Matthias W. Hoffmann

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.

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.

Production of cytokines (TNF-alpha, IL-1-beta) and endothelial cell activation in human liver allograft rejection.

Intragraft production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) was determined in rejecting human liver grafts during acute rejection and in chronic graft dysfunction. The localization of cytokine-producing cells was then correlated with the distribution of monocytes and macrophages as their main producers, as well as with effector functions such as endothelial cell activation. In selected patients collateral TNF-alpha plasma levels were measured. In normal liver and biopsies taken during an uncomplicated course, few TNF-alpha and even fewer IL-1-beta positive macrophages were found. During acute rejection episodes of all degrees of severity liver grafts were infiltrated by large numbers of TNF-alpha-positive monocytes, and concomitant TNF-alpha plasma levels were elevated compared with uncomplicated controls. In marked contrast IL-1-beta production by macrophages and vascular and sinus endothelial cells was restricted to the most severe, irreversible rejection episodes. The localization of cytokine-positive cells coincided with areas of maximum induction of ICAM-1 and von Willebrand Factor. In chronic graft dysfunction increased numbers of mature macrophages were found. A large proportion of these were positive for TNF-alpha as well as IL-1-beta. Distinct from acute rejection episodes, however, parallel TNF-alpha plasma levels were not elevated, suggesting cytokine storage rather than secretion. The present results indicate an important local role of TNF-alpha and IL-1-beta in the early phase of the rejection process. They presumably activate endothelial cells to upregulate the expression of adhesion molecules, thereby facilitating mononuclear cell adhesion and extravasation. Therefore, specific inactivation of cyto-kines or of their actions may prove to be a powerful tool in the prevention and treatment of allograft rejection in the future.

Improvement of acute and chronic renal dysfunction in liver transplant patients after substitution of calcineurin inhibitors by mycophenolate mofetil.

BACKGROUND Renal dysfunction caused by treatment with the calcineurin inhibitors (CNI) is a major problem in the long-term course after liver transplantation. PATIENTS In 22 liver graft recipients with renal dysfunction and stable graft function between 3 weeks and 12 years after transplantation, CNI were substituted by MMF at a final dose of 1.5-3 g/day between October 1996 and October 1998. METHODS In a prospective non-randomized study, the development of renal function, the side effects of MMF medication, and the stability of liver function were analyzed for a mean follow-up of 15 months. Results. (1) MMF was withdrawn in four patients for major side effects between 1 and 7 months after study entry; eight patients had minor side effects. (2) Six months after study entry, renal function had improved in 17 of the 22 study patients; mean serum creatinine +/-SD (micromol/L) was 201+/-77 at entry and 153+/-65 after 3 months (P<0.001). (3) Improvement occurred in 11 of 15 patients with creatinine elevation > or =12 months and in 6 of 6 patients with creatinine elevation < or =6 months. (4) One patient developed transient liver dysfunction and a second required retransplantation for progressive cholestasis but without signs of rejection. CONCLUSIONS In patients who undergo liver transplantation, substitution of CNI by MMF leads to improvement of acute as well as chronic renal dysfunction in most cases. Side effects of MMF may be limiting in some patients, and the immunological consequences remain to be studied.

Chemokine Receptor CXCR5 Supports Solitary Intestinal Lymphoid Tissue Formation, B Cell Homing, and Induction of Intestinal IgA Responses

Solitary intestinal lymphoid tissue (SILT) comprises a spectrum of phenotypically diverse lymphoid aggregates interspersed throughout the small intestinal mucosa. Manifestations of SILT range from tiny lymphoid aggregates almost void of mature lymphocytes to large structures dominated by B cells. Large SILT phenotypically resemble a single Peyer’s patch follicle, suggesting that SILT might contribute to intestinal humoral immune responses. In this study, we track the fate of individual SILT in vivo over time and analyze SILT formation and function in chemokine receptor CXCR5-deficient mice. We show that, in analogy to Peyer’s patches, formation of SILT is invariantly determined during ontogeny and depends on CXCR5. Young CXCR5-deficient mice completely lack SILT, suggesting that CXCR5 is essential for SILT formation during regular postnatal development. However, microbiota and other external stimuli can induce the formation of aberrant SILT distinguished by impaired development of B cell follicles in CXCR5-deficient mice. Small intestinal transplantation and bone marrow transplantation reveal that defect follicle formation is due to impaired B cell homing. Moreover, oral immunization with cholera toxin or infection with noninvasive Salmonella fail to induce efficient humoral immune responses in CXCR5-deficient mice. Bone marrow transplantation of CXCR5-deficient recipients with wild-type bone marrow rescued B cell follicle formation in SILT but failed to restore full humoral immune responses. These results reveal an essential role of CXCR5 in Peyer’s patch and SILT development and function and indicate that SILT do not fully compensate for the lack of Peyer’s patches in T cell-dependent humoral immune responses.

Necrotic Tumor Cell Death In Vivo Impairs Tumor-Specific Immune Responses

The manner in which cells die is believed to have a major impact on the nature of immune responses to their released Ags. In this study, we present the first direct analysis of tumor-specific immune responses to in vivo occurring tumor cell death through apoptosis or necrosis. Mice bearing thymidine kinase-transfected tumors were treated either with ganciclovir to induce tumor cell apoptosis in vivo or a vascular targeting agent, ZD6126, to induce tumor cell necrosis in vivo. In contrast to tumor apoptosis, induction of necrosis reduced the frequency and impaired the function of tumor-specific CD8+ T cells. Adoptive transfer of lymphocytes from mice with apoptotic tumors into tumor-challenged mice resulted in a significant tumor protection, which was absent when splenocytes were transferred from mice with necrotic tumors. Anti-CD40 treatment reversed impaired Ag-specific CD8+ T cell responses in these mice. These observations have not only fundamental importance for the development of immunotherapy protocols but also help to understand the underlying mechanism of in vivo immune responses to tumor cell death.

