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Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant lipoproteins within the lung alveoli. Alveolar macrophages (AMs) are crucial for surfactant clearance, and their differentiation depends on colony-stimulating factor 2 (CSF2), which regulates the establishment of an AM-characteristic gene regulatory network. Here, we report that the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) is essential for the development of the AM identity, as demonstrated by transcriptome and chromatin accessibility analysis. Furthermore, C/EBPβ-deficient AMs showed severe defects in proliferation, phagocytosis, and lipid metabolism, collectively resulting in a PAP-like syndrome. Mechanistically, the long C/EBPβ protein variants LAP* and LAP together with CSF2 signaling induced the expression of Pparg isoform 2 but not Pparg isoform 1, a molecular regulatory mechanism that was also observed in other CSF2-primed macrophages. These results uncover C/EBPβ as a key regulator of AM cell fate and shed light on the molecular networks controlling lipid metabolism in macrophages.

The IκB kinase (IKK)‐NF‐κB pathway is activated as part of the DNA damage response and controls both inflammation and resistance to apoptosis. How these distinct functions are achieved remained unknown. We demonstrate here that DNA double‐strand breaks elicit two subsequent phases of NF‐κB activation in vivo and in vitro, which are mechanistically and functionally distinct. RNA‐sequencing reveals that the first‐phase controls anti‐apoptotic gene expression, while the second drives expression of senescence‐associated secretory phenotype (SASP) genes. The rapidly activated first phase is driven by the ATM‐PARP1‐TRAF6‐IKK cascade, which triggers proteasomal destruction of inhibitory IκBα, and is terminated through IκBα re‐expression from the NFKBIA gene. The second phase, which is activated days later in senescent cells, is on the other hand independent of IKK and the proteasome. An altered phosphorylation status of NF‐κB family member p65/RelA, in part mediated by GSK3β, results in transcriptional silencing of NFKBIA and IKK‐independent, constitutive activation of NF‐κB in senescence. Collectively, our study reveals a novel physiological mechanism of NF‐κB activation with important implications for genotoxic cancer treatment., DNA damage induces two kinetically and functionally distinct phases of NF‐κB activation, the first being anti‐apoptotic and the non‐canonical second wave inducing the senescence‐associated secretory phenotype (SASP).

Interleukin 17-producing helper T (Th17) cells have been widely defined by the lineage transcription factor retinoid-related orphan receptor (ROR)γt. Pathophysiologically, these cells play a crucial role in autoimmune diseases and have been linked to dysregulated germinal center (GC) reactions and autoantibody production. In this study, we used gene expression and flow cytometric analyses for the characterization of Rorγt(-/-) and Rorγt(-/-)Il21(RFP/+) mice to demonstrate a previously unknown transcriptional flexibility in the development of IL-17-producing Th-cell subsets. We found an accumulation of follicular Th (Tfh) cells by 5.2-fold, spontaneous 13-fold higher GC formation, decreased frequency of follicular Foxp3(+) T-regulatory (Treg) cells (50%), and a 3.4-fold increase in the number of proliferating follicular B cells in RORγt-deficient vs. wild-type mice. Dysregulated B-cell responses were associated with enhanced production of IL-17 (6.4-fold), IL-21 (2.2-fold), and B-cell-activating factor (BAFF) (2-fold) and were partially rescued by adoptive transfer of Treg cells. In an unexpected finding, we detected RORγt-independent IL-17 expression in ICOS(+)CXCR5(+)Tfh and in ICOS(+)CXCR5(-)Th cells. Based on the observed high Irf4 and Batf gene expression, we suggest that CD4(+) T-cell transcription factors other than RORγt can cooperatively induce differentiation of IL-17-producing Th cells, including Th17-like Tfh-cell subsets. We conclude that the occurrence of aberrant Tfh and follicular Treg cells support spontaneous GC formation and dysregulated B-cell responses in RORγt-deficient mice.

Microenvironmental regulation in lymphoid tissues is essential for the development of chronic lymphocytic leukemia. We identified cellular and molecular factors provided by the splenic marginal zone (MZ), which alter the migratory and adhesive behavior of leukemic cells. We used the Cxcr5-/-Eµ-Tcl1 leukemia mouse model, in which tumor cells are excluded from B cell follicles and instead accumulate within the MZ. Genes involved in MZ B cell development and genes encoding for adhesion molecules were upregulated in MZ-localized Cxcr5-/-Eµ-Tcl1 cells. Likewise, surface expression of the adhesion and homing molecules, CD49d/VLA-4 and CXCR7, and of NOTCH2 was increased. In vitro, exposing Eµ-Tcl1 cells or human CLL cells to niche-specific stimuli, like B cell receptor- or Toll-like receptor ligands, caused surface expression of these molecules characteristic for a follicular or MZ-like microenvironment, respectively. In vivo, inhibition of VLA-4-mediated adhesion and CXCL13-mediated follicular homing displaced leukemic cells not only from the follicle, but also from the MZ and reduced leukemia progression. We conclude that MZ-specific factors shape the phenotype of leukemic cells and facilitate their niche-specific retention. This strong microenvironmental influence gains pathogenic significance independent from tumor-specific genetic aberrations.

