Lymph node stromal cells: subsets and functions in health and disease.

Lymph nodes (LNs) aid the interaction between lymphocytes and antigen-presenting cells, resulting in adequate and prolonged adaptive immune responses. LN stromal cells (LNSCs) are crucially involved in steering adaptive immune responses at different levels. Most knowledge on LNSCs has been obtained from mouse studies, and few studies indicate similarities with their human counterparts. Recent advances in single-cell technologies have revealed significant LNSC heterogeneity among different subsets with potential selective functions in immunity. This review provides an overview of current knowledge of LNSCs based on human and murine studies describing the role of these cells in health and disease.

The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms.

Mucosal surfaces are constantly exposed to environmental antigens, colonized by commensal organisms and used by pathogens as points of entry. As a result, the immune system has devoted the bulk of its resources to mucosal sites to maintain symbiosis with commensal organisms, prevent pathogen entry, and avoid unnecessary inflammatory responses to innocuous antigens. These functions are facilitated by a variety of mucosal lymphoid organs that develop during embryogenesis in the absence of microbial stimulation as well as ectopic lymphoid tissues that develop in adults following microbial exposure or inflammation. Each of these lymphoid organs samples antigens from different mucosal sites and contributes to immune homeostasis, commensal containment, and immunity to pathogens. Here we discuss the mechanisms, mostly based on mouse studies, that control the development of mucosal lymphoid organs and how the various lymphoid tissues cooperate to maintain the integrity of the mucosal barrier.

Colonic patch and colonic SILT development are independent and differentially regulated events.

Intestinal lymphoid tissues have to simultaneously ensure protection against pathogens and tolerance toward commensals. Despite such vital functions, their development in the colon is poorly understood. Here, we show that the two distinct lymphoid tissues of the colon-colonic patches and colonic solitary intestinal lymphoid tissues (SILTs)-can easily be distinguished based on anatomical location, developmental timeframe, and cellular organization. Furthermore, whereas colonic patch development depended on CXCL13-mediated lymphoid tissue inducer (LTi) cell clustering followed by LTα-mediated consolidation, early LTi clustering at SILT anlagen did not require CXCL13, CCR6, or CXCR3. Subsequent dendritic cell recruitment to and gp38(+)VCAM-1(+) lymphoid stromal cell differentiation within SILTs required LTα; B-cell recruitment and follicular dendritic cell differentiation depended on MyD88-mediated signaling, but not the microflora. In conclusion, our data demonstrate that different mechanisms, mediated mainly by programmed stimuli, induce the formation of distinct colonic lymphoid tissues, therefore suggesting that these tissues may have different functions.

CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor.

Expression of CD200, the gene encoding the ligand for the inhibitory immune receptor CD200R, is an independent prognostic factor for various forms of leukemia predicting worse overall survival of the patients. The enhanced expression of CD200 on the tumors implies that anti-tumor responses can be enhanced by blockage of the CD200-CD200R interaction. Indeed, antibody-mediated blockade of the CD200-CD200R inhibitory axis is currently evaluated in clinical tests to boost immune responses against CD200-expressing tumors. Here, we show that mice lacking CD200, the exclusive ligand for CD200R, are resistant to chemical skin carcinogenesis. Importantly, CD200R controls tumor outgrowth independently of CD200 expression by the tumor cells themselves. Furthermore, Cd200(-/-) mice do not become tolerant to intranasally administered antigens, suggesting that tumor rejection is normally suppressed through CD200-induced immune tolerance. Decreased tumor outgrowth is accompanied by increased expression of the proinflammatory cytokines interleukin (IL)-1ß and IL-6 by the lymph node (LN) dendritic cells. During carcinogenesis, skin-draining LNs of Cd200(-/-) mice contain increased numbers of IL-17-producing FoxP3(+) cells, which preferentially home to the tumors. Thus, the CD200-CD200R axis induces tolerance to external and tumor antigens and influences the T-regulatory/Th17 cell ratio. We demonstrate for the first time that the absence of CD200R signaling inhibits outgrowth of an endogenous tumor irrespective of CD200 expression by the tumor cells. This important paradigm shift leads to a much broader applicability of CD200-blockade in the treatment of tumors.

