RAG-2 KO Mice Research Articles

T cells are necessary for resolution of inflammation-induced depression-like behavior

Depression is one of the most common mental disorders worldwide. Accumulating evidence indicates a role of the innate immune system in the onset of depression. However, how T cells contribute to depression is unknown. Recently, we showed that T cells are required for resolution of neuropathic pain. Here, we hypothesized that T cells are also required for resolution of inflammation-induced depression. We compared the duration of depression-like behavior of C57Bl/6 WT and T cell deficient (Rag1-/- and Rag2-/-) mice in response to lipopolysaccharide or complete Freund’s adjuvant. Depression-like behavior was measured as increased immobility time in the forced swim test. In both models, the onset of depression was similar between WT and T cell deficient mice, but depression was drastically prolonged in T cell deficient mice. This was associated with a prolonged upregulation of indoleamine 2,3-dioxygenase (Ido1) in the brain. Reconstitution of Rag-KO mice with T cells normalized duration of depression-like behavior and expression of brain Ido1. T cells migrated to the meninges during the resolution. The expression of Ido1 inducing cytokines such as IFNg, TNF and IL1b was T cell independent. In contrast, T cells were required for induction of IL-10 in the brain. Inhibition of brain IL-10-signaling prolonged depression-like behavior. However, the T cells were not the source of IL-10. In conclusion, T cells prevent chronicity of depression via an IL-10-dependent pathway.

MDR1A deficiency restrains tumor growth in murine colitis-associated carcinogenesis.

Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.

TNF signalling drives expansion of bone marrow CD4+ T cells responsible for HSC exhaustion in experimental visceral leishmaniasis.

Visceral leishmaniasis is associated with significant changes in hematological function but the mechanisms underlying these changes are largely unknown. In contrast to naïve mice, where most long-term hematopoietic stem cells (LT-HSCs; LSK CD150+ CD34- CD48- cells) in bone marrow (BM) are quiescent, we found that during Leishmania donovani infection most LT-HSCs had entered cell cycle. Loss of quiescence correlated with a reduced self-renewal capacity and functional exhaustion, as measured by serial transfer. Quiescent LT-HSCs were maintained in infected RAG2 KO mice, but lost following adoptive transfer of IFNγ-sufficient but not IFNγ-deficient CD4+ T cells. Using mixed BM chimeras, we established that IFNγ and TNF signalling pathways converge at the level of CD4+ T cells. Critically, intrinsic TNF signalling is required for the expansion and/or differentiation of pathogenic IFNγ+CD4+ T cells that promote the irreversible loss of BM function. These findings provide new insights into the pathogenic potential of CD4+ T cells that target hematopoietic function in leishmaniasis and perhaps other infectious diseases where TNF expression and BM dysfunction also occur simultaneously.

Abstract 1015: Directionality of T-cell-mediated cross-protection against the same tumor in lungs versus skin

Abstract Immunotherapy is a new pillar of cancer therapy. One theoretical advantage of immunotherapy of cancer is that effector cells induced at one site should be able to kill metastatic cancer cells in other sites or tissues. On the other hand, it has been recognized that each tissue has unique immune components that play critical roles in protection against pathogens. However, very little is known whether effector T cells induced against tumors in one tissue can work against the same tumors in other tissues. To address this question, we compared the effect of effector cells induced against the same tumor cells growing in either the skin or the lung by using CT26 murine tumor models. Rejection of s.c. CT26 tumors was achieved by pretreatment with anti-CD25, which blocks the function of Treg cells. Both CD4 and CD8 T cells were necessary for the protection. When anti-CD25-pretreated mice challenged with s.c. CT26 were simultaneously inoculated i.v. with CT26, they also rejected tumors in the lung, while anti-CD25-pretreated mice without s.c. CT26 did not. This observation suggested that T cell mediated anti-tumor protective immunity induced against s.c. tumors can also protect against lung metastases of the same tumors. In contrast to Treg depletion which allowed for the induction of protective immunity in the s.c. tumor model, NKT cell-deficiency in CD1d KO mice induced significant CD8 T cell-mediated protection against lung metastasis of CT26 but had no effect on the growth of s.c. CT26 tumors. When CD1d KO mice rejecting i.v. CT26 were simultaneously challenged with s.c. CT26, the development of s.c. tumors was not affected, indicating that tumor rejection induced against the CT26 in the lung did not confer protection for the same tumor cells in the skin. Since the protection against CT26 in the lung in CD1d KO mice is mediated by CD8 T cells, we transferred T cells from CD1d KO mice inoculated i.v. with CT26 into RAG1 KO recipients, and challenged the recipient mice with the CT26 tumor cells. The recipient RAG1 KO mice were highly resistant to CT26 inoculated i.v. but not s.c. confirming that the CD8 T cells protective in the lung are not protective in the skin. These data indicate the effector cells against the same tumor do not work in all tissues, and the induction site of the effector T cells is critical to control metastasis. Citation Format: Jessica J. O'Konek, Elena Ambrosino, Anja Bloom, Liat Izhak, Jay A. Berzofsky, Masaki Terabe. Directionality of T-cell-mediated cross-protection against the same tumor in lungs versus skin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1015. doi:10.1158/1538-7445.AM2017-1015

