TNF and lymphotoxin beta cooperate in the maintenance of secondary lymphoid tissue microarchitecture but not in the development of lymph nodes

Inactivation of genes encoding members of TNF and TNF receptor families reveal their divergent roles in the formation and function of secondary lymphoid organs. Most lymphotoxin alpha (ltalpha)- and all lymphotoxin beta receptor (ltbetar)-deficient mice are completely devoid of lymph nodes (LNs); however, most lymphotoxin beta (ltbeta)-deficient mice develop mesenteric LNs. Tnf- and tnfrp55-deficient mice develop a complete set of LNs, while ltbeta/tnfrp55 double-deficient mice lack all LNs, demonstrating cooperation between LTbeta and TNFRp55 in LN development. Now we report that ltbeta/tnf double-deficient mice develop the same set of mucosal LNs as do ltbeta-deficient mice, suggesting that ligands other than TNF signal through TNFRp55 during LN development. These LNs retain distinct T and B cells areas; however, they lack follicular dendritic cell networks. Structures resembling germinal centers can be found in the LNs from immunized ltbeta-deficient mice but not in ltbeta/tnf double-deficient mice. Additionally, stromal components of the spleen and LNs appear to be more severely disturbed in ltbeta/tnf double-deficient mice as compared with ltbeta-deficient mice. We conclude that LTbeta and TNF cooperate in the establishment of the correct microarchitecture of lymphoid organs.     

The role of lymphotoxin signaling in the development of autoimmune pancreatitis and associated secondary extra-pancreatic pathologies

The pathogenic mechanisms of autoimmune pancreatitis (AIP), an increasingly recognized, immune-mediated form of chronic pancreatitis, have so far remained elusive. Treatment options for AIP are currently limited and disease relapse is frequent. Still, AIP can be characterized by specific clinical and histologic features. It has turned out that as described in other autoimmune diseases the generation of tertiary lymphoid organs is also a hallmark of patients with AIP. We have recently demonstrated that pancreata derived from human AIP patients display overexpression of lymphotoxin (LT) α and β and LTβR-target genes expressed by immune cells but also by irradiation resistant cells of the pancreas (e.g. acinar cells). Expression of LT α and β on acinar cells in murine pancreata Tg(Ela1-Lta,b) mice led to chronic pancreatitis and sufficed to reproduce key features of human AIP including the development of autoimmunity and AIP associated secondary extra pancreatic pathologies. Here, we review how aberrant and ectopic expression of LT α and β can induce inflammation and autoimmune diseases in general and how this knowledge might specifically lead to an alternative treatment for patients suffering from autoimmune pancreatitis.     

The development of lymphoid and haemopoietic tissues in pig fetuses

This paper presents the results of the development of lymphatic and haemopoietic organs in pig fetuses of various ages. The thymus appears to be the first lymphatic organ in these fetuses as well as in other animal species so far studied. On the 77th day the thymus is fully morphologically developed. The accumulations of lymphocytes in the spleen appear on the 70th day. The development of periarteriolar formations takes place around the 84th day of gestation. Further development of lymphoid tissue in the lymph nodes, tongue (tonsilla lingualis) and intenstine is described. Lymphatic follicles were observed both in the tongue and the small intestine on the 77th day. The dynamics of haemopoietic activity in the liver and bone marrow is characterized. The germinal centers in lymphoid folicles were never observed as well as cells of the plasmatic series.     

Expression of the lymphotoxin beta receptor on follicular stromal cells in human lymphoid tissues

The lymphotoxin beta receptor (LTbetaR), and its ligand, LTalpha1beta2, have been proposed to play a key role in the development and organization of lymphoid tissues. The LTbetaR is expressed on a variety of human primary and transformed cells, but strikingly absent on T or B lymphocytes and primary monocytes or peripheral dendritic cells, although LTbetaR is detected on some myeloid leukemic lines. In the developing thymus LTbetaR is prominent along the trabeculae and into the medulla upto corticomedullary junction. In the spleen, LTbetaR is prominently expressed by cells in the red pulp and along the borders of red and white pulp which colocalizes with reticular stromal cells. The LTbetaR is expressed on a human follicular dendritic cell line, FDC-1, and signals expression of CD54 when ligated with the LTalpha1beta2 complex. These results support the concept that directional interactions between LTalpha1beta2 bearing lymphocytes and LTbetaR bearing stromal cells are involved in the organization of lymphoid tissue.     

