Chemokine CXCL13 is essential for lymph node initiation and is induced by retinoic acid and neuronal stimulation.

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.

Vagal innervation is required for the formation of tertiary lymphoid tissue in colitis.

Tertiary lymphoid tissue (TLT) is lymphoid tissue that forms in adult life as a result of chronic inflammation in a tissue or organ. TLT has been shown to form in a variety of chronic inflammatory diseases, though it is not clear if and how TLT develops in the inflamed colon during inflammatory bowel disease. Here, we show that TLT develops as newly formed lymphoid tissue in the colon following dextran sulphate sodium induced colitis in C57BL/6 mice, where it can be distinguished from the preexisting colonic patches and solitary intestinal lymphoid tissue. TLT in the inflamed colon develops following the expression of lymphoid tissue-inducing chemokines and adhesion molecules, such as CXCL13 and VCAM-1, respectively, which are produced by stromal organizer cells. Surprisingly, this process of TLT formation was independent of the lymphotoxin signaling pathway, but rather under neuronal control, as we demonstrate that selective surgical ablation of vagus nerve innervation inhibits CXCL13 expression and abrogates TLT formation without affecting colitis. Sympathetic neuron denervation does not affect TLT formation. Hence, we reveal that inflammation in the colon induces the formation of TLT, which is controlled by innervation through the vagus nerve.

Vitamin A metabolism and mucosal immune function are distinct between BALB/c and C57BL/6 mice.

The vitamin A metabolite retinoic acid (RA) has been reported to suppress Th1 responses and enhance Th2 responses. Here, we investigated whether differences in vitamin A metabolism could underlie the differences between C57BL/6 and BALB/c mice, which are reportedly seen as Th1 and Th2 responders, respectively. BALB/c mice were shown to have higher intestinal epithelial expression of RALDH1 (where RALDH is retinaldehyde dehydrogenase), and, consequently, higher RALDH activity in MLN-DCs, leading to an increased ability to induce IgA class switching in B cells. Furthermore, within BALB/c mice, induction of IgA secretion as well as increased accumulation of regulatory T cells (Treg) in the intestinal lamina propria was observed. Additionally, as BALB/c mice are more resistant to dextran sulphate sodium (DSS) induced colitis, mice that lacked vitamin A in their diet had a more severe form of DSS-induced colitis compared to control mice. Therefore, the level of RA production and consequently the degree of RA-mediated signaling is crucial for the efficiency of the mucosal immune system.

Effective collaboration between marginal metallophilic macrophages and CD8+ dendritic cells in the generation of cytotoxic T cells.

The spleen is the lymphoid organ that induces immune responses toward blood-borne pathogens. Specialized macrophages in the splenic marginal zone are strategically positioned to phagocytose pathogens and cell debris, but are not known to play a role in the activation of T-cell responses. Here we demonstrate that splenic marginal metallophilic macrophages (MMM) are essential for cross-presentation of blood-borne antigens by splenic dendritic cells (DCs). Our data demonstrate that antigens targeted to MMM as well as blood-borne adenoviruses are efficiently captured by MMM and exclusively transferred to splenic CD8(+) DCs for cross-presentation and for the activation of cytotoxic T lymphocytes. Depletion of macrophages in the marginal zone prevents cytotoxic T-lymphocyte activation by CD8(+) DCs after antibody targeting or adenovirus infection. Moreover, we show that tumor antigen targeting to MMM is very effective as antitumor immunotherapy. Our studies point to an important role for splenic MMM in the initial steps of CD8(+) T-cell immunity by capturing and concentrating blood-borne antigens and the transfer to cross-presenting DCs which can be used to design vaccination strategies to induce antitumor cytotoxic T-cell immunity.

LTbetaR signaling induces cytokine expression and up-regulates lymphangiogenic factors in lymph node anlagen.

The formation of lymph nodes is a complex process crucially controlled through triggering of LTbetaR on mesenchymal cells by LTalpha(1)beta(2) expressing lymphoid tissue inducer (LTi) cells. This leads to the induction of chemokines to attract more hematopoietic cells and adhesion molecules to retain them. In this study, we show that the extravasation of the first hematopoietic cells at future lymph node locations occurs independently of LTalpha and that these cells, expressing TNF-related activation-induced cytokine (TRANCE), are the earliest LTi cells. By paracrine signaling the first expression of LTalpha(1)beta(2) is induced. Subsequent LTbetaR triggering on mesenchymal cells leads to their differentiation to stromal organizers, which now also start to express TRANCE, IL-7, as well as VEGF-C, in addition to the induced adhesion molecules and chemokines. Both TRANCE and IL-7 will further induce the expression of LTalpha(1)beta(2) on newly arrived immature LTi cells, resulting in more LTbetaR triggering, generating a positive feedback loop. Thus, LTbetaR triggering by LTi cells during lymph node development creates a local environment to which hematopoietic precursors are attracted and where they locally differentiate into fully mature, LTalpha(1)beta(2) expressing, LTi cells. Furthermore, the same signals may regulate lymphangiogenesis to the lymph node through induction of VEGF-C.

