Chain Knockout Mice Research Articles

Serum lipidomic study of long-chain fatty acids in psoriasis patients prior to and after anti-IL-17A monoclonal antibody treatment by quantitative GC‒MS analysis with in situ extraction

BackgroundLong-chain fatty acids (LCFAs) are involved in regulating multiple physiological processes as signalling molecules. Gas chromatography–mass spectrometry (GC–MS) is widely used to quantify LCFAs. However, current quantitative methods for LCFAs using GC–MS have demonstrated complicated issues. Psoriasis is a chronic inflammatory skin disease, and its pathogenesis may be related to the overproduction of interleukin-17A (IL-17A). Clinical efficacy of anti-IL-17A monoclonal antibody (mAb) treatment in psoriasis patients has been demonstrated. Recent studies suggest that LCFAs play varying roles in the pathogenesis of psoriasis. However, more comprehensive research is needed to illuminate the mechanism of LCFAs in psoriasis.MethodsThe established in situ derivatization method for analysing LCFAs with a GC–MS platform was utilized to conduct serum lipidomics analysis of healthy volunteers and psoriasis patients receiving pretherapy and posttreatment with of anti-IL-17A mAb. Imiquimod (IMQ)-treated wild type (WT) and T-cell receptor delta chain knock-out (Tcrd−/−) mice were used to investigate the correlation between IL-17A and abnormal changes in LCFAs in psoriasis patients.ResultsA rapid and sensitive in situ extraction derivatization method for quantifying LCFAs using GC–MS was established. Serum lipidomic results showed that psoriasis patients had higher levels of saturated fatty acids (SFAs) and ω-6 polyunsaturated fatty acids (PUFAs) but lower levels of monounsaturated fatty acids (MUFAs) and ω-3 PUFAs than healthy individuals, indicating impaired serum LCFA metabolism. Anti-IL-17A mAb treatment affected most of these LCFA changes. Analysis of LCFAs in IMQ-treated mice showed that LCFAs increased in the serum of WT mice, while there were no significant changes in the Tcrd−/− mice. SFAs increased in IMQ-treated WT mice, while MUFAs showed the opposite trend, and PUFAs did not change significantly.ConclusionsThis study presented a dependable method for quantifying LCFAs that enhanced sensitivity and reduced analysis time. The lipidomic analysis results showed that anti-IL-17A mAb not only ameliorated skin lesions in psoriasis patients but also affected abnormal LCFAs metabolism. Furthermore, the study indicated a potential correlation between IL-17A and abnormal LCFA metabolism in psoriasis patients, which was supported by the alterations in serum LCFAs observed in IMQ-treated WT and Tcrd−/− mice.

Adipose knockout of H-ferritin improves energy metabolism in mice

ObjectiveFerritin, the principal iron storage protein, is essential to iron homeostasis. How iron homeostasis affects the adipose tissue is not well understood. We investigated the role of ferritin heavy chain in adipocytes in energy metabolism. MethodsWe generated adipocyte-specific ferritin heavy chain (Fth, also known as Fth1) knockout mice, herein referred to as FthAKO. These mice were analyzed for iron homeostasis, oxidative stress, mitochondrial biogenesis and activity, adaptive thermogenesis, insulin sensitivity, and metabolic measurements. Mouse embryonic fibroblasts and primary mouse adipocytes were used for in vitro experiments. ResultsIn FthAKO mice, the adipose iron homeostasis was disrupted, accompanied by elevated expression of adipokines, dramatically induced heme oxygenase 1(Hmox1) expression, and a notable decrease in the mitochondrial ROS level. Cytosolic ROS elevation in the adipose tissue of FthAKO mice was very mild, and we only observed this in the brown adipose tissue (BAT) but not in the white adipose tissue (WAT). FthAKO mice presented an altered metabolic profile and showed increased insulin sensitivity, glucose tolerance, and improved adaptive thermogenesis. Interestingly, loss of ferritin resulted in enhanced mitochondrial respiration capacity and a preference for lipid metabolism. ConclusionsThese findings indicate that ferritin in adipocytes is indispensable to intracellular iron homeostasis and regulates systemic lipid and glucose metabolism.

Unraveling Tumorigenicity in iPSC-Derived Immune Cells: The Impact of Chromosomal Abnormalities and Proliferative Intermediates