Postnatal development of mucosa-associated lymphoid tissues in chickens

Summary The postnatal development of chicken mucosa-associated lymphoid tissues of the eyes, lungs, and intestines were investigated with monoclonal antibodies specific for either all leucocytes, B lymphocytes, mononuclear phagocytes, IgM, IgG, or IgA. Attention has been paid to the relation of lymphoid infiltrates with their surrounding mucosae, the segregation into B-cell and T-cell areas, development of germinal centers, and secretory immunoglobulins. Abudant secretory IgM and IgA was detected in the epithelium of the Harderian glands in the orbits, even though they lacked large leucocyte infiltrates with germinal centers. Lymphoid tissues in the mucosae of lungs and intestines developed separate B-cell and T-cell areas. The proventriculus, Meckel's diverticulum, and Peyer's patches generally contained germinal centers from 12 weeks of age on. Because chickens as young as 2 weeks old had germinal centers in bronchus-associated lymphoid tissue and cecal tonsils, these areas were probably highly stimulated by antigens. Isotype-specific monoclonal antibodies were used to detect IgM-, IgG-, and IgA-bearing follicular cells in the same germinal center.     

The role of lymphotoxin receptor signaling in diseases

LT, LIGHT, and TNF are core family members of the TNFR superfamily of cytokines. LT and LIGHT, produced primarily by lymphocytes, interact with LTbetaR expressed by stromal and epithelial cells. Extensive studies over the last decade have revealed a critical role of LT-LTbetaR interactions for organogenesis and maintenance of the secondary lymphoid organs and in the generation of an efficient humoral immune response to various pathogens. LTbetaR's function beyond the lymphoid organs shows valuable potential yet remains largely undefined. Recent studies indicate that LTbetaR signaling is required for liver regeneration, hepatitis, and hepatic lipid metabolism. The balance of beneficial and detrimental effects of LTbetaR is critical for understanding the mechanisms of autoimmune disease and liver function and may open a new avenue for therapeutic intervention. This review will discuss recent advances in understanding LTbetaR's role in various human and murine disease models while focusing on its regulation of and implications in various liver related diseases.     

Defining early human NK cell developmental stages in primary and secondary lymphoid tissues

A better understanding of human NK cell development in vivo is crucial to exploit NK cells for immunotherapy. Here, we identified seven distinctive NK cell developmental stages in bone marrow of single donors using 10-color flow cytometry and found that NK cell development is accompanied by early expression of stimulatory co-receptor CD244 in vivo. Further analysis of cord blood (CB), peripheral blood (PB), inguinal lymph node (inLN), liver lymph node (liLN) and spleen (SPL) samples showed diverse distributions of the NK cell developmental stages. In addition, distinctive expression profiles of early development marker CD33 and C-type lectin receptor NKG2A between the tissues, suggest that differential NK cell differentiation may take place at different anatomical locations. Differential expression of NKG2A and stimulatory receptors (e.g. NCR, NKG2D) within the different subsets of committed NK cells demonstrated the heterogeneity of the CD56^brightCD16^+/− and CD56^dimCD16⁺ subsets within the different compartments and suggests that microenvironment may play a role in differential in situ development of the NK cell receptor repertoire of committed NK cells. Overall, differential in situ NK cell development and trafficking towards multiple tissues may give rise to a broad spectrum of mature NK cell subsets found within the human body.     

The role of secondary heart field in cardiac development

Although de la Cruz and colleagues showed as early as 1977 that the outflow tract was added after the heart tube formed, the source of these secondarily added cells was not identified for nearly 25 years. In 2001, three pivotal publications described a secondary or anterior heart field that contributed to the developing outflow tract. This review details the history of the heart field, the discovery and continuing elucidation of the secondarily adding myocardial cells, and how the different populations identified in 2001 are related to the more recent lineage tracing studies that defined the first and second myocardial heart fields/lineages. Much recent work has focused on secondary heart field progenitors that give rise to the myocardium and smooth muscle at the definitive arterial pole. These progenitors are the last to be added to the arterial pole and are particularly susceptible to abnormal development, leading to conotruncal malformations in children. The major signaling pathways (Wnt, BMP, FGF8, Notch, and Shh) that control various aspects of secondary heart field progenitor behavior are discussed.     