Lymph node stromal cells support dendritic cell-induced gut-homing of T cells.

T cells are imprinted to express tissue-specific homing receptors upon activation in tissue-draining lymph nodes, resulting in their migration to the site of Ag entry. Expression of gut-homing molecules alpha(4)beta(7) and CCR9 is induced by retinoic acid, a vitamin A metabolite produced by retinal dehydrogenases, which are specifically expressed in dendritic cells as well as stromal cells in mucosa-draining lymph nodes. In this study, we demonstrate that mesenteric lymph node stromal cell-derived retinoic acid can directly induce the expression of gut-homing molecules on proliferating T cells, a process strongly enhanced by bone marrow-derived dendritic cells in vitro. Therefore, cooperation of sessile lymph node stromal cells with mobile dendritic cells warrants the imprinting of tissue specific homing receptors on activated T cells.

Amino acid regions 572-579 and 657-666 of the spacer domain of ADAMTS13 provide a common antigenic core required for binding of antibodies in patients with acquired TTP.

Antibodies directed against ADAMTS13 have been detected in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). We have previously localized a major antigenic determinant within the spacer domain of ADAMTS13. To identify the amino acid residues of the spacer domain that are involved in binding of anti-ADAMTS13 antibodies, we constructed a series of fifteen hybrids (designated A-O) in which 5-10 amino acids of the spacer domain were exchanged for the corresponding region of ADAMTS1. Plasma from six patients with antibodies directed against the spacer domain was analyzed for reactivity with the ADAMTS13/ADAMTS1 chimeras. Exchange of amino acid residues 572-579 (hybrid C) and 657-666 (hybrid M) completely abolished the binding of antibodies from all six patients analyzed. Regions 580-587 (D), 602-620 (G, H), 629-638 (J), and 667-767 (N) contributed to binding of antibodies from patients 2, 4, and 5 (epitope present within regions CDGHJMN). Antibodies derived from patient 1 required region 602-620 (G, H) for binding (CGHM-epitope). For antibodies of patient 3, residues 564-571 (B), 580-587 (D), and 629-638 (J) were required (BCDJM-epitope), whereas replacement of residues 602-610 (G) and 629-638 (J) greatly diminished binding of antibodies from patient 6 (CGJM-epitope). Despite the presumably polyclonal origin of the antibodies present in plasma of patients, our results suggest that residues 572-579 (C) and 657-666 (M) comprise a common antigenic core region that is crucial for binding of anti-ADAMTS13 antibodies. Other regions that spatially surround this antigenic core further modulate binding of antibodies to the spacer domain.

Lymph node stromal cells constrain immunity via MHC class II self-antigen presentation.

Non-hematopoietic lymph node stromal cells shape immunity by inducing MHC-I-dependent deletion of self-reactive CD8+ T cells and MHC-II-dependent anergy of CD4+ T cells. In this study, we show that MHC-II expression on lymph node stromal cells is additionally required for homeostatic maintenance of regulatory T cells (Tregs) and maintenance of immune quiescence. In the absence of MHC-II expression in lymph node transplants, i.e. on lymph node stromal cells, CD4+ as well as CD8+ T cells became activated, ultimately resulting in transplant rejection. MHC-II self-antigen presentation by lymph node stromal cells allowed the non-proliferative maintenance of antigen-specific Tregs and constrained antigen-specific immunity. Altogether, our results reveal a novel mechanism by which lymph node stromal cells regulate peripheral immunity.

Different mechanisms regulate CD4(+) T cell independent induction of oral and nasal tolerance of CD8(+) T cells.

Mucosal administration of antigens is known to induce antigen specific regulatory CD4(+) T cells, but less is known about the effects on CD8(+) T cell function. Using a murine model for mucosal tolerance induction, we show that both oral and nasal OVA (ovalbumin) application reduced OVA specific CD8(+) T effector cell numbers and suppressed in vivo cytotoxicity in response to subsequent immunisation. To investigate whether CD4(+) T cells are essential for oral or nasal CD8(+) T cell tolerance, we used MHC class II deficient mice. Normal CD8(+) T cell tolerance was observed in MHC class II deficient mice, indicating that CD4(+) T cells are not required for both oral and nasal CD8(+) T cell tolerance induction. To study the direct effects of mucosal antigen application on naive CD8(+) T cells, we adoptively transferred OVA specific transgenic CD8(+) T cells and analysed their fate after mucosal antigen application. Oral OVA application reduced the numbers of OVA specific CD8(+) T cells, whereas nasal OVA application induced proliferation. However, the expanded population of OVA specific CD8(+) T cells from nasally treated mice was unable to proliferate upon re-stimulation. In conclusion, our studies point to suppressive effects of the mucosal immune system directly on CD8(+) T cells and indicate multiple mechanisms of tolerance induction. More insight in these mechanisms of tolerance induction is necessary for the development of mucosal tolerance therapies for autoimmune diseases and allergy.

Lymph sacs are not required for the initiation of lymph node formation.