Infection with helminths and exposure to antigens induce a strong type 2 immune response resulting in the secretion of the cytokines IL-4 and IL-13 by CD4(+) T cells and several innate cell types. IL-4 and IL-13 promote class switch recombination to IgG1 and IgE while their role for germinal center (GC) formation is poorly understood. We found a dramatic reduction in the numbers of GC B cells when investigating different type 2 immune responses in IL-4/IL-13-deficient mice. IL-4/IL-13 from T cells located outside B-cell follicles was sufficient for GC formation. We further revealed that IL-4/IL-13 acts directly on B cells for the formation of a robust GC response. The frequency of apoptotic GC B cells was not altered in the absence of IL-4/IL-13 and proliferation was even enhanced. However, deficiency of signal transducer and activator of transcription 6 signaling in B cells resulted in failure to downregulate the chemotactic receptor Gpr183 (Ebi2) and downregulation of this receptor has been shown to be essential for proper GC B-cell differentiation. Thus, T-cell-derived extrafollicular IL-4/IL-13 and signal transducer and activator of transcription 6-regulated genes in B cells play a critical role for orchestration of the GC response in type 2 immunity.

Multiple myeloma (MM) is a B cell neoplasm that originates from the malignant transformation of plasma cells, with treatment strategies that include chemotherapeutic agents and immunomodulatory drugs. Recently, significant effort has been applied to the development of monoclonal antibody (mAb) and chimeric antigen receptor (CAR) T cell therapies for the treatment of advanced MM. Anti-CD38 mAb therapy is at the forefront of these efforts, with clearly demonstrated clinical benefit and availability of a FDA-approved mAb in daratumumab. Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism of action of CD38-targeted mAbs; however, high CD38 expression on natural killer (NK) cells results in fratricide, which depletes the NK cells necessary for ADCC. In addition to CD38, targeting of other MM-associated cell-surface proteins has been explored. Of these antigens, the TNF-superfamily member BCMA is among the most researched and is under development by multiple groups as a CAR target. Several clinical trials in MM have shown promising initial results targeting BCMA with CAR T cells, however there remains significant opportunity to improve both relapse rates and treatment of relapsed patients. Collectively, clinical data would suggest that combinatorial targeting of both CD38 and BCMA may improve clinical efficacy compared with targeting either antigen alone. We have developed a multiple-target, adoptive NK cell immunotherapy approach for the treatment of MM. The strategy utilizes our off-the-shelf NK cell platform with four engineered attributes: 1) an anti-BCMA CAR for direct MM targeting, 2) high affinity non-cleavable CD16 (hnCD16) for enhanced ADCC in combination with anti-CD38 mAbs, 3) CD38 deletion for resistance to anti-CD38 mAb induced NK cell depletion, and 4) IL-15/IL-15 receptor α fusion protein (IL-15RF; IL-15 fused to IL-15Rα) for enhanced NK cell persistence. The anti-BCMA CAR consists of a unique single chain variable fragment (scFv) targeting domain with a BCMA binding affinity in the low nanomolar range, providing high functional avidity and efficacy in disease settings where BCMA antigen density is low. Our approach utilizes NK cells derived from a genetically engineered, clonally-derived master pluripotent stem cell line with uniform expression of anti-BCMA CAR, IL-15RF, hnCD16, and CD38 bi-allelic knockout. The engineered master pluripotent stem cell line serves as the starting material for consistent and repeatable manufacture of off-the-shelf NK cells that contain all described attributes in a homogenous manner (termed FT576) and that can be produced at a scale to support multi-dose treatment strategies and on-demand dose availability. In preclinical studies, FT576 NK cells exhibited uniform expression of CD16, CAR, and IL15-RF and did not express CD38 (99% cytotoxicity in a 3D-spheroid culture model. Preclinical studies are ongoing to support the advancement of FT576 as the first-of-kind cellular therapeutic for the combination of anti-BCMA CAR and mAb-directed targeting of MM. Disclosures Bjordahl: Fate Therapeutics, Inc.: Employment. Gaidarova:Fate Therapeutics, Inc: Employment. Goodridge:FATE THERAPEUTICS: Employment. Mahmood:Fate Therapeutics, Inc: Employment. Bonello:Fate Therapeutics, Inc.: Employment. Robinson:Fate Therapeutics, Inc.: Employment. Ruller:Fate Therapeutics, Inc.: Employment. Pribadi:Fate Therapeutics, Inc.: Employment. Lee:Fate Therapeutics, Inc.: Employment. Abujarour:Fate Therapeutics, Inc.: Employment. Dinella:Fate Therapeutics, Inc.: Employment. Huffman:Fate Therapeutics, Inc.: Employment. Chu:FATE THERAPEUTICS: Employment. Valamehr:Fate Therapeutics, Inc: Employment.

Autoimmunity is associated with a strong genetic component, but onset and persistence of clinically apparent autoimmune diseases often require an additional environmental trigger. The balance between immunity and tolerance is regulated by numerous molecular factors including nuclear hormone and homeostatic chemokine receptors. The nuclear hormone receptor ROR{gamma}t and the chemokine receptor CCR7 are both essentially involved in functional lymphoid organogenesis and maintenance of lymphocyte homeostasis. Lack of one or the other impairs thymic T cell development and alters T cell homeostasis. Mice deficient for both, Ccr7-/-Ror{gamma}t-/-, succumbed early to acute destructive inflammation, characterized by massive recruitment of inflammatory leukocytes, pro-inflammatory cytokine and autoantibody production, and wasting disease. Antibiotic-treatment of mice before disease onset reduced the overall gut microflora and abrogated the development of fatal mucosal inflammation. Hence, commensal bacteria and a confined tissue-specific inflammatory milieu serve as complementary trigger to initiate the lethal pathophysiologic process in Ccr7-/-Ror{gamma}t-/- mice.

