Control of the specificity of T cell-mediated anti-idiotype immunity by natural regulatory T cells

The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2Kd-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy

Master Thesis: Pathogenese und Keimzentrumsreaktion im Typ 1 Diabetes Mausmodell (Pathogenesis and germinal center reaction in the NOD T1D mouse model)

Type1 diabetes (T1D) is a chronic autoimmune disease leading to the destruction of the insulin-producing β-cells of the pancreas, thereby causing a disturbance of glucose homeostasis in the body. In industrialized countries T1D represents one of the most common endocrine disorders. Interactions between T and B cells are critical for the development of many autoimmune diseases (AID) and therapeutic manipulation, for example by B-cell-depleting antibodies, is an effective treatment for many AID. An important messenger in the T-B cell interaction is the cytokine IL-21, which is crucial for the development of T1D in the NOD mouse model. Using the NOD mouse, I wanted to find a correlation between disease progression, tissue destruction and germinal center reactions in correlation to IL-21. On tissue sections of the pancreas it could be demonstrated that the destruction of the islets progressing from a state with intact insulin producing β-cells, without the presence of lymphocytes over a stage of a peri-insulitis to destructive insulitis with the total loss of insulinproducing cells. Using immunohistochemical staining it was shown that the disease is accompanied by the presence of T and B cells and FDC. The presence of these cells could be an indication for the formation of germinal center like structures at the site of inflammation in the pancreas. Interestingly, numerous regulatory T cells could also be found in the pancreas during fulminant inflammation. In secondary lymphoid organs like spleen, lymph nodes and Peyer patches a slight tendency towards the development of inflammation could be assessed in NOD mice with increasing age, with a slight decrease in regulatory cells and the inverse rise of activated effector cells. Overall, the highest immune response could be observed in the PP, suggesting a continuous activation by the commensal flora. IL-21 expression of CD4 + cells was induced by the stimulation via IL-6. However, the selected intracellular cytokine staining showed IL-21 expression independently of IL-6 which is not compatible with the literature. In summary, several differences in the T-B cell interactions could be shown and the further understanding of the interactions in the germinal center and the role of the cytokines IL-6 and IL-21 in the induction of diabetes in the NOD model could possibly provide new therapies for diabetes patients

Different adaptations of IgG effector function in human and non-human primates and implications for therapeutic antibody treatment

Safety of human therapeutic antibodies is generally assessed in non-human primates. While IgG1 shows identical FcγR interaction and effector function profile in both species, fundamental differences in the IgG2 and IgG4 antibody subclasses were found between the two species. Granulocytes, the main effector cells against IgG2 and IgG4 opsonized bacteria and parasites, do not express FcγRIIIb, but show higher levels of FcγRII in cynomolgus monkey. In humans, IgG2 and IgG4 adapted a silent Fc region with weak binding to FcγR and effector functions, whereas in contrast cynomolgus monkey IgG2 and IgG4 display strong effector function as well as differences in IgG4 Fab arm exchange. To balance this shift toward activation, the cynomolgus inhibitory FcγRIIb shows strongly increased affinity for IgG2. In view of these findings, in vitro and in vivo results for human IgG2 and IgG4 obtained in the cynomolgus monkey have to be cautiously interpreted, whereas effector function related effects of human IgG1 antibodies are expected to be predictable for man

A whole blood in vitro cytokine release assay with aqueous monoclonal antibody presentation for the prediction of therapeutic protein induced cytokine release syndrome in humans