ENU-induced C-terminal truncation of Runx3 results in Th17-associated autoimmune colitis

Abstract Inflammatory bowel disease (IBD) is a common, chronic, progressive inflammatory intestinal disease in the world. The etiology of IBD is due to the imbalance of host immune responses to intestinal flora. Many human genes play the important role to induce improper immune responses, including IL-10, STAT3, IRGM, AGT16L1, NOD2 and RUNX3. It has been reported that homozygous Runx3-ko mice develop colitis spontaneously. However, homozygous Runx3-ko mice show very high mortality rate in the first 2 week of life, and are hard to study the role of Runx3 in the spontaneous colitis. A mouse pedigree (spontaneous colitis, SC) carried an ENU-induced Tyr301stop point mutation of Runx3, which resulted in a short form truncated form of Runx3. SC mice started to show spontaneous diarrhea after weaning. SC mice exhibited prominant enlargement of colon that revealed large amounts of inflammatory cells infiltration from mucosal to muscular layers. Immunofluorescent staining showed large amounts of CD4+ T cells involved in the inflammatory colon. Moreover, inflammatory colons of SC mice expressed higher mRNA levels of IL-17A, and serum IL-17A level were also upregulated in SC mice. In addition, SC CD4+ T cells expressed stronger IL-17A than WT CD4+ T cells after in vitro activation. Transfer of total SC CD4+ T cells into Rag1-ko host mice resulted in autoimmune colitis. In summary, C-terminal truncated Runx3 results in autoimmune colitis through elevated Th17 immune responses. Thus, the SC mouse model is a valuable tool for investigations on the effect of immune system upon spontaneous colitis.

Th17 cells confer both systemic and mucosal protection against Trypanosoma cruzi infection

Abstract Chagas disease is caused by chronic infection with the Trypanosoma cruzi parasite. It affects 11 million people, a third of whom will develop life-threatening cardiac and gastrointestinal morbidities. Optimal immunity to this intracellular pathogen requires CD8+ and CD4+ T cell responses. Recently, we demonstrated that CD4+ Th17 cells can confer even stronger protection against T. cruzi infection than CD4+ Th1 cells, typically considered most important for intracellular immunity. To study systemic protection, we adoptively transferred RAG KO mice with naive polyclonal CD8+ T cells and parasite-specific Th1 or Th17 cells, then infected them subcutaneously. Th17 cell co-transfer resulted in 100% long-term survival, compared to 38% with Th1 co-transfer. Mechanistically, the Th17 cells produced high levels of IL-21, which provided help for the activation of CD8+ T cells. Th17 cells given with IL-21R KO CD8+ T cells failed to protect mice, underscoring the importance of IL-21 signaling. Although CD8+ T cells were required for systemic immunity, Th17 cells alone protected against mucosal infections. Mice reconstituted with Th17 cells prior to orogastric challenge had decreased parasite burdens in the stomach and lower levels of dissemination to other organs, indicating a direct protective effect. In vitro, we discovered that Th17 cells can directly suppress infection in macrophages via the IL-17A-dependent triggering of NADPH oxidase, important for the phagocyte respiratory burst response. In summary, we have shown that Th17 cells drive robust systemic and mucosal immune responses against an intracellular parasite through both direct and indirect means, highlighting important new roles for Th17 cells in intracellular immunity.