An ENU-induced mutation in the lymphotoxin alpha gene impairs organogenesis of lymphoid tissues in C57BL/6 mice

The TNF family is critical for development of lymphoid organs and plays significant roles in regulation of innate and adoptive immune responses. Here, we describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutant strain, HLB382, with a point mutation in the Lta gene, which encodes lymphotoxin (LT) α, a member of the TNF family. Mutant mice had no lymph nodes and no Peyer’s patches. Microscopically, the spleens had disordered follicles and no germinal centers or discernible marginal zones (MZ). While the development of T cells and follicular B cells was normal, the numbers of NK and MZ B cells were significantly reduced. Interestingly, the numbers of peritoneal B1b cells were significantly increased. Genomic DNA sequences revealed a single base pair insertion in the coding region of Lta resulting in a frame shift and a premature stop codon. This new strain provides opportunities for understanding the full range of Lta gene function on a pure C57BL/6 background.     

Effector T cell differentiation and memory T cell maintenance outside secondary lymphoid organs

Naive T cell circulation is restricted to secondary lymphoid organs. Effector and memory T cells, in contrast, acquire the ability to migrate to nonlymphoid tissues. In this study we examined whether nonlymphoid tissues contribute to the differentiation of effector T cells to memory cells and the long-term maintenance of memory T cells. We found that CD4, but not CD8, effector T cell differentiation to memory cells is impaired in adoptive hosts that lack secondary lymphoid organs. In contrast, established CD4 and CD8 memory T cells underwent basal homeostatic proliferation in the liver, lungs, and bone marrow, were maintained long-term, and functioned in the absence of secondary lymphoid organs. CD8 memory T cells found in nonlymphoid tissues expressed both central and effector memory phenotypes, whereas CD4 memory T cells displayed predominantly an effector memory phenotype. These findings indicate that secondary lymphoid organs are not necessary for the maintenance and function of memory T cell populations, whereas the optimal differentiation of CD4 effectors to memory T cells is dependent on these organs. The ability of memory T cells to persist and respond to foreign Ag independently of secondary lymphoid tissues supports the existence of nonlymphoid memory T cell pools that provide essential immune surveillance in the periphery.     

Developmental stage-dependent collaboration between the TNF receptor-associated factor 6 and lymphotoxin pathways for B cell follicle organization in secondary lymphoid organs

Signal transduction pathways regulating NF-kappaB activation essential for microenvironment formation in secondary lymphoid organs remain to be determined. We investigated the effect of a deficiency of TNFR-associated factor 6 (TRAF6), which activates the classical NF-kappaB pathway, in splenic microenvironment formation. Two-week-old TRAF6-deficient mice showed severe defects in B cell follicle and marginal zone formation, similar to mutant mice defective in lymphotoxin (Lt) beta receptor (LtbetaR) signal induction of nonclassical NF-kappaB activation. However, analysis revealed a TRAF6 role in architecture formation distinct from its role in the early neonatal Lt signaling pathway. LtbetaR signal was essential for primary B cell cluster formation with initial differentiation of follicular dendritic cells (FDCs) in neonatal mice. In contrast, TRAF6 was dispensable for progression to this stage but was required for converting B cell clusters to B cell follicles and maintaining FDCs through to later stages. Fetal liver transfer experiments suggested that TRAF6 in radiation-resistant cells is responsible for follicle formation. Despite FDC-specific surface marker expression, FDCs in neonatal TRAF6-deficient mice had lost the capability to express CXCL13. These data suggest that developmentally regulated activation of TRAF6 in FDCs is required for inducing CXCL13 expression to maintain B cell follicles.     