Autoimmune gastritis is a common autoimmune disorder characterized by chronic inflammatory cell infiltrates, atrophy of the corpus and fundus, and the occurrence of autoantibodies to parietal cell antigen. In CCR7-deficient mice, autoimmune gastritis developed spontaneously and was accompanied by metaplasia of the gastric mucosa and by the formation of tertiary lymphoid organs at gastric mucosal sites. T cells of CCR7-deficient mice showed an activated phenotype in the gastric mucosa, mesenteric lymph nodes, and peripheral blood. In addition, elevated serum IgG levels specific to gastric parietal cell antigen were detected. Because the role of organized lymphocytic aggregates at this inflammatory site is not completely understood, we first analyzed the cellular requirements for the formation of these structures. Autoreactive CD4(+) T cells were pivotal for tertiary lymphoid follicle formation, most likely in cooperation with dendritic cells, macrophages, and B cells. Second, we analyzed the necessity of secondary lymph nodes and tertiary lymphoid organs for the development of autoimmune gastritis using CCR7 single- and CCR7/lymphotoxin α double-deficient mice. Strikingly, manifestation of autoimmune gastritis was observed in the absence of secondary lymph nodes and preceded the development of tertiary lymphoid organs. Taken together, these findings identify an inflammatory process where gastric autoreactive T cells independent of organized tertiary lymphoid organs and classic lymph nodes can induce and maintain autoimmune gastritis.

Lymphoma cell survival and progression are putatively dependent on a specific microanatomic localization within secondary lymphoid organs. Despite compelling data correlating homeostatic chemokine receptor expression and human lymphoma pathogenesis, genetic models that either mimic lymphoma dissemination or dissect a crosstalk of lymphoma and stromal cells are missing. Applying the genetically tractable Eμ-Myc transgenic mouse model, we show that the chemokine receptor CCR7 regulates Eμ-Myc lymphoma homing to lymph nodes and distinctive microanatomic sites of the spleen. CCR7-controlled access of lymphoma cells to the splenic T-cell zone led to a significant survival advantage compared with CCR7-deficient lymphoma cells, which were excluded from this zone. Within the niche, lymphoma cells stimulated a reciprocal cross-talk with gp38+ fibroblastic reticular cells. This reciprocal cooperation program was mediated by lymphoma B cell–presented lymphotoxin, which acted on lymphotoxin-β–receptor-bearing stromal cells followed by alteration of stromal cellular composition. Cross-talk inhibition by lymphotoxin-α deletion and using a lymphotoxin-β receptor-immunoglobulin fusion protein impaired lymphoma growth. Thus, abrogation of CCR7-governed migration and of sustained lymphotoxin signaling could provide new targets in lymphoma therapy.

CCR7 plays a crucial role in lymphocyte exit from the peritoneal cavity. T and B lymphocytes recirculate among blood, lymph, and extralymphoid tissues to ensure immune surveillance and the establishment of self-tolerance. The underlying mechanisms regulating homeostatic lymphocyte recirculation through body cavities are not fully understood. Here, we demonstrate that the homeostatic chemokine receptor CCR7 regulates homeostatic recirculation of lymphocytes through body cavities. CCR7 deficiency results in massive accumulation of CD4+ and CD8+ T cells and B-2 B cells in the peritoneal and pleural cavities. The increase in B-2 B and T lymphocytes is not associated with an altered maturation and/or activation status of these cells. Mechanistically, an increase in peritoneal lymphocyte numbers is caused by impaired egress of CCR7-deficient lymphocytes from body cavities. These results establish that CCR7 plays a crucial role in lymphocyte exit from the PerC.

Thymus-independent type 2 (TI-2) antigens are repetitive antigens capable of eliciting antibody responses without T cell help. They are important in the immune response against encapsulated bacteria and as a rapid first line of defense against pathogens. TI-2 antigens induce strong proliferation in extrafollicular foci. However, any germinal centers forming in response to TI-2 antigens involute synchronously 5 days after immunization. This is thought to be caused by the lack of T cell help. Surprisingly, immunization of mice deficient for the homeostatic chemokine receptor CCR7 with TI-2 antigens resulted not only in the expected, vigorous extrafollicular plasma cell response but also in persisting splenic germinal centers. This was observed for two different TI-2 antigens, heat-killed Streptococcus pneumoniae and (4-hydroxy-3-nitrophenyl)acetyl-Ficoll (NP-Ficoll). Germinal centers induced by TI-2 and thymus-dependent (TD) antigens were located in the periarteriolar area of the white pulp in CCR7 knockout mice, corresponding to the T zone of wild-type (WT) mice. The TI-2-induced germinal centers contained peripheral rings of follicular dendritic cells and unusually for TI-2-induced germinal centers, T cells. The licensing responsible for their atypical persistence did not endow TI-2-induced germinal centers with the full range of characteristics of classic germinal centers induced by TD antigens. Thus, class-switching, affinity maturation, and memory B cell generation were not increased in CCR7-deficient mice. It seems unlikely that a defect in regulatory T cell (Treg) location was responsible for the atypical persistence of TI-2-induced germinal centers, as Tregs were comparably distributed in germinal centers of CCR7-deficient and WT mice.