P007 Oral tyrosine kinase 2 inhibitor ameliorates T cell transfer colitis

Background: Several Janus Kinase (JAK) inhibitors selectively targeting one JAK-family member are currently under development for the treatment of inflammatory bowel diseases (IBD). Tyrosine Kinase 2, one of the JAK family members, mediates signalling of pro-inflammatory cytokines involved in the pathogenesis of IBD including IL6, IL12, IL23 and interferon gamma (IFNγ). The aim of this study was to investigate the potency of an oral TYK2 inhibitor (TYK2i) in vivo in a murine colitis model. Methods: T cell transfer colitis was induced by adoptive transfer of wild type CD45RBhigh T lymphocytes into RAG1KO mice. After onset of endoscopic disease (day 36), animals were administered with placebo or TYK2i (10, 30, 70 mg/kg/day) daily by oral gavage. Upon sacrifice, colon weight, colon length and disease activity index (DAI, consisting of diarrhoea, oedema and occult blood, score 0–7) were recorded. Colon tissue was analysed by histology (score 0–12) and protein and transcriptional analyses of various cytokines were performed. Results: In the T cell transfer colitis model, daily administration of TYK2i prevented loss of bodyweight at all doses tested. Both endoscopic and clinical disease activity were decreased by TYK2i in a dose-dependent manner, with animals receiving 70mg/kg displaying disease activity comparable to healthy controls (median activity 0, 1.75 and 0 for healthy controls, placebo and 70mg/kg respectively). Histologically, animals receiving 70 mg/kg showed significantly decreased colitis when compared to placebo treated animals, although some residual inflammation was apparent (median score 1, 5.25 and 1.5 for healthy controls, placebo and 70mg/kg respectively). Analysis of the affected colon revealed decreased expression of IFNγ and IL6 both at the mRNA and protein level, as well as decreased protein expression of TNFα. Conclusions: Our results show that oral administration of a TYK2 inhibitor ameliorates the course of T cell transfer colitis, suggesting TYK2 as a potential therapeutic target in the treatment of IBD.

P2.03b-074 NSCLC Homing Nanoparticles Selectively Transfect Lung Cancer: Topic: Biomarkers

Gene therapy could treat lung cancer by transfecting cancer cells with plasmid DNA encoding the genes of interest. Unfortunately, it is not clinically feasible to perform repetitive intratumoral injections due to the anatomic location of lung cancer. To circumvent this problem, we created a dendrimer consisting of a fifth generation nanoparticle attached to a peptide with binding specificity for non-small cell lung cancer called NSCLC-NP. NSCLC-NP shows selective binding and transfection of human lung cells in vivo. In vivo, systemically applied NSCLC-NP home to human lung cancer cells growing in RAG2 KO mice and cause expression of the gene encoded by the attached plasmid. Dendrimers can accumulate at tumors due to the Enhanced Permeability and Retention effect. Using a corneal model growing human lung cancer cells, NSCLC-NP show binding to human lung cancer cells is based on the lung cancer homing peptide and not the EPR. The NSCLC-NP reagent is a vehicle to cause expression of any gene by lung cancer cells after systemic administration.

Macrophage-mediated psoriasis can be suppressed by regulatory T lymphocytes.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1KO mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1KO or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1KO or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deficiency of stearoyl-CoA desaturase-1 aggravates colitogenic potential of adoptively transferred effector T cells.

Stearoyl-CoA desaturase-1 (SCD1) is a lipogenic enzyme involved in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayers of cell membranes. Accordingly, Scd1KO mice display substantially reduced oleate in cell membranes. An altered SCD1 level was observed during intestinal inflammation; however, its role in modulating inflammatory bowel disease remains elusive. Herein, we investigated the colitogenic capacity of Scd1KO effector T cells by employing the adoptive T-cell transfer colitis model. Splenic effector T cells (CD4+CD25-) from age- and sex-matched wild-type (WT) and Scd1KO mice were isolated by FACS and intraperitoneally administered to Rag1KO mice, which were monitored for the development of colitis. At day 60 postcell transfer, Rag1KO mice that received Scd1KO CD4+CD25- T cells displayed accelerated and exacerbated colitis than mice receiving WT CD4+CD25- T cells. Intriguingly, Scd1KO CD4+CD25- T cells display augmented inflammatory cytokine profile and cellular membrane fluidity with a concomitant increase in proinflammatory saturated fatty acids, which we postulate to potentially underlie their augmented colitogenic potential.

