Neurofibromatosis Type 1 (NF1) is a genetic disease caused by mutations in Neurofibromin 1 (NF1). NF1 patients present with a variety of clinical manifestations and are predisposed to cancer development. Many NF1 animal models have been developed, yet none display the spectrum of disease seen in patients and the translational impact of these models has been limited. We describe a minipig model that exhibits clinical hallmarks of NF1, including café au lait macules, neurofibromas, and optic pathway glioma. Spontaneous loss of heterozygosity is observed in this model, a phenomenon also described in NF1 patients. Oral administration of a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor suppresses Ras signaling. To our knowledge, this model provides an unprecedented opportunity to study the complex biology and natural history of NF1 and could prove indispensable for development of imaging methods, biomarkers, and evaluation of safety and efficacy of NF1-targeted therapies.

Regulation of the magnitude and quality of immune responses is dependent on the integration of multiple signals which typically operate through positive and negative feedback loops. Cytokines that promote or limit T cell expansion and differentiation are often both present in the complex lymphoid environment where antigen-initiated T cell responses take place. The nature and strength of the cytokine signal received by the responding cell, as well as by surrounding regulatory cells, will determine the extent of clonal expansion and the progression towards effector and memory cell differentiation. The mechanisms that determine how much cytokine is produced and how cytokine activities are controlled by receptor expression and intracellular regulators of signaling are not fully understood. Here we discuss the opposing functions of two members of the common receptor gamma chain (γc) cytokines, IL-2 and IL-7 in the generation and regulation of immune responses in vivo.

BALB/c IL-2–deficient (IL-2–KO) mice develop systemic autoimmunity, dying within 3 to 5 wk from complications of autoimmune hemolytic anemia. Disease in these mice is Th1 mediated, and IFN-γ production is required for early autoimmunity. In this study, we show that dendritic cells (DCs) are required for optimal IFN-γ production by T cells in the IL-2–KO mouse. Disease is marked by DC accumulation, activation, and elevated production of Th1-inducing cytokines. IL-2–KO DCs induce heightened proliferation and cytokine production by naive T cells compared with wild-type DCs. The depletion of either conventional or plasmacytoid DCs significantly prolongs the survival of IL-2–KO mice, demonstrating that DCs contribute to the progression of autoimmunity. Elimination of Th1-inducing cytokine signals (type 1 IFN and IL-12) reduces RBC-specific Ab production and augments survival, indicating that cytokines derived from both plasmacytoid DCs and conventional DCs contribute to disease severity. DC activation likely precedes T cell activation because DCs are functionally activated even in an environment lacking overt T cell activation. These data indicate that both conventional and plasmacytoid DCs are critical regulators in the development of this systemic Ab-mediated autoimmune disease, in large part through the production of IL-12 and type 1 IFNs.

BALB/c IL-2-KO mice develop systemic autoimmunity, dying within 4 wks from autoimmune hemolytic anemia. Disease in these mice is Th1-mediated, and IFNγ production is required for early autoimmunity. We asked whether APC are required for IFNγ production by T cells in the IL-2-KO mouse. Our data show that APC:T cell interactions through B7, but not via B cells, are necessary for IFNγ production in the absence of IL-2, suggesting that dendritic cells (DC) mediate this alteration. Disease is marked by DC accumulation, activation and elevated cytokine production, including IL-12 and type1-interferons. The depletion of either conventional (cDC) or plasmacytoid (pDC) significantly augmented the survival of IL-2-KO mice, demonstrating that DC contribute to the progression of autoimmunity. In the absence of IL-12, T cell activation and disease is only moderately altered, thus Th1 differentiation and IFNγ induction as a part of the underlying kinetics of autoimmunity is primarily IL-12-independent. Elimination of type 1 interferon signals in addition to IL-12 further augmented survival, indicating that cytokines derived from both pDC and cDC contribute to disease severity. Enhanced DC function is not dependent on T cell activation as DC in an environment without overt T cell activation are still functionally activated. Overall, our data suggest that DC activation can be an initiating event during the development of disease and thus DC are critical regulators of autoimmune development.

It is imperative to gain a better understanding of biological changes that occur during tumor progression/evolution/recurrence to facilitated the development of new anti-recurrent therapies. METHODS: Single patient tumor cells were immediately dissociated, cultured in vitro or/and transplanted into mouse brains corresponding to the patient's tumor location. Whole genome DNA methylation, transcriptomic profiling and RNA sequencing were performed in addition to immunohistological phenotypes. Standard therapies which follow clinical regimen were conducted as well in tumor xenografts. RESULTS: We successfully established two sets of matched primary and recurrent PDOX models. Compared with the primary tumors, the tumor-take rate of the recurrent tumors (obtained 9 months after chemo-therapy and stem cell rescue) with shortened tumor onset time increased from 70% of primary tumor 19850PNET to 100% of recurrent 22909PNET (piece#1) and 23542PNET (piece#2) with significantly shorten animal survival times (4-5 months to 2-3 months). In the second set of PDOX models derived a low grade ganglioglioma (100815GGG) and a matching recurrent glioblastoma (104488GBM), progressive phenotypes with decreased tumor onset time (from 5 months to 3 months) was also noted. RNA sequencing reveals preserved and newly mutated genes in recurrent tumors. High-throughput drug screening using cultured patient tumor cells 100815GGG and 104488GBM discovered a shared and a diverse panel of therapeutic targets that could be selected further for in vivo drug treatment in future. Standard chemo-therapy were conducted in xenografts for primary 19850PNET and recurrent 23542PNET tumor. Chemo-drug administration significantly extend survival time of recurrent tumor in contrast to primary tumor. Molecular phenotypes such as genomic methylation and transcriptome profile are underway. CONCLUSION: Two sets of matched primary and recurrent pediatric brain tumors, PNET/PNET and GGG/GBM, were successfully established in pediatric orthotopic mice tumor models (PDOXs) providing clinically-relevant and biologically-accurate animal model systems for development of novel therapeutic targets.