Response to tumor cells by the immune system can determine the course of a cancer. This guiding principle and the dynamic, adaptable nature of our immune system underlies the drive to generate immune cells for cancer immunotherapy. Induced pluripotent stem cells (iPSCs) provide an unlimited resource for the generation of immune cells such as T cells and dendritic cells (DCs) for cancer immunotherapy. However, the use of iPSC-derived immune cells in clinical applications has been limited due to concerns of safety. In vitro passage of iPSCs can introduce selective pressures that promote the accumulation of mutations that confer a growth advantage. Over several passages, this can lead to the acquisition of chromosomal abnormalities. Moreover, differentiation protocols of varying efficiency permit the presence of immature cells, contaminating the differentiated cell population. However, the impact of chromosomal abnormalities and intermediate products with proliferation potential on tumorigenicity of iPSC-derived immune cells remains unclear. Here, we investigated the tumorigenic potential of iPSC-derived T cells and DCs in immunocompetent and immunodeficient mice. Pmel-1 T-cell receptor (TCR) transgenic mouse T cells were reprogrammed using Sendai virus vectors carrying Oct3/4, Sox2, Klf4 and cMyc. While our iPSCs were phenotypically normal, even at early passage numbers, various chromosomal abnormalities were found in the resulting cell population. These abnormalities included trisomy 8 and 11 and loss of X chromosome with high frequency. Furthermore, within a population of iPSCs determined to be karyotypically normal by G-banding, unbalanced translocations were found by spectral karyotyping. These findings are consistent with those reported in the literature and highlight the prevalence of chromosomal abnormalities in iPSC populations. As iPSCs can harbor chromosomal abnormalities that are undetectable by standard methods, we assessed the tumorigenic potential of iPSC-derived cells, similar to what may be used in clinical applications. Mouse iPSCs were differentiated into CD8αβ T cells by co-culture with murine OP9 bone marrow stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) and were enriched by CD8 positive selection. Activated iPSC-derived T cells were adoptively transferred to immunocompetent C57BL/6 and immunodeficient NOD/SCID, IL-2 receptor γ chain knockout mice. Immunodeficient but not immunocompetent mice developed hepatic tumors. Reverse transcription (RT)-PCR identified Pme1-l TCR gene in tumor cells, confirming tumor origin. Moreover, histology of liver tumors is suggestive of lymphoma or lymphosarcoma. Within the population of CD8+ iPSC-derived cells, a small subpopulation co-expressing CD8 and SSEA-1 was present. Sorting the cells by SSEA-1 positivity eliminated tumor formation in vivo. Mouse iPSCs were also differentiated into DCs by co-culture with OP9-DL1 cells and granulocyte macrophage colony-stimulating factor. Despite similar use of iPSCs with chromosomal abnormalities and prolonged passaging in vitro, SSEA-1+ cells were not identified when they were differentiated to DCs, and iPSC-derived DCs did not form tumors in immunodeficient mice. Taken together, this study revealed the potential risk of tumorigenicity of iPSC-derived immune cells in our preclinical model. The presence of chromosomal abnormalities did not definitively determine risk of tumorigenesis upon transplantation. The relationship between SSEA-1 positivity and tumor formation suggests contaminating cells may carry tumorigenic characteristics. Further work is required to develop differentiation methods that generate pure populations, free of tumorigenic intermediates, to potentiate the clinical use of iPSC-derived immunotherapy.

The CMV-encoded G protein-coupled receptors M33 and US28 play pleiotropic roles in immune evasion and alter host T cell responses.

Human cytomegalovirus (HCMV) is a global health threat due to its ubiquity and lifelong persistence in infected people. During latency, host CD8+ T cell responses to HCMV continue to increase in a phenomenon known as memory inflation. We used murine CMV (MCMV) as a model for HCMV to characterize the memory inflation response to wild-type MCMV (KP) and a latency-defective mutant (ΔM33stop), which lacks M33, an MCMV chemokine receptor homolog. M33 is essential for normal reactivation from latency and this was leveraged to determine whether reactivation in vivo contributes to T cell memory inflation. Mice were infected with wild-type or mutant MCMV and T cell responses were analyzed by flow cytometry at acute and latent time points. Ex vivo reactivation and cytotoxicity assays were carried out to further investigate immunity and virus replication. Quantitative reverse-transcriptase polymerase chain reaction (q-RTPCR) was used to examine gene expression during reactivation. MHC expression on infected cells was analyzed by flow cytometry. Finally, T cells were depleted from latently-infected B cell-deficient mice to examine the in vivo difference in reactivation between wild-type and ΔM33stop. We found that ΔM33stop triggers memory inflation specific for peptides derived from the immediate-early protein IE1 but not the early protein m164, in contrast to wild-type MCMV. During ex vivo reactivation, gene expression in DM33stop-infected lung tissues was delayed compared to wild-type virus. Normal gene expression was partially rescued by substitution of the HCMV US28 open reading frame in place of the M33 gene. In vivo depletion of T cells in immunoglobulin heavy chain-knockout mice resulted in reactivation of wild-type MCMV, but not ΔM33stop, confirming the role of M33 during reactivation from latency. Further, we found that M33 induces isotype-specific downregulation of MHC class I on the cell surface suggesting previously unappreciated roles in immune evasion. Our results indicate that M33 is more polyfunctional than previously appreciated. In addition to its role in reactivation, which had been previously described, we found that M33 alters viral gene expression, host T cell memory inflation, and MHC class I expression. US28 was able to partially complement most functions of M33, suggesting that its role in HCMV infection may be similarly pleotropic.