Abstract # 1720 T lymphocytes are required for resolution of inflammatory pain and depression-like behavior

Transient pain and depressed mood commonly develop in response to inflammation. While normally reversible once the peripheral inflammation has resolved, depression and pain persist in some individuals. On the basis of previous studies showing that T lymphocytes contribute to homeostasis and repair in the nervous system, we hypothesized that T lymphocytes promote endogenous resolution of comorbid depression-like behavior and pain. Transient pain and depression-like behavior were induced by systemic administration of lipopolysaccharide (LPS) or by intraplantar injection of complete Freund’s adjuvant (CFA). In both models, depression-like behavior, mechanical allodynia and ongoing pain (measured by analgesic-induced conditioned place preference) were substantially prolonged in mice lacking mature lymphocytes (Rag1 and Rag2 KO mice). Reconstitution of Rag KO mice with T cells normalized resolution of depression-like behavior, mechanical allodynia and ongoing pain. Increased expression of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in the brain which mediates depression-like behavior in response to peripheral inflammation was also prolonged in absence of T cells. The anti-inflammatory cytokine IL-10 and the ATP-degrading enzyme CD73 were upregulated in the prefrontal cortex (PFC) of WT mice after they had recovered, but not in Rag KO mice at the same time point. These findings demonstrate an important role for T lymphocytes in the endogenous resolution of inflammatory pain and depression.

Interleukin-21 signaling in B cells, but not in T cells, is indispensable for the development of collagen-induced arthritis in mice.

BackgroundInterleukin-21 (IL-21) is a T-cell-derived cytokine whose receptor is expressed on a variety of cells and therefore might have pleiotropic roles in the pathogenesis of rheumatoid arthritis (RA). In this study, we investigated the involvement of IL-21 signaling in the development of collagen-induced arthritis (CIA), an animal model of RA, using IL-21 receptor knockout (Il21r KO) mice.MethodsIl21r KO mice or wild-type (WT) C57BL/6 mice were immunized with chicken type II collagen (CII) emulsified in complete Freund adjuvant on day 0 and were given a boost injection on day 21. The production of anti-CII antibody, development of T-cell and B-cell subsets, and T-cell responses to CII were analyzed. CIA was induced in Rag2 KO mice to which combinations of WT or Il21r KO CD4 T cells and WT or Il21r KO B cells had been transferred, in order to examine the role of IL-21 signaling in each cell subset.ResultsIl21r KO mice were resistant to the development of CIA. CII-specific IgG but not IgM production was impaired in Il21r KO mice. This is consistent with a reduction of germinal center B cells in the draining lymph nodes. In contrast, CII-specific Th1 and Th17 responses were unaffected in Il21r KO mice. There was also no difference in the number of CII-specific follicular helper T cells between WT and Il21r KO mice. By analyzing the development of CIA in T-cell and B-cell mixed transfer experiments, we confirmed that IL-21 receptor expression on B cells, but not on T cells, was essential for the development of CIA.ConclusionIL-21 signaling in B cells, but not in T cells, plays essential roles in the production of pathogenic autoantibodies that induce CIA development.

The role of the adaptive immune system in burn-induced heterotopic ossification and mesenchymal cell osteogenic differentiation