Preferential expression of IgG2b in nose draining cervical lymph nodes and its putative role in mucosal tolerance induction.

Induction of intranasal tolerance prevents the body from eliciting unwanted immune responses against harmless antigens that enter the body through the nasal mucosa. To study the intrinsic capacities of the cervical, nose draining lymph nodes (CLN), which are essential for tolerance induction, genes that are differentially expressed in CLN and not in peripheral lymph nodes (PLN) were characterized. The gene that is predominantly overexpressed in CLN codes for IgG2b. This is confirmed by a higher percentage of IgG2b+ B220+ cells in CLN compared with any PLN. However, this predominance of IgG2b-positive B cells in the CLN is not specific for the lymph node itself but rather determined by the region drained by lymph nodes at the cervical site, as transplanted PLN at these locations show a comparable predominance. It was demonstrated that IgG2b, when compared with IgG1, led to differential activation of dendritic cells (DC) through Fc receptor signalling. The results point to a unique local combination of cells and factors in the nose draining CLN leading to highly specialized immune reactivity. The results point out that predominance of a distinct IgG isotype in a lymphoid environment may lead to highly specialized immune reactivity.

The fetal liver counterpart of adult common lymphoid progenitors gives rise to all lymphoid lineages, CD45+CD4+CD3- cells, as well as macrophages.

We identified an IL-7Ralpha(+)Sca-1(low)c-Kit(low) population in E14 fetal liver, which is the phenotypical analog of common lymphoid progenitors (CLP) in adult bone marrow. After transfer into newborn mice, the IL-7Ralpha(+)Sca-1(low)c-Kit(low) population rapidly differentiated into CD45(+)CD4(+)CD3(-) cells, which are candidate cells for initiating lymph node and Peyer's patch formation. In addition, this population also gave rise to B, T, NK, and CD8alpha(+) and CD8alpha(-) dendritic cells. The fetal liver precursors expressed a significantly lower level of the myeloid-suppressing transcription factor Pax-5, than adult CLP, and retained differentiation activity for macrophages in vitro. We propose that the transition from fetal liver IL-7Ralpha(+)Sca-1(low)c-Kit(low) cells to adult CLP involves a regulated restriction of their developmental potential, controlled, at least in part, by Pax-5 expression.

Regulation of peripheral lymph node genesis by the tumor necrosis factor family member TRANCE.

Proper lymph node (LN) development requires tumor necrosis factor-related activation-induced cytokine (TRANCE) expression. Here we demonstrate that the defective LN development in TRANCE(-/)- mice correlates with a significant reduction in lymphotoxin (LT)alphabeta(+)alpha(4)beta(7)(+)CD45(+)CD4(+)CD3(-) cells and their failure to form clusters in rudimentary mesenteric LNs. Transgenic TRANCE overexpression in TRANCE(-/)- mice results in selective restoration of this cell population into clusters, and results in full LN development. Transgenic TRANCE-mediated restoration of LN development requires LTalphabeta expression on CD45(+) CD4(+)CD3(-) cells, as LNs could not be induced in LTalpha(-/)- mice. LTalpha(-/)- mice also showed defects in the fate of CD45(+)CD4(+)CD3(-) cells similar to TRANCE(-/)- mice. Thus, we propose that both TRANCE and LTalphabeta regulate the colonization and cluster formation by CD45(+) CD4(+)CD3(-) cells in developing LNs, the degree of which appears to correlate with the state of LN organogenesis.

Requirement for RORgamma in thymocyte survival and lymphoid organ development.

Most developing thymocytes undergo apoptosis because they cannot interact productively with molecules encoded by the major histocompatibility complex. Here, we show that mice lacking the orphan nuclear hormone receptor RORgamma lose thymic expression of the anti-apoptotic factor Bcl-xL. RORgamma thus regulates the survival of CD4+8+ thymocytes and may control the temporal window during which thymocytes can undergo positive selection. RORgamma was also required for development of lymph nodes and Peyer's patches, but not splenic follicles. In its absence, there was loss of a population of CD3-CD4+CD45+ cells that normally express RORgamma and that are likely early progenitors of lymphoid organs. Hence, RORgamma has critical functions in T cell repertoire selection and lymphoid organogenesis.