Blocking lymphotoxin signaling abrogates the development of ectopic lymphoid tissue within cardiac allografts and inhibits effector antibody responses

Tertiary lymphoid organs (TLOs) may develop within allografts, but their contribution to graft rejection remains unclear. Here, we study a mouse model of autoantibody-mediated cardiac allograft vasculopathy to clarify the alloimmune responses mediated by intragraft TLOs and whether blocking lymphotoxin-β-receptor (LTβR) signaling, a pathway essential for lymphoid organogenesis, abrogates TLO development. TLOs (defined as discrete lymphoid aggregates associated with high endothelial venules) were detectable in 9 of 13 heart allografts studied and were predominantly B cell in composition, harboring germinal-center activity. These are most likely manifestations of the humoral autoimmunity triggered in this model after transplantation; TLOs did not develop if autoantibody production was prevented. Treatment with inhibitory LTβR-Ig fusion protein virtually abolished allograft TLO formation (mean TLOs/heart: 0.2 vs. 2.2 in control recipients; P=0.02), with marked attenuation of the autoantibody response. Recipients primed for autoantibody before transplantation rejected grafts rapidly, but this accelerated rejection was prevented by postoperative administration of LTβR-Ig (median survival time: 18 vs. >50 d, respectively, P=0.003). Our results provide the first demonstration that TLOs develop within chronically rejecting heart allografts, are predominantly B cell in origin, and can be targeted pharmacologically to inhibit effector humoral responses.     

The effect of primary and secondary immunization on the lymphoid tissues of the carp, Cyprinus carpio L

Mirror carp immunized with human gamma globulin (HGG) in Freund's complete adjuvant (FCA) show a proliferative response involving cells whose cytoplasm stains deep red with methyl green-pyronin (pyroninophilic cells). This response occurs particularly in the haemopoietic parenchyma of the pronephros and mesonephros. It peaks at week 3, with the formation of clusters of pyroninophilic cells in the pronephros. Immunization with Aeromonas salmonicida elicited a less intense pyroninophilic response but caused a larger increase in pigment-containing cells. After a secondary immunization with HGG in FCA, a distinct response was observed in the spleen: Pyroninophilic cells collected within the ellipsoid sheaths in large numbers and formed nodules. The reticulum of such nodules acquired spherical proportions and resembled the white pulp reticulum of the tetrapod spleen. The roles of such pyroninophilic cells and the possibility that aggregations of them may be functionally analogous to homoiotherm germinal centres are discussed.     

Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire

Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance. In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT. We studied the mechanism of bacteria-induced GALT development. Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits). We then identified specific members of the intestinal flora that promote GALT development. The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells. Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis. B. fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response. To identify bacterial pathways required for GALT development, we introduced B. fragilis along with stress-response mutants of B. subtilis into germfree-appendix rabbits. We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development. We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.     

Organogenesis of lymphoid tissues

The development of lymphoid organs depends on the correct expression of several molecules within a defined timeframe during ontogeny. Although this is an extremely complex process, with each secondary lymphoid tissue requiring subtly different signals, a common framework for lymphoid development is beginning to emerge. Drawing on studies of lymph nodes, Peyer's patches and nasal-associated lymphoid tissue, an integrative model of lymphoid-tissue development, involving adhesion molecules, cytokines and chemokines, which emphasizes the role of interactions between CD3-CD4+CD45+ 'inducer' cells and VCAM1+ICAM1+ stromal 'organizer' cells is presented.     

The role of syndecans in lymphoid systems

Syndecans, transmembrane heparan sulfate proteoglycans, play an important role in cell-cell and cell-matrix interactions, as receptors/co-receptors of matrix elements, cytokines, growth factors. These functions are partly non-specific and due to the heparan sulfate chains attached to the ectodomain, and partly specific related to the transmembrane and cytoplasmic domains of the core protein. In hemopoietic cells syndecan-1 is expressed in certain B cells, in pre-B cells and plasma cells. In lymphoproliferative diseases this normal syndecan-1 expression of plasma cells is retained in myelomas/plasmocytomas, other lymphoplasmocytic NHL subtypes and primary effusional lymphomas. Syndecan-1 expression is probably gained in B-CLL, and lost in other NHLs of pre- or post-follicular origin. These results suggest that the expression of syndecan is essential for some NHLs, probably ensuring the required connections to the microenvironment. From a diagnostic point of view, syndecan-1 is a very useful phenotypic marker to identify cells with plasmocytic differentiation. The importance of syndecan expression in CLL and Hodgkin-lymphoma still requires further studies.     

