Overlapping Roles of CXCL13, Interleukin 7 Receptor α, and CCR7 Ligands in Lymph Node Development

Lymphoid tissue development is associated with local accumulation of CD4+ CD3− IL-7Rαhi hematopoietic cells that deliver lymphotoxin (LT)α1β2 signals to resident stromal cells. Previous studies have established an important role for CXCL13 (BLC) in the development of Peyer's patches (PP) and some peripheral lymph nodes (LNs), but the chemokine requirements for several LN types, including mesenteric LNs, remain undefined. Using CXCL13−/− mice that additionally carry the paucity of LN T cell mutation (plt/plt), we discovered that CCR7 ligands function in peripheral LN development. We also tested for a genetic interaction during LN development between CXCL13 and a cytokine receptor required in PP development, IL-7Rα. Mice deficient for both CXCL13 and IL-7Rα displayed a striking absence of LNs, including mesenteric LNs. These data extend the role of CXCL13 to the development of all LNs and establish a previously unappreciated role for IL-7Rα in this process. Both circulating and LN CD4+ CD3− IL-7Rαhi cells are shown to express LTα1β2 in an IL-7Rα–dependent manner. Furthermore, CXCL13 was found to be sufficient to mediate CD4+ CD3− IL-7Rαhi cell recruitment in vivo to an ectopic site. These findings indicate that CXCL13 and CCR7 ligands promote accumulation of CD4+ CD3− IL-7Rαhi cells, delivering IL-7Rα–dependent LTα1β2 signals critical for LN development

BLC Expression in Pancreatic Islets Causes B Cell Recruitment and Lymphotoxin-Dependent Lymphoid Neogenesis

AbstractCXCR5, the receptor for B lymphocyte chemoattractant (BLC), is required for normal development of Peyer's patches, inguinal lymph nodes, and splenic follicles. To test the in vivo activity of BLC in isolation of other lymphoid organizers, transgenic mice were generated expressing BLC in the pancreatic islets. In addition to attracting B cells, BLC expression led to development of lymph node–like structures that contained B and T cell zones, high endothelial venules, stromal cells, and the chemokine SLC. Development of these features was strongly dependent on B lymphocytes and on lymphotoxin α1β2 and could be reversed by blocking lymphotoxin α1β2. These findings establish that BLC is sufficient to activate a pathway of events leading to formation of organized lymphoid tissue

Fibroblastic Reticular Cells From Lymph Nodes Attenuate T Cell Expansion by Producing Nitric Oxide

Adaptive immune responses are initiated when T cells encounter antigen on dendritic cells (DC) in T zones of secondary lymphoid organs. T zones contain a 3-dimensional scaffold of fibroblastic reticular cells (FRC) but currently it is unclear how FRC influence T cell activation. Here we report that FRC lines and ex vivo FRC inhibit T cell proliferation but not differentiation. FRC share this feature with fibroblasts from non-lymphoid tissues as well as mesenchymal stromal cells. We identified FRC as strong source of nitric oxide (NO) thereby directly dampening T cell expansion as well as reducing the T cell priming capacity of DC. The expression of inducible nitric oxide synthase (iNOS) was up-regulated in a subset of FRC by both DC-signals as well as interferon-γ produced by primed CD8+ T cells. Importantly, iNOS expression was induced during viral infection in vivo in both LN FRC and DC. As a consequence, the primary T cell response was found to be exaggerated in Inos−/− mice. Our findings highlight that in addition to their established positive roles in T cell responses FRC and DC cooperate in a negative feedback loop to attenuate T cell expansion during acute inflammation

Malt1 protease inactivation efficiently dampens immune responses but causes spontaneous autoimmunity.

The protease activity of the paracaspase Malt1 has recently gained interest as a drug target for immunomodulation and the treatment of diffuse large B-cell lymphomas. To address the consequences of Malt1 protease inactivation on the immune response in vivo, we generated knock-in mice expressing a catalytically inactive C472A mutant of Malt1 that conserves its scaffold function. Like Malt1-deficient mice, knock-in mice had strong defects in the activation of lymphocytes, NK and dendritic cells, and the development of B1 and marginal zone B cells and were completely protected against the induction of autoimmune encephalomyelitis. Malt1 inactivation also protected the mice from experimental induction of colitis. However, Malt1 knock-in mice but not Malt1-deficient mice spontaneously developed signs of autoimmune gastritis that correlated with an absence of Treg cells, an accumulation of T cells with an activated phenotype and high serum levels of IgE and IgG1. Thus, removal of the enzymatic activity of Malt1 efficiently dampens the immune response, but favors autoimmunity through impaired Treg development, which could be relevant for therapeutic Malt1-targeting strategies