Drainage of central nervous system (CNS) antigens to the brain-draining cervical lymph nodes (CLN) is likely crucial in the initiation and control of autoimmune responses during multiple sclerosis (MS). We demonstrate neuronal antigens within CLN of MS patients. In monkeys and mice with experimental autoimmune encephalomyelitis (EAE) and in mouse models with non-inflammatory CNS damage, the type and extent of CNS damage was associated with the frequencies of CNS antigens within the cervical lymph nodes. In addition, CNS antigens drained to the spinal-cord-draining lumbar lymph nodes. In human MS CLN, neuronal antigens were present in pro-inflammatory antigen-presenting cells (APC), whereas the majority of myelin-containing cells were anti-inflammatory. This may reflect a different origin of the cells or different drainage mechanisms. Indeed, neuronal antigen-containing cells in human CLN did not express the lymph node homing receptor CCR7, whereas myelin antigen-containing cells in situ and in vitro did. Nevertheless, CLN from EAE-affected CCR7-deficient mice contained equal amounts of myelin and neuronal antigens as wild-type mice. We conclude that the type and frequencies of CNS antigens within the CLN are determined by the type and extent of CNS damage. Furthermore, the presence of myelin and neuronal antigens in functionally distinct APC populations within MS CLN suggests that differential immune responses can be evoked.

Multiple myeloma (MM) is an aggressive incurable plasma cell malignancy with a median life expectancy of less than seven years. Antibody-based therapies have demonstrated substantial clinical benefit for patients with hematological malignancies, particular in B cell Non-Hodgkin's lymphoma. The lack of immunotherapies specifically targeting MM cells led us to develop a human-mouse chimeric antibody directed against the B cell maturation antigen (BCMA), which is almost exclusively expressed on plasma cells and multiple myeloma cells. The high affinity antibody blocks the binding of the native ligands APRIL and BAFF to BCMA. This finding is rationalized by the high resolution crystal structure of the Fab fragment in complex with the extracellular domain of BCMA. Most importantly, the antibody effectively depletes MM cells in vitro and in vivo and substantially prolongs tumor-free survival under therapeutic conditions in a xenograft mouse model. A BCMA-antibody-based therapy is therefore a promising option for the effective treatment of multiple myeloma and autoimmune diseases.

Homeostatic trafficking of lymphocytes through extralymphoid tissues has been recently observed, and a potential role in immune surveillance and the establishment of peripheral tolerance are considered. However, the mechanisms regulating lymphocyte recirculation through peripheral tissues under noninflammatory conditions are not well understood. Here, we demonstrate that the chemokine receptor CCR7 controls not only lymphocyte trafficking to and within secondary lymphoid organs but also homeostatic migration of T and B lymphocytes through nonlymphoid tissues. Lack of CCR7 results in a massive accumulation of lymphocytes in epithelial tissues. In particular, the gastrointestinal mucosal tissue of CCR7−/− mice is highly permissive for the formation of lymphoid aggregates, which develop into ectopic follicular structures with major topologic characteristics of lymph nodes. Flow cytometry analysis of CD4+ T cells derived from ectopic follicles revealed that CD44hiCD62Llo effector memory T cells predominate in the gastric lymphoid aggregates. In aged mice, lack of CCR7 induced age-dependent histomorphologic changes in the stomach with profound cystic hyperplasia and an increased rate of mucosal proliferation resembling Menetrier disease. Thus, CCR7 regulates the cellular organization of visceral tissue by governing life-long recirculation of naive and memory lymphocytes under homeostatic conditions.

Dendritic cells (DC) either induce T cell tolerance or contribute to the initiation and modulation of T and B cell responses. Since many of the variables determining the thresholds of naive T cell priming were defined in vitro using a homogeneously matured DC population, we here focused on partially mature DC which might reflect the occurrence of tumor-infiltrating and thymic DC. To predict how those DC regulate the induction of antigen-specific T cell proliferation and T cell tolerance, we co-cultured ovalbumin-pulsed murine DC at different ratios with antigen-specific DO11.10 transgenic T cells. Whereas partially mature DC at a DC/T cell ratio of 1 : 10 supported proliferation, a DC/T cell ratio of 1 : 2 induced proliferation arrest in naive CD4+ T cells. The acquisition of the NK cell inhibitory markers NK1.1 and KLRG on T cells exposed to high numbers of DC suggests a role for these molecules in the protection of antigen-responsive T cells from exhaustion by overstimulation. Mechanistically, abortive T cell proliferation upon encounter of high numbers of partially mature DC is caused by an apoptosis-related pathway, suggesting that excessive antigen density without sufficient costimulation results in activation-induced cell death.

Effective priming of T cell responses depends on cognate interactions between naive T cells and professional antigen-presenting cells (APCs). This contact is the result of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, play a central role. We used the murine Listeria monocytogenes infection model to characterize the role of the CCR7/CCR7 ligand system in the generation of T cell responses during bacterial infection. We demonstrate that efficient priming of naive major histocompatibility complex (MHC) class Ia–restricted CD8+ T cells requires CCR7. In contrast, MHC class Ib–restricted CD8+ T cells and MHC class II–restricted CD4+ T cells seem to be less dependent on CCR7; memory T cell responses are independent of CCR7. Infection experiments with bone marrow chimeras or mice reconstituted with purified T cell populations indicate that CCR7 has to be expressed on CD8+ T cells and professional APCs to promote efficient MHC class Ia–restricted T cell priming. Thus, different T cell subtypes and maturation stages have discrete requirements for CCR7.

