Marsilius Mues

Commensal microbiota and myelin autoantigen cooperate to trigger autoimmune demyelination

Active multiple sclerosis lesions show inflammatory changes suggestive of a combined attack by autoreactive T and B lymphocytes against brain white matter. These pathogenic immune cells derive from progenitors that are normal, innocuous components of the healthy immune repertoire but become autoaggressive upon pathological activation. The stimuli triggering this autoimmune conversion have been commonly attributed to environmental factors, in particular microbial infection. However, using the relapsing–remitting mouse model of spontaneously developing experimental autoimmune encephalomyelitis, here we show that the commensal gut flora—in the absence of pathogenic agents—is essential in triggering immune processes, leading to a relapsing–remitting autoimmune disease driven by myelin-specific CD4+ T cells. We show further that recruitment and activation of autoantibody-producing B cells from the endogenous immune repertoire depends on availability of the target autoantigen, myelin oligodendrocyte glycoprotein (MOG), and commensal microbiota. Our observations identify a sequence of events triggering organ-specific autoimmune disease and these processes may offer novel therapeutic targets.

Functional and Pathogenic Differences of Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis

Background There is consensus that experimental autoimmune encephalomyelitis (EAE) can be mediated by myelin specific T cells of Th1 as well as of Th17 phenotype, but the contribution of either subset to the pathogenic process has remained controversial. In this report, we compare functional differences and pathogenic potential of “monoclonal” T cell lines that recognize myelin oligodendrocyte glycoprotein (MOG) with the same transgenic TCR but are distinguished by an IFN-γ producing Th1-like and IL-17 producing Th17-like cytokine signature. Methods and Findings CD4+ T cell lines were derived from the transgenic mouse strain 2D2, which expresses a TCR recognizing MOG peptide 35–55 in the context of I-Ab. Adoptive transfer of Th1 cells into lymphopenic (Rag2−/−) recipients, predominantly induced “classic” paralytic EAE, whereas Th17 cells mediated “atypical” ataxic EAE in approximately 50% of the recipient animals. Combination of Th1 and Th17 cells potentiated the encephalitogenicity inducing classical EAE exclusively. Th1 and Th17 mediated EAE lesions differed in their composition but not in their localization within the CNS. While Th1 lesions contained IFN-γ, but no IL-17 producing T cells, the T cells in Th17 lesions showed plasticity, substantially converting to IFN-γ producing Th1-like cells. Th1 and Th17 cells differed drastically by their lytic potential. Th1 but not Th17 cells lysed autoantigen presenting astrocytes and fibroblasts in vitro in a contact-dependent manner. In contrast, Th17 cells acquired cytotoxic potential only after antigenic stimulation and conversion to IFN-γ producing Th1 phenotype. Conclusions Our data demonstrate that both Th1 and Th17 lineages possess the ability to induce CNS autoimmunity but can function with complementary as well as differential pathogenic mechanisms. We propose that Th17-like cells producing IL-17 are required for the generation of atypical EAE whereas IFN-γ producing Th1 cells induce classical EAE.

Real-time in vivo analysis of T cell activation in the central nervous system using a genetically encoded calcium indicator

To study T cell activation in vivo in real time, we introduced a newly developed fluorescence resonance energy transfer–based, genetically encoded calcium indicator into autoantigen-specific and non–autoantigen-specific CD4+ T cells. Using two-photon microscopy, we explored the responses of retrovirally transduced calcium indicator–expressing T cells to antigen in the lymph nodes and the central nervous system. In lymph nodes, the administration of exogenous antigen caused an almost immediate arrest of T cells around antigen-presenting cells and an instant rise of cytosolic calcium. In contrast, encephalitogenic T cells entering the leptomeningeal space, one main portal into the central nervous system parenchyma during experimental autoimmune encephalomyelitis, showed elevated intracellular calcium concentrations while still meandering through the space. This approach enabled us to follow the migration and activation patterns of T cells in vivo during the course of the disease.

An autoimmunity odyssey: how autoreactive T cells infiltrate into the CNS

Summary:  Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model of multiple sclerosis (MS), a human autoimmune disease. To explore how EAE and ultimately MS is induced, autoantigen‐specific T cells were established, were labeled with fluorescent protein by retroviral gene transfer, and were tracked in vivo after adoptive transfer. Intravital imaging with two‐photon microscopy was used to record the entire entry process of autoreactive T cells into the CNS: a small number of T cells first appear in the CNS leptomeninges before onset of EAE, and crawl on the intraluminal surface of blood vessels, which is integrin α4 and αL dependent. After extravasation, the T cells continue into the perivascular space, meeting local antigen‐presenting cells (APCs), which present endogenous antigens. This interaction activates the T cells and guides them to penetrate the CNS parenchyma. As the local APCs in the CNS are not saturated with endogenous antigens, exogenous antigens stimulate the autoreactive T cells more strongly and, as a result, exacerbate the clinical outcome. Currently, we are attempting to visualize T‐cell activation in vivo in both rat T‐cell‐mediated EAE and mouse spontaneous EAE models.