Isolation of the intact white pulp. Quantitative and qualitative analysis of the cellular composition of the splenic compartments.

The spleen is anatomically and functionally divided into two compartments: the red pulp, where particles are effectively removed from the blood, and the white pulp, where specific immune responses are generated. Here the isolation of white pulp from red pulp is described, allowing a detailed analysis of the cellular components of both red and white pulp separately. A striking abundance of memory T cells was found in the white and red pulp with an overall ratio of T and B cells in the white pulp being similar to that in lymph nodes. Both NK and gamma delta T cells can be found in white pulp and lymph nodes, but granulocytes are absent. The distribution of dendritic cell subsets showed significant differences between white pulp and lymph nodes. Furthermore, short-term homing experiments showed that migration of lymphocytes into the white pulp greatly exceeded that into lymph nodes, with significant differences in migration of various lymphocytes subsets. This suggests a different migration and retention mechanism in the white pulp. This new isolation technique will allow further analysis of the functional capacities of the splenic compartments.

Intranasally induced immunological tolerance is determined by characteristics of the draining lymph nodes: studies with OVA and human cartilage gp-39.

Mucosal tolerance is a naturally occurring immunological phenomenon that prevents harmful inflammatory responses to ingested or inhaled environmental, predominantly nondangerous, Ags. The nasal mucosa is an extremely efficient compartment in the induction of immunological tolerance which can be exploited in Ag-specific treatment of autoimmune disease. With the use of a model Ag (OVA) and an Ag implicated in the autoimmune disease rheumatoid arthritis (human cartilage gp-39), we here show in a mouse model that the superficial cervical and internal jugular lymph nodes that drain the nasal mucosa are instrumental in the induction of tolerance. Removal of these lymph nodes abrogates tolerance induction, which can be restored by transplantation of superficial cervical lymph nodes, but not of peripheral lymph nodes. The results indicate that lymph nodes that directly drain the nasal mucosa constitute a unique microenvironment which favors the induction of immunological tolerance.

L-selectin-mediated lymphocyte aggregation: role of carbohydrates, activation and effects on cellular interactions.

L-selectin on lymphocytes reacts with glycosylated ligands on high endothelial venule walls in lymphoid organs. Through this carbohydrate-dependent interaction, rolling and initial attachment of lymphocytes to endothelium is mediated. Here we have studied an earlier described L-selectin-induced homotypic aggregation, to further elucidate the events that occur after engagement of L-selectin. It was found that the interaction of L-selectin with fucoidan, but not with other carbohydrates, or with monoclonal antibodies directed against the carbohydrate recognition domain of L-selectin, resulted in homotypic aggregation among both B- or T lymphocytes. Importantly, this aggregation was shown to be both lymphocyte function-associated antigen-1 (LFA-1) and calcium-independent. Furthermore, for aggregation metabolic energy was required, and signalling via protein tyrosine kinase appeared to be involved. Neither de novo protein synthesis, protein kinase C mediated signalling, Gi-protein mediated signal transduction, nor calcium mobilization were required for aggregation. During aggregation, L-selectin was not shed from the lymphocyte's cell surface. Finally, it was found that the lymphocyte binding capacity to high endothelial venules on cryostat sections was not altered upon triggering these lymphocytes via L-selectin. Interestingly, L-selectin-triggered cells showed increased binding to paracortical areas in peripheral lymph nodes. Our data suggest that signals via L-selectin, might lead to altered expression of cell surface molecules, important in interactions other than the first stage of lymphocyte rolling.

MAdCAM-1 dependent colonization of developing lymph nodes involves a unique subset of CD4+CD3- hematolymphoid cells.

During fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules briefly express the Peyer's patch addressin MAdCAM-1. This allows initial seeding by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta 7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. It was found that the CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1. They can differentiate into natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. In addition to LN, CD4+CD3- cells can also be found in fetal spleen starting at 13.5 dpc, while absent from fetal liver. In view of the necessity of lymphotoxin in lymphoid organ development, it is thought that the novel subset of CD4+CD3- LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

Regulation of fucosyltransferase-VII expression in peripheral lymph node high endothelial venules.