The development of secondary lymphoid organs is a complex process dependent on a coordinated interaction of cells of hematopoietic and non-hematopoietic origin. In this context, chemokines and cytokines belonging to the tumor necrosis factor (TNF)/lymphotoxin (LT) family are critical signaling molecules during the initial steps of lymph node and Peyer's patch organogenesis. Homeostatic chemokines, such as CXCL13, CCL21, and CCL19, as well as their corresponding receptors, CXCR5 and CCR7, have now been shown to closely cooperate in the development of lymphoid organs and the maintenance of lymphoid tissue microarchitecture. We summarize recent data on the function of CXCR5 and CCR7 and their intricate connection to the TNF/LT system in order to refine the current model of lymphoid organ development.

Antigen-pulsed dendritic cells (DCs) are used as natural adjuvants for vaccination, but the factors that influence the efficacy of this treatment are poorly understood. We investigated the parameters that affect the migration of subcutaneously injected mouse-mature DCs to the draining lymph node. We found that the efficiency of DC migration varied with the number of injected DCs and that CCR7+/+ DCs migrating to the draining lymph node, but not CCR7−/− DCs that failed to do so, efficiently induced a rapid increase in lymph node cellularity, which was observed before the onset of T cell proliferation. We also report that DC migration could be increased up to 10-fold by preinjection of inflammatory cytokines that increased the expression of the CCR7 ligand CCL21 in lymphatic endothelial cells. The magnitude and quality of CD4+ T cell response was proportional to the number of antigen-carrying DCs that reached the lymph node and could be boosted up to 40-fold by preinjection of tumor necrosis factor that conditioned the tissue for increased DC migration. These results indicate that DC number and tissue inflammation are critical parameters for DC-based vaccination.

Chemokines regulate both homeostatic leukocyte recirculation and trafficking to sites of infection and inflammation. Apart from the well-established physiological functions, chemokines receive growing interest for their role in pathophysiological processes such as autoimmune diseases, cancer, and allograft rejection. The chemokine receptor CCR7, which is responsible for directing T cells, B cells, and dendritic cells (DCs) into secondary lymphoid organs and their precise positioning therein, has already been implicated in lymphoid organ infiltration by neoplastic cells and the localization of metastasis formation. We have shown that the differential expression of CCR7 by neoplastic cells in two entities of Hodgkin's disease (HD), classic HD (cHD) and the nodular lymphocyte predominant HD (NLPHD), may account for the differences observed in tumor cell dissemination within the affected lymph nodes. Because of the prominent role of the chemokine receptors CCR7 and CXCR5 in lymphocyte homing to secondary lymphoid organs, we hypothesized that they may also be involved in the action of FTY720, a synthetic immunosuppressant inducing lymphopenia. By using CXCR5 and CCR7 knockout mice, we have tested for a possible function of these receptors in the FTY720-induced migration of lymphocytes into Peyer's patches (PPs) and peripheral lymph nodes (PLNs). Lymphopenia is noticeably delayed in mice lacking CCR7, whereas CXCR5 knockout mice show a significant reduction of lymphocyte accumulation in secondary lymphoid organs that are infrequently present in these mice. However, FTY720-induced lymphocyte sequestration appears to be essentially independent of CCR7 and CXCR5.

B lymphocytes are a highly dynamic cell population. During their lifetime they travel over distances that are many times larger than their own size. Mature B cells constantly travel throughout the body, patrolling for antigens. This migration process is tightly regulated through a system of chemokines and adhesion molecules, which act like address codes to bring the B cells to their destination in various tissues. Moreover, already during their maturation process in the bone marrow, B cells of various developmental stages are undergoing distinct changes in their localization. This chapter focuses on the localization of various B cell subsets and on how their migration, adhesion, and interactions with tissue-resident stromal cells are regulated on a molecular level to ensure an efficient humoral immune response against pathogens. Apart from their role in protective immune responses, the same mechanisms that guide the motility of B cells can be detrimental under certain circumstances (e.g., in chronic inflammation and lymphoma dissemination).

Chemokines and chemokine receptors are key mediators for regulating cell traffic and positioning in both homeostatic and inflammatory conditions. It is also presumed that chemokines and their receptors are likely to play a critical role in the localization of malignant hematopoietic cells in their target organs. This study analyzed chemokine and chemokine receptor expression in several Hodgkin disease (HD)–derived cell lines and in HD tumors. All HD-derived cell lines expressed functional CCR7 and CXCR4 receptors. CCR7 up-regulation was mediated by constitutive NF-κB activity. Lymphoid tissues in HD revealed differential expression levels of CCR7, CXCR4, and CXCR5, depending on the distinct subtypes of HD. HD of the classical subtypes, predominantly located in the interfollicular zone, showed strong CCR7 and CXCR4 expression and moderate CXCR5 expression. In contrast, the nodular lymphocyte-predominant HD (NLP) subtype, regularly associated with follicular structures, exhibited no CCR7 reactivity but abundant CXCR4 staining. Their respective chemokine ligands showed marked expression by reactive cells within the tumors of classical HD and outside of the tumor nodules in NLPHD. Functionally, such differential chemokine receptor expression might contribute to specific localization and confinement of neoplastic cells within the target organs in the distinct HD entities.