The lymphatic vasculature drains lymph fluid from the tissue spaces of most organs and returns it to the blood vasculature for recirculation. Before reaching the circulatory system, antigens and pathogens transported by the lymph are trapped by the lymph nodes. As proposed by Florence Sabin more than a century ago and recently validated, the mammalian lymphatic vasculature has a venous origin and is derived from primitive lymph sacs scattered along the embryonic body axis. Also as proposed by Sabin, it has been generally accepted that lymph nodes originate from those embryonic primitive lymph sacs. However, we now demonstrate that the initiation of lymph node development does not require lymph sacs. We show that lymph node formation is initiated normally in E14.5 Prox1-null mouse embryos devoid of lymph sacs and lymphatic vasculature, and in E17.5 Prox1 conditional mutant embryos, which have defective lymph sacs. However, subsequent clustering of hematopoietic cells within these developing lymph nodes is less efficient.

Blockade of IDO inhibits nasal tolerance induction.

The amino acid tryptophan is essential for the proliferation and survival of cells. Modulation of tryptophan metabolism has been described as an important regulatory mechanism for the control of immune responses. The enzyme IDO degrades the indole moiety of tryptophan, not only depleting tryptophan but also producing immunomodulatory metabolites called kynurenines, which have apoptosis-inducing capabilities. In this study, we show that IDO is more highly expressed in nonplasmacytoid dendritic cells of the nose draining lymph nodes (LNs), which form a unique environment to induce tolerance to inhaled Ags, when compared with other peripheral LNs. Upon blockade of IDO during intranasal OVA administration, Ag-specific immune tolerance was abrogated. Analysis of Ag-specific T cells in the LNs revealed that inhibition of IDO resulted in enhanced survival at 48 h after antigenic stimulation, although this result was not mediated through alterations in apoptosis or cell proliferation. Furthermore, no differences were found in CD4(+) T cells expressing FoxP3. Our data suggest that the level of IDO expression in dendritic cells, present in nose draining LNs, allows for the generation of a sufficient number of regulatory T cells to control and balance effector T cells in such a way that immune tolerance is induced, whereas upon IDO blockade, effector T cells will outnumber regulatory T cells, leading to immunity.

Secretory leukoprotease inhibitor in mucosal lymph node dendritic cells regulates the threshold for mucosal tolerance.

The notion that the mucosal immune system maintains a tolerogenic response to harmless Ags while continually being challenged with microbial products seems an enigma. The aim of this study was to unravel mechanisms that are involved in regulating the development of tolerance under constant microbial pressure. The tolerogenic response to Ags administered via the nasal mucosa is dependent on the organized lymphoid tissue of the cervical lymph nodes (LN). We show that cervical LN differentially express secretory leukoprotease inhibitor (SLPI) compared with peripheral LN. SLPI was expressed by dendritic cells (DCs) and because SLPI is known to suppress LPS responsiveness, it was hypothesized that its expression in mucosal DCs may be required to regulate cellular activation to microbial products. Indeed, compared with wild-type controls, bone marrow-derived DCs from SLPI(-/-) mice released more inflammatory cytokines and enhanced T cell proliferation after stimulation with low dose LPS. This increased sensitivity to LPS was accompanied by increased NF-kappaB p65 activation in SLPI(-/-) DCs. In vivo, nasal application of OVA with LPS to SLPI(-/-) mice resulted in enhanced DC activation in the cervical LN reflected by increased costimulatory molecule expression and release of inflammatory cytokines. This led to failure to maintain tolerance to nasal OVA application in the presence of low doses of LPS. We propose that expression of SLPI functions as a rheostat by controlling the level of bacterial stimuli that induce mucosal DC activation. As such, it regulates the quality of the ensuing Ag-specific immune response in the mucosa draining LN.

Initiation of cellular organization in lymph nodes is regulated by non-B cell-derived signals and is not dependent on CXC chemokine ligand 13.

The molecular and cellular events that initiate the formation of T and B cell areas in developing lymph nodes are poorly understood. In this study we show that formation of the lymphoid architecture in murine neonatal lymph nodes evolves through a series of distinct stages. The initial segregation of T and B cells is regulated in a CXCL13-independent manner, characterized by the localization of B cells in a ring-like pattern in the outer cortex on day 4. However, during this CXCL13-independent phase of lymph node modeling, CXCL13 is expressed and regulated in a lymphotoxin-alpha1beta2 (LTalpha1beta2)-dependent manner. Surprisingly, neonatal B cells are unable to respond to this chemokine and also lack surface LTalpha1beta2 expression. At this time, CD45+CD4+CD3- cells are the predominant LTalpha1beta2-expressing cells and are also capable of responding to CXCL13. From day 4 on, architectural changes become CXCL13 dependent, and B cells become fully CXCL13 responsive, express LTalpha1beta2, and cluster in anatomically distinct follicles. Because the initial induction of CXCL13 is dependent on LTalpha1beta2, a role for CD45+CD4+CD3- cells in inducing chemokine expression in the developing lymph nodes is proposed and, as such, a role in initiation of the shaping of the microenvironment.