The administration of several monoclonal antibodies (mAbs) to humans has been associated with acute adverse events characterized by clinically significant release of cytokines in the blood. The limited predictive value of toxicology species in this field has triggered intensive research to establish human in vitro assays using peripheral blood mononuclear cells or blood to predict cytokine release in humans. A thorough characterization of these assays is required to understand their predictive value for hazard identification and risk assessment in an optimal manner, and to highlight potential limitations of individual assay formats. We have characterized a whole human blood cytokine release assay with only minimal dilution by the test antibodies (95% v/v blood) in aqueous presentation format, an assay which so far received less attention in the scientific world with respect to evaluation of its suitability to predict cytokine release in humans. This format was compared with a human PBMC assay with immobilized mAbs presentation already well-characterized by others. The test mAbs were anti-CD28 superagonist TGN1412-like material (TGN1412L), another anti-CD28 superagonistic mAb (ANC28.1), a T-cell depleting mAb (Orthoclone™), and a TGN1412 isotype-matched control (Tysabri™) not associated with clinically-relevant cytokine release. The whole blood assay was found suitable for hazard identification due to the production of the expected clinical cytokine signature for the tested mAbs and showed markedly lower assay background and cytokine release with the isotype-matched control mAb Tysabri™. However, lower donor response rates to TGN1412L for most tested cytokines were observed when compared to the PBMC assay. Importantly, quantitative and qualitative differences in the relative cytokine responses to the individual mAbs, in the concentration-response relationships and the prominent cytokine signatures for individual mAbs in the two formats reflect diverging mechanisms of cytokine release and different levels of dependency on high density coating even for two anti-CD28 superagonistic antibodies. These results clearly show that one generic approach to assessment of cytokine release using in vitro assays is not sufficient, but rather the assay format needs to be optimized considering the target characteristics and the mechanistic features of the therapeutic mAbs being evaluated

Immune cell landscaping reveals a protective role for Tregs during kidney injury and fibrosis

Acute kidney injury (AKI) and chronic kidney diseases (CKD) are associated with high mortality and morbidity in hospitalized patients. Although the underlying mechanisms determining the transition from acute to irreversible chronic injury are not well understood, immune mediated inflammatory processes are critical in renal injury. We have performed a comparison of two mouse models leading to either kidney regeneration or fibrosis, to better characterize the cellular and molecular events leading to both outcomes. Global gene expression profiling and histological analysis revealed a major upregulation of immune system related pathways during fibrosis. Further unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed that tissue resident macrophages and T cells were major altered populations. In fibrotic kidneys, there was a marked increase in tissue resident IL33R+ and IL2Ra+ regulatory T cells (Tregs). Indeed, prophylactic expansion of this population resulted in protection from kidney injury and fibrosis. Transcriptional profiling of Tregs showed a differential up-regulation of regenerative and pro-angiogenic set of genes in the regeneration model, whereas they expressed markers of hyper activation and fibrosis in the fibrosis model. This suggests that Tregs could exert different functions in the same tissue, dictated by environmental cues. Overall, we have provided a detailed cellular and molecular characterization of murine kidneys after injury and identified key changes in immune cell populations during fibrosis development

Immune cell landscaping reveals a protective role for regulatory T cells during kidney injury and fibrosis

Acute kidney injury (AKI) and chronic kidney diseases are associated with high mortality and morbidity. Although the underlying mechanisms determining the transition from acute to chronic injury are not completely understood, immune-mediated processes are critical in renal injury. We have performed a comparison of 2 mouse models leading to either kidney regeneration or fibrosis. Using global gene expression profiling we could identify immune-related pathways accounting for the majority of the observed transcriptional changes during fibrosis. Unbiased examination of the immune cell composition, using single-cell RNA sequencing, revealed major changes in tissue-resident macrophages and T cells. Following injury, there was a marked increase in tissue-resident IL-33R+ and IL-2Ra+ regulatory T cells (Tregs). Expansion of this population before injury protected the kidney from injury and fibrosis. Transcriptional profiling of Tregs showed a differential upregulation of regenerative and proangiogenic pathways during regeneration, whereas in the fibrotic environment they expressed markers of hyperactivation and fibrosis. Our data point to a hitherto underappreciated plasticity in Treg function within the same tissue, dictated by environmental cues. Overall, we provide a detailed cellular and molecular characterization of the immunological changes during kidney injury, regeneration, and fibrosis