Most T lymphocytes are generated within the thymus. It is unclear, however, how newly generated T cells relocate out of the thymus to the circulation. The present study shows that a CC chemokine CCL19 attracts mature T cells out of the fetal thymus organ culture. Another CC chemokine CCL21, which shares CCR7 with CCL19 but has a unique C-terminal extension containing positively charged amino acids, failed to show involvement in thymic emigration. Neonatal appearance of circulating T cells was defective in CCL19-neutralized mice as well as in CCR7-deficient mice but not in CCL21-neutralized mice. In the thymus, CCL19 is predominantly localized in the medulla including endothelial venules. These results indicate a CCL19- and CCR7-dependent pathway of thymic emigration, which represents a major pathway of neonatal T cell export.

We recently demonstrated that gene-targeted disruption of the C5a anaphylatoxin receptor prevented lung injury in immune complex–mediated inflammation. In this study, we compare the effect of C5aR deficiency in immune complex–induced inflammation in the peritoneal cavity and skin with the results derived from our immune complex alveolitis model. C5aR- deficient mice exhibit decreased migration of neutrophils and decreased levels of TNF-α and interleukin 6 in the peritoneal reverse passive Arthus reaction compared to their wild-type littermates. In the reverse passive Arthus reaction in the skin the C5aR was also required for the full expression of neutrophil influx and edema formation; C5aR-deficient mice showed reduced neutrophil migration and microvascular permeability changes. In contrast to our studies in immune complex–induced lung inflammation, C5aR deficiency does not completely prevent injury in the peritoneal cavity and skin. These data indicate a dominant role for the C5aR and its ligand in the reverse passive Arthus reaction in the lung and a synergistic role together with other inflammatory mediators in immune complex–mediated peritonitis and skin injury.

The formation of intrapulmonary immune complexes in mice generates a vigorous inflammatory response characterized by microvascular permeability and polymorphonuclear neutrophil influx. Gene-targeted disruption of the substance P receptor (NK-1R) protected the lung from immune complex injury, as did disruption of the C5a anaphylatoxin receptor. Immunoreactive substance P was measurable in fluids lining the lung at time points before neutrophil influx and may thus be involved in an early step in the inflammatory response to immune complexes in the lung.

Migration of dendritic cells to secondary lymphatic organs is a key event in acquired immunity. The role of the multifunctional ectoenzyme CD38 in humoral immune responses has now been revisited, suggesting that CD38 links innate and adaptive immunity by triggering chemokine-mediated dendritic cell chemotaxis.

The chemokine receptor CCR7 is a central regulator in the maintenance of cellular homeostasis of mucosal tissues. CCR7⁻/⁻ mice develop autoimmune gastritis and exocrinopathy accompanied by the formation of mucosal tertiary lymphoid follicles. Here we found that CCR7-deficient mice frequently suffered from chronic diarrhea linked with increased gastrointestinal motility and the development of severe anorectal prolapse. Enhanced formation of intestinal lymphoid follicles was associated with an elevated proportion of activated colonic T cells and increased production of the cytokine interleukin (IL)-1β. To uncover the pathomechanisms of diarrhea in CCR7⁻/⁻ mice, colonic epithelial barrier and ion channel activities were analyzed in Ussing chambers. Although overt acute colitis was absent, CCR7 deficiency resulted in reduced electrogenic sodium absorption and colonic chloride secretion. As it is known that IL-1β regulates epithelial sodium channel (ENaC) activity, these data imply a causal link between CCR7 expression, IL-1β level, and Na⁺ malabsorption owing to altered ENaC expression and diarrhea.

In this issue of Blood, Fhu et al report that Reed-Sternberg cell-derived lymphotoxin-{alpha} activates endothelial cells to enhance T-cell recruitment in classical Hodgkin lymphoma (cHL), a process that is regulated by cyclooxygenase/nuclear factor-{kappa}B/activator protein 1 signaling pathways.

Ectopic lymphoid follicles are a key feature of chronic inflammatory autoimmune and infectious diseases, such as rheumatoid arthritis, Sjogren's syndrome, and Helicobacter pylori-induced gastritis. Homeostatic chemokines are considered to be involved in the formation of such tertiary lymphoid tissue. High expression of CXCL13 and its receptor, CXCR5, has been associated with the formation of ectopic lymphoid follicles in chronic infectious diseases. Here, we defined the role of CXCR5 in the development of mucosal tertiary lymphoid tissue and gastric inflammation in a mouse model of chronic H. pylori infection. CXCR5-deficient mice failed to develop organized gastric lymphoid follicles despite similar bacterial colonization density as infected wild-type mice. CXCR5 deficiency altered Th17 responses but not Th1-type cellular immune responses to H. pylori infection. Furthermore, CXCR5-deficient mice exhibited lower H. pylori-specific serum IgG and IgA levels and an overall decrease in chronic gastric immune responses. In conclusion, the development of mucosal tertiary ectopic follicles during chronic H. pylori infection is strongly dependent on the CXCL13/CXCR5 signaling axis, and lack of de novo lymphoid tissue formation attenuates chronic immune responses.

The location of embryonic lymph node development is determined by the initial clustering of lymphoid tissue-inducer (LTi) cells. Here we demonstrate that both the chemokine CXCL13 and the chemokine CCL21 attracted LTi cells at embryonic days 12.5-14.5 and that initial clustering depended exclusively on CXCL13. Retinoic acid (RA) induced early CXCL13 expression in stromal organizer cells independently of lymphotoxin signaling. Notably, neurons adjacent to the lymph node anlagen expressed enzymes essential for RA synthesis. Furthermore, stimulation of parasymphathetic neural output in adults led to RA receptor (RAR)-dependent induction of CXCL13 in the gut. Therefore, our data show that the initiation of lymph node development is controlled by RA-mediated expression of CXCL13 and suggest that RA may be provided by adjacent neurons.