Distinct oncogenic Ras signals characterized by profound differences in flux through the RasGDP/RasGTP cycle.

ABSTRACT T cell acute lymphoblastic leukemia/lymphoma (T-ALL) is an aggressive bone marrow cancer in children and adults, and chemotherapy often fails for relapsing patients. Molecularly targeted therapy is hindered by heterogeneity in T-ALL and mechanistic details of the affected pathways in T-ALL are needed. Deregulation of Ras signals is common in T-ALL. Ras is genetically mutated to a constitutively active form in about 15% of all haematopoietic malignancies, but there is a range of other ways to augment signaling through the Ras pathway. Several groups including our own uncovered that RasGRP1 overexpression leads to T-ALL in mouse models and in pediatric T-ALL patients, and we reported that this Ras guanine nucleotide exchange factor, RasGRP1, cooperates with cytokines to drive leukemogenesis. In our recent study by Ksionda et al. we analyzed the molecular details of cytokine receptor-RasGRP1-Ras signals in T-ALL and compared these to signals from mutated Ras alleles, which yielded several surprising results. Examples are the striking differences in flux through the RasGDP/RasGTP cycle in distinct T-ALL or unexpected differences in wiring of the Ras signaling pathway between T-ALL and normal developing T cells, which we will discuss here.

Visualizing context-dependent calcium signaling in encephalitogenic T cells in vivo by two-photon microscopy.

Significance Before invading the central nervous system, encephalitogenic T cells cross a series of microenvironments where they interact with local cells. T-cell activation was visualized by specific calcium signals using a combination of a genetic calcium reporter, Twitch1, and in vivo two-photon microscopy. In peripheral immune organs, short-lived calcium signaling indicated antigen-independent interactions. By contrast, in the CNS, saturated long-lived calcium signaling was induced by endogenous autoantigens presented by a subset of local antigen-presenting cells. Because T-cell trafficking is controlled at serial checkpoints, our findings may help to identify therapeutic targets for preventing CNS inflammation. In experimental autoimmune encephalitis (EAE), autoimmune T cells are activated in the periphery before they home to the CNS. On their way, the T cells pass through a series of different cellular milieus where they receive signals that instruct them to invade their target tissues. These signals involve interaction with the surrounding stroma cells, in the presence or absence of autoantigens. To portray the serial signaling events, we studied a T-cell–mediated model of EAE combining in vivo two-photon microscopy with two different activation reporters, the FRET-based calcium biosensor Twitch1 and fluorescent NFAT. In vitro activated T cells first settle in secondary (2°) lymphatic tissues (e.g., the spleen) where, in the absence of autoantigen, they establish transient contacts with stroma cells as indicated by sporadic short-lived calcium spikes. The T cells then exit the spleen for the CNS where they first roll and crawl along the luminal surface of leptomeningeal vessels without showing calcium activity. Having crossed the blood–brain barrier, the T cells scan the leptomeningeal space for autoantigen-presenting cells (APCs). Sustained contacts result in long-lasting calcium activity and NFAT translocation, a measure of full T-cell activation. This process is sensitive to anti-MHC class II antibodies. Importantly, the capacity to activate T cells is not a general property of all leptomeningeal phagocytes, but varies between individual APCs. Our results identify distinct checkpoints of T-cell activation, controlling the capacity of myelin-specific T cells to invade and attack the CNS. These processes may be valuable therapeutic targets.

Comprehensive analysis of T cell leukemia signals reveals heterogeneity in the PI3 kinase-Akt pathway and limitations of PI3 kinase inhibitors as monotherapy