The role of proton dynamics in the development and maintenance of multidrug resistance in cancer

With a projected 382.4 per 100,000 people expected to suffer from some form of malignant neoplasm in 2015, improving treatment is an essential focus of cancer research today. Multi-drug resistance (MDR) is the leading cause of chemotherapeutic failure in the treatment of cancer, the term denoting a characteristic of the disease-causing agent to avoid damage by drugs designed to bring about their destruction. MDR is also characterised by a reversal of the pH gradient across cell membranes leading to an acidification of the outer milieu and an alkalinisation of the cytosol that is maintained by the proton pump vacuolar-type ATPase (V-ATPase) and the proton transporters: Na⁺/H⁺ exchanger (NHE1), Monocarboxylate Transporters (MCTs), Carbonic anhydrases (CAs) (mainly CA-IX), adenosinetriphosphate synthase, Na⁺/HCO₃^? co-transporter and the Cl^?/HCO₃^?exchanger. This review aims to give an introduction to MDR. It will begin with an explanation for what MDR actually is and go on to look at the proposed mechanisms by which a state of drug resistance is achieved. The role of proton-pumps in creating an acidic extracellular pH and alkaline cytosol, as well as key biomechanical processes within the cell membrane itself, will be used to explain how drug resistance can be sustained.     

Neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues

Antigen-transporting cells take up pathogens, and then migrate from sites of inflammation to secondary lymphoid tissues to induce an immune response. Among antigen-transporting cells, dendritic cells (DCs) are believed to be the most potent and professional antigen-presenting cells that can stimulate naïve T cells. However, the cells that transport antigens, tumor cell antigens in particular, have not been clearly identified. In this study we have analyzed what types of cells transport tumor cell antigens to secondary lymphoid tissues. We show that neutrophils, monocytes and macrophages but not DCs engulf X-irradiated P388 leukemic cells after their injection into the peritoneal cavity, and that neutrophils and monocytes but not macrophages migrate to the parathymic lymph nodes (pLN), the blood, and then the spleen. The monocytes in the pLN comprise Gr-1⁻ and Gr-1⁺ ones, and some of these cells express CD11c. Overall, this study demonstrates that neutrophils and monocytes transport tumor cell antigens from the peritoneal cavity to secondary lymphoid tissues.     

The abundant NK cells in human secondary lymphoid tissues require activation to express killer cell Ig-like receptors and become cytolytic

Natural killer cells are important cytolytic cells in innate immunity. We have characterized human NK cells of spleen, lymph nodes, and tonsils. More than 95% of peripheral blood and 85% of spleen NK cells are CD56(dim)CD16(+) and express perforin, the natural cytotoxicity receptors (NCRs) NKp30 and NKp46, as well as in part killer cell Ig-like receptors (KIRs). In contrast, NK cells in lymph nodes have mainly a CD56(bright)CD16(-) phenotype and lack perforin. In addition, they lack KIRs and all NCR expression, except low levels of NKp46. The NK cells of tonsils also lack perforin, KIRs, NKp30, and CD16, but partially express NKp44 and NKp46. Upon IL-2 stimulation, however, lymph node and tonsilar NK cells up-regulate NCRs, express perforin, and acquire cytolytic activity for NK-sensitive target cells. In addition, they express CD16 and KIRs upon IL-2 activation, and therefore display a phenotype similar to peripheral blood NK cells. We hypothesize that IL-2 can mobilize the NK cells of secondary lymphoid tissues to mediate natural killing during immune responses. Because lymph nodes harbor 40% and peripheral blood only 2% of all lymphocytes in humans, this newly characterized perforin(-) NK cell compartment in lymph nodes and related tissues probably outnumbers perforin(+) NK cells. These results also suggest secondary lymphoid organs as a possible site of NK cell differentiation and self-tolerance acquisition.