Foxp3+CD25+CD4+ Treg play a fundamental role in the maintenance of self tolerance and the control of inflammatory reactions. Previous data demonstrated a division of labor between naive- and effector/memory-like Treg subsets, which is largely based on their lymph node-recirculating and inflammation-seeking migration behavior, respectively. The chemokine receptor CCR7 is expressed on both types of Treg subsets, albeit at different levels. Whether it fulfills similar or distinct roles in these subsets has not been studied so far. We here show that the recirculation of naive-like Treg through LN and, to some extent, the gut is dependent on CCR7. Lack of CCR7 not only prevents recirculation, but also almost completely abolishes the ability of naive-like Treg to control the priming phase of an immune response. In contrast, CCR7 deficiency in effector/memory-like Treg promotes their accumulation in inflamed sites, compatible with a role of CCR7 for exit from the tissue. Local Treg accumulation was accompanied by an enhanced suppression of inflammation. Together, our findings provide conclusive evidence that CCR7 expression on Treg differentially controls in vivo function of the naive- and effector/memory-like subsets.

The presence of an anti-bacterial T cell response and evidence of bacterial products in inflamed joints of reactive arthritis patients suggests an antigen transportation role in this disease for macrophages and dendritic cells. We have investigated the functional properties and in vivo migration of macrophages and DC after infection with Salmonella enterica serovar Typhimurium (S. typhimurium). BM-derived macrophages and DC displayed enhanced expression of costimulatory molecules (CD40 and CD86) and increased production of pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-12p40) and nitric oxide after infection. Upon adoptive transfer into mice, infected DC migrated to lymphoid tissues and induced an anti-Salmonella T cell response, whereas infected macrophages did not. Infection of DC with S. typhimurium was associated with strong up-regulation of the chemokine receptor CCR7 and acquisition of responsiveness to chemokines acting through this receptor. Moreover, S. typhimurium-infected CCR7-deficient DC were unable to migrate to lymph nodes after adoptive transfer, although they did reach the spleen. Our data demonstrate distinct roles for macrophages and DC as antigen transporters after S. typhimurium infection and a dependence on CCR7 for migration of DC to lymph nodes after bacterial infection.

The hallmark of pulmonary tuberculosis is the granuloma, which consists predominantly of lymphocytes and macrophages and promotes immune-cell interaction with the causative pathogen, Mycobacterium tuberculosis. Granuloma formation is a highly organized process, which depends on leukocyte recruitment facilitated by adhesion molecules and chemokines. Thus, during chronic experimental tuberculosis, granulomata display characteristics of lymphoid structures comprising follicular aggregation of B cells, formation of high endothelial venules, presence of follicular dendritic cells, and expression of the homeostatic chemokines CXCL13 and CCL19. CCR7-/- mice, which are deficient in CCL19 and CCL21 signaling, exhibit increased local inflammatory infiltrates but no follicular B-cell aggregation within those lymphoid structures. However, CCR7-deficient mice are fully capable to control pulmonary tuberculosis; at time points later than 6 weeks postinfection, they carry a lower bacterial load in peripheral organs. Our results show that, during chronic pulmonary tuberculosis in mice, the homeostatic chemokine signaling-network contributes to spatial organization of the granulomatous response, which participates in both containment of M. tuberculosis and the latter's dissemination to other organs.

The chemokine receptor CCR7 and its ligands regulate migration and colocalization of T cells and mature dendritic cells to and within secondary lymphoid organs. The requirement of CCR7 in efficient priming of allospecific cytotoxic CD8(+) T cells is poorly characterized. Here, we demonstrate a role for CCR7 in the initiation of an alloimmune response and in the development of transplant rejection. Remarkably, in a model of acute allogeneic tumor rejection, CCR7(-/-) mice completely failed to reject subcutaneously injected MHC class I mismatched tumor cells and cytotoxic activity of allospecific T cells was severely compromised. When solid tumors derived from wild-type mice were transplanted, recipient CCR7(-/-) mice were capable of rejecting the allografts. In contrast, tumor allografts transplanted from CCR7(-/-) donors onto CCR7(-/-) recipients showed allograft survival up to 28 days, suggesting a critical function of CCR7 on donor-type passenger leukocytes in the initiation of cytotoxic CD8(+) T cell responses. In a heterotopic heart transplantation model CCR7 deficiency resulted in significantly prolonged but not indefinite allograft survival. Additional prolongation of graft survival was observed when hearts from CCR7(-/-) mice were used as donor organs. Our results define a key role for CCR7 in allogeneic T cell priming within the context of draining lymph nodes.

