopy to define lymphocyte trafficking routes within the spleen, an environment of open blood circulation and shear forces unlike other lymphoid organs. Upon release from arterioles into the red pulp sinuses, T cells latched onto perivascular stromal cells in a manner that was independent of the chemokine receptor CCR7 but sensitive to Gi protein-coupled receptor inhibitors. This latching sheltered T cells from blood flow and enabled unidirectional migration to the bridging channels and then to T zones, entry into which required CCR7. Inflammatory responses modified the chemotactic cues along the perivascular homing paths, leading to rapid block of entry. Our findings reveal a role for vascular structures in lymphocyte recirculation through the spleen, indicating the existence of separate entry and exit routes and that of a checkpoint located at the gate to the T zone.

IgA production requires B cell interaction with subepithelial dendritic cells in Peyer's patches.

Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-ß receptor (LTßR)-dependent maintenance of DCs. PP DCs augment IgA production by integrin αvß8-mediated activation of transforming growth factor-ß (TGFß). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGFß activation and induction of mucosal IgA responses.

Blood, sphingosine-1-phosphate and lymphocyte migration dynamics in the spleen.

The spleen, the largest secondary lymphoid organ, has long been known to play important roles in immunity against blood-borne invaders. Yet how cells migrate within the spleen to ensure fast and effective responses is only now coming to light. Chemokines and oxysterols guide lymphocytes from sites of release at terminal arterioles into the lymphocyte-rich white pulp. Sphingosine-1-phosphate (S1P) and S1P-receptor-1 (S1PR1) promote lymphocyte egress from white to red pulp and back to circulation. Intravital two-photon microscopy has shown that marginal zone (MZ) B cells that are enriched between white and red pulps undergo continual oscillatory migration between the MZ and follicles, ferrying antigens. Cycles of G-protein-coupled receptor kinase-2 (GRK2) mediated S1PR1 desensitization and resensitization underlie this remarkable behavior. The findings discussed in this review have implications for understanding how splenic antibody and T-cell responses are mounted, how the immunosuppressant drug FTY720 (fingolimod) affects the spleen, and how cell shuttling behaviors contribute to immunity.

Visualization of splenic marginal zone B-cell shuttling and follicular B-cell egress.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is sphingosine-1-phosphate receptor-1 (S1PR1)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S1PR1-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.

GRK2-dependent S1PR1 desensitization is required for lymphocytes to overcome their attraction to blood.

Lymphocytes egress from lymphoid organs in response to sphingosine-1-phosphate (S1P); minutes later they migrate from blood into tissue against the S1P gradient. The mechanisms facilitating cell movement against the gradient have not been defined. Here, we show that heterotrimeric guanine nucleotide-binding protein-coupled receptor kinase-2 (GRK2) functions in down-regulation of S1P receptor-1 (S1PR1) on blood-exposed lymphocytes. T and B cell movement from blood into lymph nodes is reduced in the absence of GRK2 but is restored in S1P-deficient mice. In the spleen, B cell movement between the blood-rich marginal zone and follicles is disrupted by GRK2 deficiency and by mutation of an S1PR1 desensitization motif. Moreover, delivery of systemic antigen into follicles is impaired. Thus, GRK2-dependent S1PR1 desensitization allows lymphocytes to escape circulatory fluids and migrate into lymphoid tissues.

Cannabinoid receptor 2 positions and retains marginal zone B cells within the splenic marginal zone.

Specialized B cells residing in the splenic marginal zone (MZ) continuously survey the blood for antigens and are important for immunity to systemic infections. However, the cues that uniquely attract cells to the MZ have not been defined. Previous work demonstrated that mice deficient in cannabinoid receptor 2 (CB2) have decreased numbers of MZ B cells but it has been unclear whether CB2 regulates MZ B cell development or positioning. We show that MZ B cells are highly responsive to the CB2 ligand 2-arachidonylglycerol (2-AG) and that CB2 antagonism rapidly displaces small numbers of MZ B cells to the blood. Antagonism for longer durations depletes MZ B cells from the spleen. In mice deficient in sphingosine-1-phosphate receptor function, CB2 antagonism causes MZ B cell displacement into follicles. Moreover, CB2 overexpression is sufficient to position B cells to the splenic MZ. These findings establish a role for CB2 in guiding B cells to the MZ and in preventing their loss to the blood. As a consequence of their MZ B cell deficiency, CB2-deficient mice have reduced numbers of CD1d-high B cells. We show that CB2 deficiency results in diminished humoral responses to a CD1d-restricted systemic antigen.

The crystal structure of CHIR-AB1: a primordial avian classical Fc receptor.

