Günter Bernhardt

Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells

Oral tolerance induction is a key feature of intestinal immunity, generating systemic nonresponsiveness to ingested antigens. In this study, we report that orally applied soluble antigens are exclusively recognized in the intestinal immune system, particularly in the mesenteric lymph nodes. Consequently, the initiation of oral tolerance is impeded by mesenteric lymphadenectomy. Small bowel transplantation reveals that mesenteric lymph nodes require afferent lymph to accomplish the recognition of orally applied antigens. Finally, oral tolerance cannot be induced in CCR7-deficient mice that display impaired migration of dendritic cells from the intestine to the mesenteric lymph nodes, suggesting that immunologically relevant antigen is transported in a cell-bound fashion. These results demonstrate that antigen transport via afferent lymphatics into the draining mesenteric lymph nodes is obligatory for oral tolerance induction, inspiring new therapeutic strategies to exploit oral tolerance induction for the prevention and treatment of autoimmune diseases.

Development and functional specialization of CD103+ dendritic cells

Summary:  CD103 (αE) integrin expression distinguishes a population of dendritic cells (DCs) that can be found in many if not all lymphoid and non‐lymphoid organs. CD103+ DCs display distinct functional activities. Migratory CD103+ DCs derived from skin, lung, and intestine efficiently present exogenous antigens in their corresponding draining lymph nodes to specific CD8+ T cells through a mechanism known as cross‐presentation. On the T cells they prime, intestinal CD103+ DCs can drive the induction of the chemokine receptor CCR9 and α4β7 integrin, both known as gut‐homing receptors. CD103+ DCs also contribute to control inflammatory responses and intestinal homeostasis by fostering the conversion of naive T cells into induced Foxp3+ regulatory T cells, a mechanism that relies on transforming growth factor‐β and retinoic acid signaling. This review discusses recent findings that identify murine CD103+ DCs as important regulators of the immune response.

Sphingosine-1-Phosphate Mediates Migration of Mature Dendritic Cells

Sphingosine-1-phosphate (S1P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S1P (S1P1–5) belong to the family of G protein-coupled receptors. Upon binding S1P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S1P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S1P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S1P, a phenomenon that correlated to the up-regulation of S1P1 and S1P3 in maturing DC. The same pattern of S1P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S1P could be severely hampered by application of the S1P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S1P-mediated signaling plays a pivotal role in the life cycle of DC.

Cryptopatches and isolated lymphoid follicles: dynamic lymphoid tissues dispensable for the generation of intraepithelial lymphocytes

In comparison to secondary lymphoid organs, gut‐associated lymphoid tissues such as isolated lymphoid follicles (ILF) and cryptopatches (CP) have been less intensively studied. To gain a better insight into processes regulating organization and function of these structures, which are believed to participate in immune responses and extrathymic T cell development, we characterized the lymphoid structures of the murine small intestine in more detail. The size and cellular composition of small intestinal lymphoid aggregations were analyzed in C57BL/6 and BALB/c wild‐type and lymphotoxin (LT)‐deficient mice, by flow cytometry, histology and automated multi‐color immunofluorescence microscopy evaluating large coherent areas of the intestine. These evaluations demonstrate that aggregated lymphoid structures in the small intestine vary in size and cellular composition, with a majority of structures not matching the current definitions of CP or ILF. Accordingly, significant variations depending on species, age and mouse strain were observed. Furthermore, small bowel transplantation revealed a rapid exchange of B but not T cells between host and grafted tissue. Moreover, LT‐deficient animals lack any intestinal lymphoid aggregations yet possess the complete panel of intraepithelial lymphocytes (IEL). In summary, our observations disclose intestinal lymphoid aggregations as dynamic structures with a great deal of inborn plasticity and demonstrate their dispensability for the generation of IEL.

Generalized multi‐organ autoimmunity in CCR7‐deficient mice

Development of autoimmunity is a multi‐factorial process involving genetic predisposition as well as environmental and stochastic factors. Although the mechanisms responsible for the initiation of autoimmunity remain only partially understood, several studies have demonstrated that genetic predisposition plays a major role in this process. In the present study, we analyzed the influence of CCR7 signaling in the development of autoimmunity, because this chemokine receptor is essentially involved in the functional organization of thymus architecture. We demonstrate that CCR7‐deficient mice are prone to develop generalized multi‐organ autoimmunity. The autoimmune phenotype of CCR7–/– mice encompasses the presence of lymphocyte infiltrates in several peripheral organs, circulating autoantibodies against a multitude of tissue‐specific antigens and IgG deposition on renal glomeruli. Additionally, CCR7‐deficient mice show increased susceptibility to streptozotocin‐induced diabetes and spontaneously display signs of chronic autoimmune renal disease. Thus, this study identifies CCR7 as a genetic factor involved in the regulation of autoimmunity.