BALB/c IL-2-KO mice develop systemic autoimmunity, dying by 4 wks from complications of autoimmune anemia. Disease in these mice is Th1-mediated, and IFNγ production is required for early disease (Blood 2008). We asked whether APC are required for IFNγ production by T cells in the IL-2-KO mouse. Our data show that APC:T cell interactions through B7, but not via B cells, are necessary for IFNγ production in the absence of IL-2, suggesting that dendritic cells (DC) mediate this alteration. Disease in IL-2-KO mice is marked by DC accumulation, activation and elevated cytokine production, including IL-12. Surprising, in the absence of IL-12 T cell activation and disease is unchanged, thus, IL-12 is dispensable for IFNγ induction as a part of the underlying kinetics of autoimmunity. This led us to ask whether DC are necessary for IFNγ induction and autoimmunity in these mice. The depletion of either CD11c-high-DC or plasmacytoid DC significantly augmented the survival of IL-2-KO mice, demonstrating that DC contribute to the propagation of autoimmunity. IL-2-KO DC induced heightened proliferation and cytokine production by naïve T cells compared to WT DC. Also, DC from mice without overt T cell activation still increased the proliferation of naïve T cells, suggesting that enhanced DC function is not dependent on T cell activation. Overall, our data suggest that DC are directly activated in the absence of functional Tregs, and are critical regulators of autoimmune development.

TCRαβ+CD4−CD8αα+ intestinal intraepithelial lymphocytes (CD8αα IEL) descend from thymic precursors. To better define this IEL precursor (IELp) population, we analyzed their maturation, localization, and emigration. Using rigorous lineage exclusion criteria, we defined two precursors among DN TCRβ+ thymocytes: a nascent PD-1+ population and a T-bet+ population that accumulates with age. Both gave rise to intestinal CD8αα IEL upon adoptive transfer. In adult mice, PD-1+ cells contained more self-reactive clones, localized to the cortex, and were dominant in S1PR1-dependent thymic egress. Gut homing α4β7 was already expressed by these IELp at a thymic stage. To understand the kinetics of CD8αα IEL seeding the intestine, we performed “time-stamp” experiments: We crossed Cd4CreERT2 with Rosa26TdT (stop-floxed tdTomato) mice. In these mice, tamoxifen or its metabolite 4-OHT permanently labels every CD4 expressing cell. As TCRαβ T cells (including CD8αα IEL) go through a CD4+CD8+ stage during thymic development, a single dose of tamoxifen or 4-OHT will label thymic IEL precursors permanently, so that they can be tracked when seeding the gut. Our results indicate that these cells enter the intestine during a narrow time window in early life and that this influx is almost completely shut down by the age of 3 weeks. These data provide an important foundation for understanding the biology of this abundant population of barrier surface T cells.

IL-2-deficient mice develop a spontaneous systemic autoimmune disease and on the BALB/c background die at 3-4 weeks due to hypoxia from autoimmune hemolytic anemia (AIHA). These mice develop progressive lymphadenopathy due to the accumulation of CD4+ and CD8+ T cells. It is known that CD4+ T cells are necessary for disease progression and we have previously shown that IL-2-KO CD4+ T cells from sick mice can transfer AIHA and lymphadenopathy to recipient mice. As CD8+ T cells also accumulate during disease, we asked whether these cells play a role in the development of autoimmune disease. To address this, we depleted CD8+ T cells by treating mice twice a week with anti-CD8α (clone 2.43). Elimination of CD8+ T cells for two weeks resulted in an augmentation of survival from 3-4 weeks to 3 months. The number and activation state of the CD4+ T cells and B cells was decreased, and the development of red blood cell (RBC)-specific antibodies delayed. Our results suggest that autoreactive CD8+ T cells contribute to the initiation and/or propagation of lymphoproliferation and pathogenesis of autoimmunity. Both CD4+ and CD8+ T cells from the IL-2-deficient mice produce IFNγ in response to RBC antigens. We are currently addressing the importance of these autoreactive CD8+ T cells relative to that of the self-reactive CD4+ T cells. We are also evaluating the possible mechanisms by which CD8+ T cells influence the disease process.