Binding of L-selectin to the highly glycosylated peripheral lymph node addressins (PNAd) plays a central role in the normal recirculation of lymphocytes between the bloodstream and the lymph node. This interaction requires correct fucosylation of the PNAd, mediated by the recently identified fucosyltransferase-VII (Fuc-TVII). Here we show that during ontogeny Fuc-TVII is absent at the day of birth, barely detectable on day 1, and clearly present from day 2 onwards. PNAd expression as detected by the MECA-79 antibody precedes the expression of Fuc-TVII. Furthermore, we demonstrate that in adult mice antigenic stimulation of peripheral lymph nodes leads to a temporary disappearance of Fuc-TVII at days 2 and 3 after stimulation, followed by a complete reappearance by day 4, while expression of MECA-79 is never completely absent during this period. Finally, occlusion of afferent lymphatics to peripheral lymph nodes resulted in a decreased expression of Fuc-TVII in the high endothelial venules by day 5, and complete disappearance within 8 days. We conclude that the activity of Fuc-TVII in cells of high endothelial venules is directly affected by afferent lymph and activation processes that occur in the lymph node after antigenic stimulation. The expression of Fuc-TVII is therefore yet another level at which the function of high endothelial venules, and thus lymphocyte trafficking, can be regulated.

Lymphocyte triggering via L-selectin leads to enhanced galectin-3-mediated binding to dendritic cells.

For proper immune surveillance, naive lymphocytes are recruited from the blood into secondary lymphoid organs. L-selectin expressed on lymphocytes plays an important role in the initial attachment of these cells to high endothelial venules (HEV) in lymph nodes. Previously, we found that triggering via L-selectin resulted in activation of lymphocytes, followed by an alteration in their adhesion capacity. This suggested that L-selectin triggering might play a role in cell-cell interactions after lymph node entry. Here, we identify a novel adhesion mechanism involving L-selectin-triggered lymphocytes and dendritic cells, and we show that enhanced binding to dendritic cells is mediated by galectin-3 and not by integrins. Furthermore, it was shown that L-selectin-triggered T lymphocytes exhibited enhanced proliferation in an allogeneic mixed lymphocyte reaction. It is concluded that, in addition to a role for L-selectin in tethering and rolling on endothelium, triggering of the molecule on the lymphocyte surface leads to changes that are pertinent for the function of the cell after passing the HEV. We argue that the described adhesion mechanism plays a role in optimizing the initial interaction between dendritic cells and lymphocytes.

Transfer of primitive stem/progenitor bone marrow cells from LT alpha-/- donors to wild-type hosts: implications for the generation of architectural events in lymphoid B cell domains.

To analyze whether the phenotypic abnormalities observed in lymphotoxin-alpha(-/-) (LT alpha-/-) mice are intrinsic to the hemolymphoid system itself or dependent on stromal elements, wild-type (WT) mice were reconstituted with bone marrow (BM) cells enriched for hemopoietic stem cells from LT alpha-/- animals. WT mice reconstituted with LT alpha-/- c-kit+ Lin- Sca-1+ BM cells do not maintain follicular dendritic cell (FDC) networks and do not form primary follicles, while clear segregation of B and T cells could be observed. Furthermore, IgM+ IgD- B cells, MOMA-1 (anti-metallophilic macrophages), ERTR-9 (anti-marginal zone macrophages), and MECA-367 (anti-MAdCAM-1) were all absent from the splenic marginal zone. Surprisingly, however, the expression of MOMA-1, ERTR-9, and MAdCAM-1 was normal in the lymph nodes of mice reconstituted with LT alpha-/- cells. In addition, peanut agglutinin-positive germinal centers were observed in both the spleen and mesenteric lymph nodes, although in the absence of detectable FDC. Furthermore, in animals reconstituted with a mixture of LT alpha-/- and WT c-kit+ Lin- Sca-1+, GC contained either predominantly LT alpha-/- B cells or WT B cells. These results suggest that although the formation of primary follicles, FDC networks, and the splenic marginal zone are all dependent on hemopoietically derived LT alpha, germinal center formation and the expression of MAdCAM-1, MOMA-1, and ERTR-9 in lymph nodes are not. Our results also suggest that the disturbed B-T cell separation in LT alpha-/- mice is unrelated to defects in the marginal zone.