BackgroundHeterotopic ossification (HO) is the pathologic process of extraskeletal bone formation. Although the exact etiology remains unknown, inflammation appears to catalyze disease progression. The goal of this study is to determine the impact of the adaptive immune system on HO. MethodsHO was induced in 8-wk-old control C57BL/6 and immunocompromised Rag1tm1Mom (Rag1 KO) male mice deficient in B- and T-lymphocytes via combined Achilles tenotomy and burn injury. Microcomputed tomography quantified the extent of HO formation at the tenotomy site. Adipose-derived mesenchymal stem cells were harvested to evaluate osteogenic differentiation potential. ResultsAreas of developing HO demonstrated substantial enrichment of CD45 + leukocytes at 3 wk after injury. HO from Rag1 KO mice was substantially less mature with foci of cartilage and disorganized trabecular bone present 12 wk after injury. Rag1 KO mice formed 60% less bone compared to immunocompetent controls (4.67 ± 1.5 mm versus 7.76 ± 0.65 mm; P = 0.001). Tartrate-resistant acid phosphatase staining and immunofluorescent analysis of osteoprotegerin and nuclear factor kappa-light-chain-enhancer of activated B cells demonstrated no appreciable difference in osteoclast number or activation. Alizarin red staining in vitro demonstrated a significant decrease in osteogenic potential in immunocompromised mice compared to controls (29.1 ± 0.54 mm versus 12.1 ± 0.14 mm; P < 0.001). ConclusionsWe demonstrate a prominent role for the adaptive immune system in the development of HO. In the absence of mature B- and T-lymphocytes, HO growth and development are attenuated. Furthermore, we demonstrate that mesenchymal populations from B- and T-cell deficient mice are inherently less osteogenic. This study identifies a potential therapeutic role for modulation of the adaptive immune system in the treatment of HO.

Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy.

Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.

Th17 cells are more protective than Th1 cells against the intracellular parasite Trypanosoma cruzi.

Abstract Th1 cells are considered the major CD4+T helper subset responsible for the control of intracellular pathogens, including Trypanosoma cruzi, the protozoan parasite of Chagas disease. Th17 cells are known for their involvement in autoimmunity and the defense against some extracellular organisms, but are generally not believed to have a significant function during intracellular infections. In contrast, we show that Th17 cells are highly protective against the intracellular pathogen T. cruzi. We reconstituted RAG KO mice lacking endogenous T cells with polyclonal CD8+ T cells and parasite-specific Th1 or Th17 cells, generated from novel T cell receptor transgenic mice recently developed by us. After adoptive cell transfer, recipients were infected with a normally lethal dose of T. cruzi. Th17 cells impressively protected 100% of mice from mortality long-term, while Th1 cells protected only 38%. Transfer of Th17 cells alone failed to protect, implicating helper effects on CD8+ T cells as the basic mechanism of Th17-mediated intracellular immunity. We confirmed this using in vitro assays, and discovered that Th17 cells help activate CD8+ T cells through IL-21 secretion, indicating a protective mechanism surprisingly independent of the main Th17 cytokine IL-17A. In vivo, co-transfer of IL-21R knock-out CD8+ T cells with Th17 cells failed to protect mice, confirming the importance of IL-21 signaling. In summary, our data define an important new role for Th17 cells in the defense against a major intracellular human pathogen and define novel mechanisms of Th17 cell effects. This work increases our understanding of the protective immune interactions in T. cruzi infection, and more broadly, illuminates the growing diversity of Th17 cell roles.

Human Factor VIII Expression and Normalization of Bleeding Following AAV Gene Therapy in a Double Knockout Mouse Model of Hemophilia