αβ T cell receptors that do not undergo major histocompatibility complex-specific thymic selection possess antibody-like recognition specificities.

Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αβ T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αβTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αβTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αβTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αβTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.

Suppression of Metastases Using a New Lymphocyte Checkpoint Target for Cancer Immunotherapy

UNLABELLED CD96 has recently been shown as a negative regulator of mouse natural killer (NK)-cell activity, with Cd96(-/-)mice displaying hyperresponsive NK cells upon immune challenge. In this study, we have demonstrated that blocking CD96 with a monoclonal antibody inhibited experimental metastases in three different tumor models. The antimetastatic activity of anti-CD96 was dependent on NK cells, CD226 (DNAM-1), and IFNγ, but independent of activating Fc receptors. Anti-CD96 was more effective in combination with anti-CTLA-4, anti-PD-1, or doxorubicin chemotherapy. Blocking CD96 in Tigit(-/-)mice significantly reduced experimental and spontaneous metastases compared with its activity in wild-type mice. Co-blockade of CD96 and PD-1 potently inhibited lung metastases, with the combination increasing local NK-cell IFNγ production and infiltration. Overall, these data demonstrate that blocking CD96 is a new and complementary immunotherapeutic strategy to reduce tumor metastases. SIGNIFICANCE This article illustrates the antimetastatic activity and mechanism of action of an anti-CD96 antibody that inhibits the CD96-CD155 interaction and stimulates NK-cell function. Targeting host CD96 is shown to complement surgery and conventional immune checkpoint blockade.

Lck Availability during Thymic Selection Determines the Recognition Specificity of the T Cell Repertoire

Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αβ T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αβTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αβTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αβTCR repertoire.

The adhesion receptor CD155 determines the magnitude of humoral immune responses against orally ingested antigens

CD155, originally known as the cellular receptor for poliovirus, is the founding member of a subfamily of immunoglobulin‐like adhesion receptors. Apart from its function in establishing adherens junctions between contacting epithelial cells, the engagement of CD155 with two recently identified ligands, CD226 and CD96, mediates immunologically relevant processes such as NK cell‐driven killing of tumor cells in humans. Here we report on the generation and immunological analysis of mice constitutively deficient of CD155. Moreover, the expression profile of CD155 on hematopoietic cells has been determined using newly established antibodies. CD155‐deficient mice develop normally without displaying an overt phenotype. However, the animals are distinguished by distinct deficits in the development of a regular humoral immune response. Whereas systemic challenges revealed no differences, orally administered antigen evoked less efficient IgG and IgA antibody responses despite of normal IgM titers when compared to wild‐type mice. Therefore, CD155 may assist in an efficient humoral immune response generated within the intestinal immune system.

The Origin and Maturity of Dendritic Cells Determine the Pattern of Sphingosine 1-Phosphate Receptors Expressed and Required for Efficient Migration

Dendritic cells (DCs) represent the most potent inducers of adaptive immune responses. Depending on their activation phenotype, DCs drive naive T cells into distinct differentiation pathways. To achieve this, DCs are present in virtually all tissues where they sample Ag and migrate to the T cell areas of lymph nodes (LNs) and spleen. Ample evidence exists demonstrating that sphingosine 1-phosphate (S1P) is an important modulator of these processes, exerting its effects by binding to the S1P receptor S1P1 and/or S1P3. However, published data are contradictory, in part. We show in this study that the expression pattern, as well as the regulation of the S1P receptors, differs among in vitro-generated DCs experiencing different kinds and duration of stimuli. Moreover, the influence of S1P1 and S1P3 on the in vivo migration of maturing DCs depends on the origin of these cells. Thus, in vitro-generated DCs require S1P1 and S1P3 to accomplish this, whereas skin-derived DCs migrate unhindered in the absence of S1P3 but not when S1P1 signaling is blocked. Migration of lamina propria DCs to the mesenteric LNs depends on S1P1 and S1P3. In contrast, relocation of maturing spleen-resident DCs to the T cell zone is independent of S1P1 and S1P3. However, intrasplenic positioning of immature DCs to the bridging channels depends on S1P1 activity, with no noticeable contribution of S1P3. These observations reveal a tissue-dependent contribution of S1P3 to DC migration and suggest a fundamental role for S1P1 for maturing DCs migrating from periphery to draining LNs.