β2‐Adrenoceptor‐mediated suppression of human intestinal mast cell functions is caused by disruption of filamentous actin dynamics

Previous studies indicated potent inhibitory effects of β2‐adrenoceptor (β2AR) activation on the immunological mediator release of mast cells (MC). Here, we studied effects of β2AR agonists on human MC mediator release, and in particular on MC proliferation, adhesion, and migration. MC were isolated from human intestinal mucosa, purified, and cultured in the presence of stem cell factor (SCF). β2AR activation by epinephrine, norepinephrine, and salbutamol suppressed the IgE receptor‐dependent release of histamine, lipid mediators, and TNF‐α, and inhibited SCF‐dependent MC proliferation and migration. Moreover, β2‐adrenergic stimulation interfered with MC adhesion to fibronectin and human endothelial cells. Using fluorescent phallacidin, we found that β2AR activation reduced the amount of filamentous actin (F‐actin) within minutes, whereas MC stimulation by either IgE receptor cross‐linking or SCF caused F‐actin accumulation. Interestingly, this activation‐induced F‐actin increase was abolished by previous β2‐adrenergic stimulation. Finally, we demonstrated that disruption of the F‐actin cytoskeleton by latrunculin B mimicked the effects of β2AR agonists on MC adhesion and migration. Our results argue for an important role of F‐actin interference in β2AR‐mediated MC inhibition. Furthermore, the data support the concept of neuroimmune interactions regulating intestinal MC distribution, density, and functionality in vivo.

Infections Requiring Hospitalization of Injection Drug Users Who Participated in an Injection Opiate Maintenance Program

A retrospective analysis of hospitalizations due to infection in 175 injection drug users was performed for the 3 years before and the period during their participation in an injection opiate maintenance program (mean duration during program, 2.6 years). Skin infections were the main reason for hospitalization. The injection opiate maintenance program did not reduce the incidence of infection leading to hospitalization among the injection drug users studied.

Tolerance induction towards cardiac allografts under costimulation blockade is impaired in CCR7-deficient animals but can be restored by adoptive transfer of syngeneic plasmacytoid dendritic cells

Deficiency of transplant recipients for the chemokine receptor CCR7 was originally described to slightly increase the survival time of vascularized solid organ grafts, probably due to a reduced priming of alloreactive T cells. Using a model of allotolerance induction by donor‐specific splenocyte transfusion (DST) in combination with anti‐CD40L mAb‐mediated costimulation blockade (CSB), we show here a striking failure of CCR7‐deficient (CCR7−/−) recipients to tolerate cardiac allografts. Furthermore, in addition to the recently described lack of Treg, CCR7−/− mice were found to harbor significantly reduced numbers of plasmacytoid dendritic cells (pDCs) within peripheral as well as mesenteric lymph nodes (LNs), but not the bone marrow or spleen. pDCs had previously been suggested to function as tolerogenic APC during allograft transplantation, and a single transfer of syngeneic WT pDCs, but not conventional DCs, was indeed sufficient to rescue graft survival in DST+CSB‐treated CCR7−/− recipients in a dose‐dependent manner. We therefore conclude that the nearly complete absence of pDCs within LNs of CCR7−/− mice prevents the successful induction of DST+CSB‐mediated allotolerance, leading to the observed acute rejection of cardiac allografts under tolerizing conditions.

IFN-γ-receptor signaling ameliorates transplant vasculopathy through attenuation of CD8+ T-cell-mediated injury of vascular endothelial cells

Occlusive transplant vasculopathy (TV) is the major cause for chronic graft rejection. Since endothelial cells (EC) are the first graft cells encountered by activated host lymphocytes, it is important to delineate the molecular mechanisms that coordinate the interaction of EC with activated T cells. Here, the interaction of CD8+ T cells with Ag‐presenting EC in vivo was examined using a transgenic heart transplantation model with β‐galactosidase (β‐gal) expression exclusively in EC (Tie2‐LacZ hearts). We found that priming with β‐gal peptide‐loaded DC failed to generate a strong systemic IFN‐γ response, but elicited pronounced TV in both IFN‐γ receptor (IFNGR)‐competent, and ifngr−/− Tie2‐LacZ hearts. In contrast, stimulation of EC‐specific CD8+ T cells with β‐gal‐recombinant mouse cytomegalovirus (MCMV‐LacZ) in recipients of ifngr+/+ Tie2‐LacZ hearts did not precipitate significant TV. However, MCMV‐LacZ infection of recipients of ifngr−/− Tie2‐LacZ hearts led to massive activation of β‐gal‐specific CD8 T cells, and led to development of fulminant TV. Further analyses revealed that the strong systemic IFN‐γ “storm” associated with MCMV infection induced upregulation of programmed death‐1 ligand 1 (PD‐L1) on EC, and subsequent attenuation of programmed death‐1 (PD‐1)‐expressing EC‐specific CD8+ T cells. Thus, IFNGR signaling in ECs activates a potent peripheral negative feedback circuit that protects vascularized grafts from occlusive TV.