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the Factor VIII coagulation protein (FVIII). Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma derived FVIII. Recently, Nathwani et al demonstrated in preclinical and clinical studies sustained expression of coagulation factor IX using AAV8 technology to deliver the human gene to the liver, driven by a liver specific promoter. The same group demonstrated FVIII expression in mice and primates using a modified B-domain truncated form of FVIII delivered in an AAV8 capsid.We have made an AAV5 construct containing a B-domain deleted FVIII gene (AAV5-SQ) with a liver specific promoter and evaluated it in a double knockout mouse model of hemophilia.The double knockout mice (DKO) were created by crossing factor VIII deficient mice with RAG2 deficient mice (RAG2 KO). The RAG2 KO mice lacked the ability to mount an adaptive immune response thereby allowing sustained expression of a human protein without the development of an antibody response.Eight week old male DKOs were randomly distributed into three groups, twenty per group, and treated via a single IV injection with either vehicle, or AAV5-SQ at 2 x 1013 or 1 x 1014 vg/kg. C57BL/6J mice comprised a fourth group and were treated with vehicle via a single IV injection to demonstrate wild type bleeding times and blood loss. Bleeding times and blood loss were assessed in these mice 8 weeks post-dose, at 16 weeks of age. In addition, forty 16 week old DKO mice were randomly divided into two groups and treated with a single IV injection of rhFVIII-SQ protein (rhSQ, Xyntha®) at either 50 or 200 IU/kg. Bleeding times were assessed in these mice 30 minutes post-dose, at 16 weeks of age.Eight weeks post dosing with either AAV5-SQ or vehicle, the tail bleeding time and blood loss were measured following transection of the tip of the tail for evaluation of the functional efficacy of AAV5-SQ gene therapy. Wild-type mice receiving vehicle had a mean of 0.040 ± 0.073 g blood loss and 5.11 ± 5.61 min bleeding time. DKO mice treated with vehicle had a mean blood loss and bleeding time of 0.741 ± 0.207 g and 28.96 ± 1.40 min, respectively. Mice receiving AAV5-SQ at 2x1013 vg/kg showed significantly reduced blood loss (0.387 ± 0.384 g, p=0.0008 vs DKO+ vehicle; p=0.0003 vs WT) and bleeding time (17.12 ± 11.58 min, p=0.00005 vs DKO+ vehicle; p=0.0013 vs WT) while 1x1014 vg/kg AAV5-SQ treatment corrected blood loss and bleeding times to wild-type levels (0.104 ± 0.203 g [p=0.192 vs WT, p= 5.49x10-12 vs DKO + vehicle] and 5.58 ± 9.32 mins [p=0.847 vs WT, p= 1.75x-13 vs DKO + vehicle], respectively). The effect of AAV5-SQ treatment on blood loss and bleeding time was comparable to the effects of rhSQ. DKO mice receiving 50 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.492 ± 0.297 g and 18.14 ± 9.39 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 2x1013 (p=0.343 for blood loss, p=0.760 for bleeding time). DKO mice receiving 200 IU/kg of rhSQ had a mean blood loss and bleeding time of 0.134 ± 0.191 g and 4.29 ± 6.16 min, respectively, which was not significantly different from mice receiving AAV5-SQ at 1x1014 (p=0.635 for blood loss, p=0.608 for bleeding time).In a separate experiment, 4 groups of DKO mice, n=10 per group, were injected with either vehicle, AAV5-SQ at 2x1013, AAV5-SQ at 2x1014 vg/kg or rhSQ at 50 IU/kg. Blood was collected 8 weeks after AAV5-SQ treatment or 5 and 30 min after rhSQ for evaluation of plasma hFVIII-SQ protein levels and activity. Expressed hFVIII-SQ levels were measured by electrochemiluminescence assay. Factor VIII-SQ protein levels at 2x1013 vg/kg AAV5-SQwere 46.8±44.0 ng/ml and 355±166ng/ml at 2x1014 vg/kg. At 50 IU/kg of rhSQ the plasma protein levels were 79.1±11.3 ng/ml at 5 min and 44.7±16.6 ng/ml at 30min post dosing. Western blot analysis of the plasma from these mice showed the expressed protein to be similar in size to rhSQ.In summary, AAV5-SQ injected into DKO hemophilic mice resulted in a dose dependent expression of B-domain deleted FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by AAV5-SQ or following exogenous administration of B-domain deleted FVIII. DisclosuresBunting:BioMarin Pharmaceutical: Employment. Zhang:BioMarin Pharmaceutical: Employment. Xie:BioMarin Pharmaceutical: Employment. Bullens:BioMarin Pharmaceutical: Employment. Mahimkar:BioMarin Pharmaceutical: Employment. Fong:BioMarin Pharmaceutical: Employment. Sandza:BioMarin Pharmaceutical: Employment. Colosi:BioMarin Pharmaceutical: Employment. Long:BioMarin Pharmaceutical: Employment. Vehar:BioMarin Pharmaceutical: Employment. Carter:BioMarin Pharmaceutical: Employment.

Reduced β-amyloid pathology in an APP transgenic mouse model of Alzheimer's disease lacking functional B and T cells.