Formation of the Intrathymic Dendritic Cell Pool Requires CCL21-Mediated Recruitment of CCR7⁺ Progenitors to the Thymus

During αβ T cell development in the thymus, migration of newly selected CD4 <sup>+</sup> and CD8 <sup>+</sup> thymocytes into medullary areas enables tolerance mechanisms to purge the newly selected αβ TCR repertoire of autoreactive specificities. Thymic dendritic cells (DC) play key roles in this process and consist of three distinct subsets that differ in their developmental origins. Thus, plasmacytoid DC and Sirpα <sup>+</sup> conventional DC type 2 are extrathymically derived and enter into the thymus via their respective expression of the chemokine receptors CCR9 and CCR2. In contrast, although Sirpα <sup>-</sup> conventional DC type 1 (cDC1) are known to arise intrathymically from immature progenitors, the precise nature of such thymus-colonizing progenitors and the mechanisms controlling their thymus entry are unclear. In this article, we report a selective reduction in thymic cDC1 in mice lacking the chemokine receptor CCR7. In addition, we show that the thymus contains a CD11c <sup>+</sup> MHC class II <sup>-</sup> Sirpα <sup>-</sup> Flt3 <sup>+</sup> cDC progenitor population that expresses CCR7, and that migration of these cells to the thymus is impaired in Ccr7 <sup>-/-</sup> mice. Moreover, thymic cDC1 defects in Ccr7 <sup>-/-</sup> mice are mirrored in plt/plt mice, with further analysis of mice individually lacking the CCR7 ligands CCL21Ser (Ccl21a <sup>-/-</sup> ) or CCL19 (Ccl19 <sup>-/-</sup> ) demonstrating an essential role for CCR7-CCL21Ser during intrathymic cDC1 development. Collectively, our data support a mechanism in which CCR7-CCL21Ser interactions guide the migration of cDC progenitors to the thymus for correct formation of the intrathymic cDC1 pool

Essential role of CCL21 in establishment of central self-tolerance in T cells

The chemokine receptor CCR7 directs T cell relocation into and within lymphoid organs, including the migration of developing thymocytes into the thymic medulla. However, how three functional CCR7 ligands in mouse, CCL19, CCL21Ser, and CCL21Leu, divide their roles in immune organs is unclear. By producing mice specifically deficient in CCL21Ser, we show that CCL21Ser is essential for the accumulation of positively selected thymocytes in the thymic medulla. CCL21Ser-deficient mice were impaired in the medullary deletion of self-reactive thymocytes and developed autoimmune dacryoadenitis. T cell accumulation in the lymph nodes was also defective. These results indicate a nonredundant role of CCL21Ser in the establishment of selftolerance in T cells in the thymic medulla, and reveal a functional inequality among CCR7 ligands in vivo

T Helper 1 (Th1) and Th2 Characteristics Start to Develop During T Cell Priming and Are Associated with an Immediate Ability to Induce Immunoglobulin Class Switching

The respective production of specific immunoglobulin (Ig)G2a or IgG1 within 5 d of primary immunization with Swiss type mouse mammary tumor virus [MMTV(SW)] or haptenated protein provides a model for the development of T helper 1 (Th1) and Th2 responses. The antibody-producing cells arise from cognate T cell B cell interaction, revealed by the respective induction of Cγ2a and Cγ1 switch transcript production, on the third day after immunization. T cell proliferation and upregulation of mRNA for interferon γ in response to MMTV(SW) and interleukin 4 in response to haptenated protein also starts during this day. It follows that there is minimal delay in these responses between T cell priming and the onset of cognate interaction between T and B cells leading to class switching and exponential growth. The Th1 or Th2 profile is at least partially established at the time of the first cognate T cell interaction with B cells in the T zone

Association of T-Zone Reticular Networks and Conduits with Ectopic Lymphoid Tissues in Mice and Humans

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node–like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin+ T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell–rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter–CXCL13–transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell–rich zone

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport.

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature

The aged lymphoid tissue environment fails to support naive T cell homeostasis.

Aging is associated with a gradual loss of naive T cells and a reciprocal increase in the proportion of memory T cells. While reduced thymic output is important, age-dependent changes in factors supporting naive T cells homeostasis may also be involved. Indeed, we noted a dramatic decrease in the ability of aged mice to support survival and homeostatic proliferation of naive T cells. The defect was not due to a reduction in IL-7 expression, but from a combination of changes in the secondary lymphoid environment that impaired naive T cell entry and access to key survival factors. We observed an age-related shift in the expression of homing chemokines and structural deterioration of the stromal network in T cell zones. Treatment with IL-7/mAb complexes can restore naive T cell homeostatic proliferation in aged mice. Our data suggests that homeostatic mechanisms that support the naive T cell pool deteriorate with age.11128Ysciescopu