CHIR-AB1 is a newly identified avian immunoglobulin (Ig) receptor that includes both activating and inhibitory motifs and was therefore classified as a potentially bifunctional receptor. Recently, CHIR-AB1 was shown to bind the Fc region of chicken IgY and to induce calcium mobilization via association with the common gamma-chain, a subunit that transmits signals upon ligation of many different immunoreceptors. Here we describe the 1.8-A-resolution crystal structure of the CHIR-AB1 ectodomain. The receptor ectodomain consists of a single C2-type Ig domain resembling the Ig-like domains found in mammalian Fc receptors such as FcgammaRs and FcalphaRI. Unlike these receptors and other monomeric Ig superfamily members, CHIR-AB1 crystallized as a 2-fold symmetrical homodimer that bears no resemblance to variable or constant region dimers in an antibody. Analytical ultracentrifugation demonstrated that CHIR-AB1 exists as a mixture of monomers and dimers in solution, and equilibrium gel filtration revealed a 2:1 receptor/ligand binding stoichiometry. Measurement of the 1:1 CHIR-AB1/IgY interaction affinity indicates a relatively low affinity complex, but a 2:1 CHIR-AB1/IgY interaction allows an increase in apparent affinity due to avidity effects when the receptor is tethered to a surface. Taken together, these results add to the structural understanding of Fc receptors and their functional mechanisms.

Harnessing soluble NK cell killer receptors for the generation of novel cancer immune therapy.

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.

Tumor and viral recognition by natural killer cells receptors.

Natural killer (NK) cells destroy hazardous cells such as tumors and virus-infected cells immediately without the need for prior antigen stimulation. The activation of NK cells largely depends on the recently identified natural cytotoxic receptors (NCRs), which include three members: NKp46, NKp44 and NKp30. The NCRs are unique in their expression pattern that is almost conclusively confined to NK cells, and in their broad specificity towards a wide range of targets. However, very little is known about the ligands identity of the NCRs and so far the only ligands known are two virally derived molecules: the hemagglutinin protein of influenza viruses that directly binds and activates two of the NCRs; NKp46 and NKp44, and the human cytomegalovirus tegument protein, pp65, which binds the NKp30 receptor and inhibits its activation thus promoting survival of the virus. In this review we describe the function of the NCRs in various pathological conditions with a special emphasis on tumor targeting.

Decidual NK cells regulate key developmental processes at the human fetal-maternal interface.

Human CD56(bright) NK cells accumulate in the maternal decidua during pregnancy and are found in direct contact with fetal trophoblasts. Several mechanisms have been proposed to explain the inability of NK cells to kill the semiallogeneic fetal cells. However, the actual functions of decidual NK (dNK) cells during pregnancy are mostly unknown. Here we show that dNK cells, but not peripheral blood-derived NK subsets, regulate trophoblast invasion both in vitro and in vivo by production of the interleukin-8 and interferon-inducible protein-10 chemokines. Furthermore, dNK cells are potent secretors of an array of angiogenic factors and induce vascular growth in the decidua. Notably, such functions are regulated by specific interactions between dNK-activating and dNK-inhibitory receptors and their ligands, uniquely expressed at the fetal-maternal interface. The overall results support a 'peaceful' model for reproductive immunology, in which elements of innate immunity have been incorporated in a constructive manner to support reproductive tissue development.

Reduced KIR2DL1 recognition of MHC class I molecules presenting phosphorylated peptides.

As initially described by K. Karre and colleagues in the missing self hypothesis, cells expressing self-MHC class I proteins are protected from NK cells attack. In contrast, reduction in the expression of MHC class I molecules due to viral infection or tumor transformation result in the killing of these "abnormal" cells by NK cells via NK-activating receptors. Thus, NK killing of target cells is determined by both negative signals coming from MHC class I proteins and by positive signals derived from the activating ligands. The bound peptide in MHC class I play an important role in the balanced recognition of NK cells. The peptide stabilizes the MHC complex and interacts directly with the NK inhibitory receptors, thus participating in the determination of the fate of the target cells. In this study we demonstrate that posttranslational modifications such as phosphorylation of the presented peptide altered the ability of NK cells to recognize MHC class I molecules. By using a consensus peptide (QYDDAVYKL) that binds HLA-Cw4 in which different positions in the bound peptide were modified by serine phosphorylation, we observed a reduction in KIR2DL1 binding that led to decreased protection from NK killing. Therefore, it might be possible that alteration in the phosphorylation pattern during tumor transformation or viral infection may result in less inhibition and, consequently, improved NK cell killing.

Lethal influenza infection in the absence of the natural killer cell receptor gene Ncr1.

The elimination of viruses and tumors by natural killer cells is mediated by specific natural killer cell receptors. To study the in vivo function of a principal activating natural killer cell receptor, NCR1 (NKp46 in humans), we replaced the gene encoding this receptor (Ncr1) with a green fluorescent protein reporter cassette. There was enhanced spread of certain tumors in 129/Sv but not C57BL/6 Ncr1(gfp/gfp) mice, and influenza virus infection was lethal in both 129/Sv and C57BL/6 Ncr1(gfp/gfp) mice. We noted accumulation of natural killer cells at the site of influenza infection by tracking the green fluorescent protein. Our results demonstrate a critical function for Ncr1 in the in vivo eradication of influenza virus.