IntroductionIn Alzheimer’s disease, accumulation and pathological aggregation of amyloid β-peptide is accompanied by the induction of complex immune responses, which have been attributed both beneficial and detrimental properties. Such responses implicate various cell types of the innate and adaptive arm of the immunesystem, both inside the central nervous system, and in the periphery. To investigate the role of the adaptive immune system in brain β-amyloidosis, PSAPP transgenic mice, an established mouse model of Alzheimer’s disease, were crossbred with the recombination activating gene-2 knockout (Rag2 ko) mice lacking functional B and T cells. In a second experimental paradigm, aged PSAPP mice were reconstituted with bone marrow cells from either Rag2 ko or wildtype control mice.ResultsAnalyses from both experimental approaches revealed reduced β-amyloid pathology and decreased brain amyloid β-peptide levels in PSAPP mice lacking functional adaptive immune cells. The decrease in brain β-amyloid pathology was associated with enhanced microgliosis and increased phagocytosis of amyloid β-peptide aggregates.ConclusionThe results of this study demonstrate an impact of the adaptive immunity on cerebral β-amyloid pathology in vivo and suggest an influence on microglia-mediated amyloid β-peptide clearance as a possible underlying mechanism.Electronic supplementary materialThe online version of this article (doi:10.1186/s40478-015-0251-x) contains supplementary material, which is available to authorized users.

IMPS-29REFUTING BRAIN IMMUNE PRIVILEGE: LONGITUDINAL MULTIPHOTON IMAGING THROUGH SKULL WINDOWS REVEALS THE IMMUNE SURVEILLANCE OF BRAIN METASTASES AND ITS REGULATION BY FRACTALKINE

INTRODUCTION: The has conventionally been considered to be immune-privileged, which has detracted from the pursuit of immunotherapies for tumors. However, whether malignancies remain insulated from the immune system is unclear. T cell migration in tissues is regulated in part by chemokines often in an organ-specific manner. We hypothesized that tumors are accessible to T cell surveillance, and that this process is regulated by a chemokine called fractalkine, which is highly expressed in the brain. METHODS: Cranial windows for intravital multiphoton microscopy were established in novel transgenic murine models in which T cells, macrophages, microglia, and dendritic cells were distinctly gene-labeled with fluorescent tags. For comparison, multicolor reporter mice deficient in fractalkine receptor (CX3CR1gfp/gfp-KO) were used. Brain metastases (BM) were induced via carotid artery injection of fluorescent MCA fibrosarcoma cells and the immune dynamics at tumor lesions was imaged longitudinally. RESULTS: BM regressed after initial progression in wild-type (WT) mice, but were lethal in Rag1-KO mice, and importantly also lethal in CX3CR1-KO mice. Intravital microscopy in WT reporter mice revealed hotspots of T cell migration at BM foci followed by tumor fragmentation. In striking contrast, T cells were reduced in BM in CX3CR1-KO reporter mice and exhibited increased meandering and decreased engagement with cancer cells. CONCLUSION: With live imaging, we directly show the existence of robust and effective immune surveillance within the contrary to the concept of brain immune privilege. Furthermore, we identify the fractalkine/CX3CR1 chemokine-receptor axis as a key regulator of T cell immune surveillance in the malignant brain.

Mammalian target of rapamycin inhibition with enterically given rapamycin alters immunity and gut flora in young and aged wild type mice and extends life of immunodeficient mice (IRC8P.452)

Abstract mTOR regulates many important immune processes. Interest in immune effects of mTOR inhibitors is heightened by recent findings of ability to boost specific immunity and vaccine response in elderly humans, treat cancers and autoimmunity, and extend life and healthspan in mice, suggesting important new clinical applications. However, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. We comprehensively analyzed immune effects of long-term rapamycin in mice. Gene expression profiling of purified T and B cells (CD4+PD1+, CD4+PD1-, CD8+PD1+, CD8+PD1-, B220+), CD11b+CD11c- myeloid cells and CD11c+ dendritic cells showed numerous, novel changes in genes affecting differentiation, function, homeostasis, migration, exhaustion, cell death and inflammation. Novel effects on Th9, Th22 and Tfh differentiation were seen as was improved function of exhausted PD1+ T cells (supported by human clinical data) and increased numbers and function of innate lymphoid cells. Immune functions relevant to cancer, infections, aging and inflammation, and innate lymphoid cell effects were validated in vitro and in vivo. eRapa prolonged life in RAG KO mice (without T or B cells) &amp;gt;12x and in IFN-γ KO mice &amp;gt;3x over wild type mice, supporting mTOR suppression immune effects mitigating disease as a mechanism for longevity extension. Metagenomic effects were linked to immune outcomes. Our data demonstrate novel mTOR immune effects meriting further investigations.

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner.

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.