Developing lymph nodes collect CD4+CD3- LTbeta+ cells that can differentiate to APC, NK cells, and follicular cells but not T or B cells.

For a brief period during fetal lymph node organogenesis in mice, lymph node postcapillary high endothelial venules surprisingly express the Peyer's patch addressin MAdCAM-1. This expression allows initial seeding of this incipient structure by two unusual lymphocyte populations selectively expressing the Peyer's patch homing receptor integrin alpha4beta7: CD4+CD3- oligolineage progenitors and TCR gammadelta+ T cells. We show here that CD4+CD3- cells are lineage-restricted progenitors that express surface lymphotoxin-beta (LTbeta) and the chemokine receptor BLR1 and that can become natural killer cells, dendritic antigen-presenting cells, and follicular cells of unknown outcome, but these cells do not become T or B lymphocytes. Since the necessity of lymphotoxin in lymphoid organ development has been shown, we propose that the novel subset of CD4+CD3-LTbeta+ fetal cells is instrumental in the development of lymphoid tissue architecture.

From stem cells to lymphocytes: biology and transplantation.

We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cells. This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.

A developmental switch in lymphocyte homing receptor and endothelial vascular addressin expression regulates lymphocyte homing and permits CD4+ CD3- cells to colonize lymph nodes.

IN adult mice, the dominant adhesion molecules involved in homing to lymph nodes are L-selectin homing receptors on lymphocytes and the peripheral lymph node addressins on specialized high endothelial venules. Here we show that, from fetal life through the first 24 hr of life, the dominant adhesion molecules are the mucosal addressin MAdCAM-1 on lymph node high endothelial venules and its counterreceptor, the Peyer's patch homing receptor, integrin alpha 4 beta 7 on circulating cells. Before birth, 40-70% of peripheral blood leukocytes are L-selectin-positive, while only 1-2% expresses alpha 4 beta 7. However, the fetal lymph nodes preferentially attract alpha 4 beta 7-expressing cells, and this can be blocked by fetal administration of anti-MAdCAM-1 antibodies. During fetal and early neonatal life, when only MAdCAM-1 is expressed on high endothelial venules, an unusual subset of CD4 + CD3- cells, exclusively expressing alpha 4 beta 7 as homing receptors, enters the lymph nodes. Beginning 24 hr after birth a developmental switch occurs, and the peripheral node addressins are upregulated on high endothelial venules in peripheral and mesenteric lymph nodes. This switch in addressin expression facilitates tissue-selective lymphocyte migration and mediates a sequential entry of different cell populations into the lymph nodes.

The role of cell adhesion molecules in the development of IDDM: implications for pathogenesis and therapy.

IDDM is a chronic inflammatory disease in which there is autoimmune-mediated organ-specific destruction of the insulin-producing beta-cells in the pancreatic islets of Langerhans. The migration of autoreactive lymphocytes and other leukocytes from the bloodstream into the target organ is of clear importance in the etiology of many organ-specific autoimmune/inflammatory disorders, including IDDM. In IDDM, this migration results in lymphocytic invasion of the islets (formation of insulitis) and subsequent destruction of beta-cells. Migration of lymphocytes from the bloodstream into tissues is a complex process involving sequential adhesion and activation events. This migration is controlled in part by selective expression and functional regulation of cell adhesion molecules (CAMs) on the surface of lymphocytes and vascular endothelial cells or in the extracellular matrix. Understanding the mechanisms that regulate lymphocyte migration to the pancreatic islets will lead to further understanding of the pathogenesis of IDDM. In this article, we summarize the recent advances regarding the function of CAMs in the development of IDDM in animal models and in humans and discuss the potential for developing CAM-based therapies for IDDM.