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic cancer. Poly-chemotherapy with cytotoxic and genotoxic drugs causes substantial toxicity and more specific therapies targeting the underlying molecular lesions are highly desired. Perturbed Ras signaling is prevalent in T-ALL and occurs via oncogenic RAS mutations or through overexpression of the Ras activator RasGRP1 in ~65% of T-ALL patients. Effective small molecule inhibitors for either target do not currently exist. Genetic and biochemical evidence link phosphoinositide 3-kinase (PI3K) signals to T-ALL, PI3Ks are activated by Ras-dependent and Ras-independent mechanisms, and potent PI3K inhibitors exist. Here we performed comprehensive analyses of PI3K-Akt signaling in T-ALL with a focus on class I PI3K. We developed a multiplex, multiparameter flow cytometry platform with pan- and isoform-specific PI3K inhibitors. We find that pan-PI3K and PI3K γ-specific inhibitors effectively block basal and cytokine-induced PI3K-Akt signals. Despite such inhibition, GDC0941 (pan-PI3K) or AS-605240 (PI3Kγ-specific) as single agents did not efficiently induce death in T-ALL cell lines. Combination of GDC0941 with AS-605240, maximally targeting all p110 isoforms, exhibited potent synergistic activity for clonal T-ALL lines in vitro, which motivated us to perform preclinical trials in mice. In contrast to clonal T-ALL lines, we used a T-ALL cancer model that recapitulates the multi-step pathogenesis and inter- and intra-tumoral genetic heterogeneity, a hallmark of advanced human cancers. We found that the combination of GDC0941 with AS-605240 fails in such trials. Our results reveal that PI3K inhibitors are a promising avenue for molecular therapy in T-ALL, but predict the requirement for methods that can resolve biochemical signals in heterogeneous cell populations so that combination therapy can be designed in a rational manner.

Abstract B22: Screening and validation of combination therapy in T cell leukemia

Oncogenic Ras mutations occur in more than 30% of metastatic cancers. There are no effective inhibitors to block oncogenic K- or N-RAS, however, blockade of Ras’ downstream RAF-MEK-ERK and PI3K-Akt-mTOR-S6 signaling cascades is currently being explored as therapy in clinical trials through the use of potent kinase inhibitors. Nonetheless, many cancers have a complex make-up of cooperating oncogenic lesions and it is therefore questionable if inhibition of single molecules in pathways will provide sufficient, long-term efficacy. To explore effective combination therapies to treat Ras-driven cancers we chose synthetic lethal screens (SLS), focusing on PI3K due to its frequent involvement in oncogenic signaling. We performed a set of SLS on T cell leukemia (T-ALL) cell lines combining specific PI3K inhibitors with high coverage shRNA libraries, initially aiming at kinases. We employed the UCSF EXPANDed RNAi library resource, which dramatically improves RNAi screening compared to commercial resources. This ultra-complex shRNA library targets each gene with >25 independent shRNAs, thus drastically minimizing experimental noise and allowing us to overcome both the common problems of high false-negative rates and high false-positive rates. Using deep sequencing of shRNAs recovered from our screening samples, we identified shRNAs that synergize with inhibitors in these drop-out SLS conditions and which represent key molecules for the survival of T-ALL lines. In addition, we also identified enriched shRNAs that confer inhibitor resistance or enhanced proliferation. To verify our screening results we used combinations of chemical inhibitors, targeting PI3K and molecules identified by our screens. To date, all inhibitor combinations tested caused strong synthetic lethality in T-ALL cells, confirming the robustness of the ultra-complex shRNA libraries used for SLS. The most promising results will be further verified in vivo, and we will expand our efforts to different leukemia and also solid cancer lines. Altogether, our high-troughput platform is a promising approach to identify effective partners for already established inhibitors, leading to novel combination therapies for the treatment of cancer. Citation Format: Marsilius Mues, Marthe F. Lindenbergh, Michael T. McManus, Jeroen P. Roose. Screening and validation of combination therapy in T cell leukemia. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B22.

Intravital imaging of T cell activation in the CNS during autoimmunity

Regulatory T (Treg) cells mediate immune tolerance to self and depend on IL-2 for homeostasis. Treg deficiency, dysfunction and instability are implicated in the pathogenesis of numerous autoimmune diseases, including relapsing–remitting multiple sclerosis (RRMS). Daclizumab high yield process (DAC HYP) is a humanized monoclonal antibody that binds the IL-2 receptor alpha subunit (IL2Ralpha or CD25) and prevents IL-2 binding. DAC HYP has demonstrated clinical efficacy in patients with RRMS, despite causing a reduction in circulating Treg cell numbers. Here we investigate the impact of DAC HYP-mediated CD25 blockade on Treg cell homeostasis in RRMS patients. Based on analysis of a large clinical sample set, we report that DAC HYP treatment caused an ~50% decrease in Treg cells by week 8 of treatment that was sustained over a 52-week period. Remaining Treg cells retained a demethylated TSDR in the FOXP3 promoter, maintained active cell cycling and had minimal production of IL-2, IFN-gamma, and IL-17. In the presence of DAC HYP, IL-2 serum concentrations increased and intermediate affinity IL-2Rbeta-gamma-signaling induced STAT5 phosphorylation and sustained FoxP3 expression. Treg cell declines did not associate with DAC HYP-related clinical benefit or cutaneous adverse events. Our results demonstrate that Treg cell phenotype and lineage stability can be maintained in the face of CD25 blockade.