Chemokines and their receptors fulfill specialized roles in inflammation and under homeostatic conditions. CCR7 and its ligands, CCL19 and CCL21, are involved in lymphocyte recirculation through secondary lymphoid organs and additionally navigate lymphocytes into distinct tissue compartments. The role of CCR7 in the migration of polarized T effector/memory cell subsets in vivo is still poorly understood. We therefore analyzed murine and human CD4+ cytokine-producing cells developed in vivo for their chemotactic reactivity to CCR7 ligands. The responses of cells producing cytokines, such as IFN-γ, IL-4, and IL-10, as well as of subsets defined by memory or activation markers were comparable to that of naive CD4+ cells, with slightly lower reactivity in cells expressing IL-10 or CD69. This indicates that CCR7 ligands are able to attract naive as well as the vast majority of activated and effector/memory T cell stages. Chemotactic reactivity of these cells toward CCL21 was absent in CCR7-deficient cells, proving that effector cells do not use alternative receptors for this chemokine. Th1 cells generated from CCR7−/− mice failed to enter lymph nodes and Peyer’s patches, but did enter a site of inflammation. These findings indicate that CD4+ cells producing effector cytokines upon stimulation retain the capacity to recirculate through lymphoid tissues via CCR7.

Human cytomegalovirus (HCMV) encodes several proteins that inhibit major histocompatibility complex (MHC) class I-dependent antigen presentation. The HCMV products US2 and US11 are each sufficient for causing the dislocation of human and murine MHC class I heavy chains from the lumen of the endoplasmic reticulum to the cytosol, where the heavy chains are readily degraded. The apparent redundancy of US2 and US11 has been probed predominantly in cultured cell lines, where differences in their specificities were shown for murine and human MHC class I locus products. Here, we expressed US11 and US2 via adenovirus vectors and show that US11 exhibits a superior ability to degrade MHC class I molecules in primary human dendritic cells. MHC class II complexes are unaffected by US2- and US11-mediated attack. We suggest that multiple HCMV-encoded immunoevasions have evolved complementary functions in response to diverse host cell types and tissues.

A FAMILY of G-protein-eoupled chemoattractant receptors is known to mediate the transport and activation of neutrophils and macrophages. This family includes receptors for chemokines, such as interleukin-8, bacterial formylated peptides, platelet-activating factor, leukotriene B4, and the complement anaphyla-toxins1–3. The apparent redundancy of these receptors suggests that they have an important underlying role in host defence. To isolate the contribution of particular molecules, we disrupted a gene that encodes a single chemoattractant receptor. Here we show that mice deficient in the chemoattractant C5a receptor, in comparison to their wild-type littermates, were unable to clear intrapulmonary-instilled Pseudomonas aeruginosa, despite a marked increase in neutrophil influx, and succumbed to pneumonia. These C5a-receptor-deficient mice challenged with sub-lethal inocula of Pseudomonas become superinfected with secondary bacterial strains. We conclude that the C5a receptor has a non-redundant function, and is required for mucosal host defence in the lung.

The complement activation fragment C5a was recently shown to induce interleukin (IL)-6 synthesis by peripheral blood mononuclear cells. To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti-C5a monoclonal antibodies (mAb) on IL-6 generation in an animal model of septic shock. Complement-depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL-6 during the 6-h observation period than complement-sufficient controls. To address specifically the role of C5a in IL-6 regulation, we produced a C5a(57-74) peptide-specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a. The mAb T13/9 does not cross-react with the precursor protein C5. The pretreatment of pigs with anti-C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75% in serum IL-6 bioactivity compared to control animals (p < 0.0001). These results indicate a role for C5a in the modulation of IL-6 synthesis in Gram-negative bacteremia.

SummaryImmunological control of infections or tumors depends on the release of effector cytokines and polarized secretion of cytotoxic granules from T cells and natural killer cells. Here we show that the sorting receptor Sortilin controlled both processes. In murine Sortilin-deficient cytotoxic T lymphocytes, regulated secretion of granzyme A and cytotoxic killing was enhanced and correlated with increased vesicle-associated membrane protein 7 availability. In contrast, loss of Sortilin reduced the release of interferon-γ upon infections and in autoimmune colitis. Exit of interferon-γ from the Golgi apparatus required the presence of Sortilin. Furthermore, we tracked the transport route of interferon-γ beyond this Sortilin-dependent Golgi to early endosome step. In wild-type T cells, trafficking of interferon-γ from the endosomal sorting platform to the plasma membrane proceeded independently of recycling endosomes, and interferon-γ remained excluded from late endosomes. Our results suggest that Sortilin modulates systemic immune responses through exocytic sorting of immunological effector molecules.

The adoptive immune response relies on a precise temporal and spatial positioning of lymphocytes within lymphoid and nonlymphoid tissues. Chemokines, constitutively expressed or induced during inflammation provide a flexible navigation system directing lymphocytes into specific microcompartments. Precision and specificity in this process are achieved by varying patterns of chemokine receptors expressed on the cell surface of lymphocytes in the course of cell differentiation. The chemokine receptors CXCR5 and CCR7 are principal regulators for targeting T cells, B cells, and dendritic cells into secondary lymphoid organs. The analyses of knockout mice have been instrumental in exploring the crucial role of these receptors for the compartmentalization of secondary lymphoid organs into functionally separated T and B cell zones. Aside from the homeostatic recirculation of lymphocytes and inflammatory processes, chemokine receptors are also involved in malignancies such as lymphoproliferative diseases and cancer metastasis. Recent results from our laboratory present evidence for the involvement of CCR7 in the dissemination of neoplastic cells in classic Hodgkin disease. There is also accumulating evidence for the involvement of CXCR5 in the formation of ectopic follicles as observed in lymphomas or autoimmune diseases. In addition, CCR7 and CXCR5 have been identified as useful markers in the classification of functionally distinct subsets of T-helper cells, which will lead to a better understanding of T cell memory and T cell effector function in lymphoid system homeostasis and disease.