Biological Analyses-Derived Translational Findings in the T Cell Receptor Alpha Chain Knockout Mouse as an Experimental Model for Ulcerative Colitis

Inflammatory bowel disease (IBD) is a group of chronic inflammatory disorders that affects many individuals throughout their lives. Ulcerative colitis (UC) and Crohn’s disease (CD) are two major forms of IBD. Until the early 1990s, a murine model of spontaneous chronic colitis was unavailable. As a major breakthrough in the basic research field of IBD, three genetically manipulated murine chronic colitis models, including interleukin (IL)-2 knockout (KO), IL-10 KO, and T cell receptor alpha chain (TCRα) KO models, were established in 1993. Since then, complicated immunobiological mechanisms during the development of UC have been gradually discovered by utilizing a wide variety of murine models of IBD, including the TCRα KO mouse model. In particular, it has been recognized that four major factors, including enteric, environmental, and immunological factors as well as enteric microbiota are highly and mutually involved in the pathogenesis of UC. As a pioneer of the TCRα KO murine model of UC, our group has identified that the interactions between the unique TCRα-β+ T cell population and antigen-presenting cells, including dendritic cells and B cells, play a key role for the development and regulation of UC-like chronic colitis, respectively. Here we have summarized clinically proven pathogenic and regulatory factors which have been identified by this novel TCRα KO murine model of UC in the past nearly three decades.

Roles of Fc receptor γ chain in inducing protective immune responses after F virus-like particles prime and FI-RSV boost against respiratory syncytial virus infection

Abstract The roles of Fc receptor (FcR) in protection or in inflammatory disease after RSV vaccination and infection remain unknown. Virus-like particles containing RSV fusion proteins (RSV F-VLPs) induce T-helper type 1 antibody responses and protection against RSV. Heterologous RSV F VLP prime and inactivated RSV (FI-RSV) boost vaccination was reported to be effective in providing protection without inflammatory disease. Here we investigated whether the FcRγ chain is important for immune protection by heterologous F VLP and FI-RSV vaccination using FcRγ chain knockout (−/−) mouse model. RSV F-VLP prime and FI-RSV boosted FcRγ−/− mice were not well protected, as shown by higher lung viral titers upon RSV virus challenge compared to RSV F-VLP prime-immunized wild-type mice. RSV F-VLP and FI-RSV immunization of FcRγ−/− mice induced significantly lower levels of neutralizing activity and interferon γ-producing CD8 T cells in bronchoalveolar lavage cells than those in in wild-type mice. In addition, FcRγ−/− mice displayed a trend of enhancing lung histopathology after RSV vaccination and infection. This study suggests that the FcR γ chain plays an important role in antiviral protection and CD8 T cell responses in RSV F-VLP and FI-RSV vaccination after RSV infections.

Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice.

Simple SummaryConsumption of alcohol increases liver damage in HIV people; however, the mechanisms remain elusive. By utilizing alcohol (ethanol) exposed HIV-infected human hepatocytes liver cells and ethanol-fed liver humanized mouse model (immunodeficient mice livers harbor human hepatocytes), we show that the combination of alcohol and HIV enhances oxidative stress and impairs lysosome activity, which in turn stimulates pathogenic nanosized extracellular vesicles. These vesicles are of great importance, as manipulating their numbers and contents will eliminate the spread of infection in other cell types.Background: Alcohol abuse is common in people living with HIV-1 and dramatically enhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomal dysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles (EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods: The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh 7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liver-humanized fumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chain knockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observed an increase in the secretion of EVs associated with a decrease in lysosomal activity and expression of lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary human hepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulated genes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressed genes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstream genes associated with increased oxidative stress, lysosomal associated disease, and function and EVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocyte-transplanted humanized mice, indicating that intensive EVs’ generation by human hepatocytes and their secretion to serum was associated with increased oxidative stress and reduction in lysosomal activities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolism-induced EVs release is tightly controlled by lysosome status in hepatocytes and participates in the development of double-insult-induced liver injury.

Efficient production of immunodeficient non-obese diabetic/Shi-scid IL2rγnull mice via the superovulation technique using inhibin antiserum and gonadotropin.

Severe immunodeficient mice are an essential tool for the examination of the efficacy and safety of new therapeutic technologies as a humanized model. Previously, non-obese diabetic (NOD)/Shi-scid IL2rγnull (NOG) mice were established as immunodeficient mice by combining interleukin-2 receptor-γ chain-knockout mice and NOD/Shi-scid mice. The NOG mice are used frequently in the research of therapeutic monoclonal antibodies and regenerative medicine for human diseases. Establishment of an efficient production system of NOG mice, using optimized reproductive techniques, is required to accelerate research. In this study, we investigated the efficacy of the superovulation technique using equine chorionic gonadotropin (eCG) and inhibin antiserum (IAS) in NOG mice of various ages (4, 8, 12, 24, or 54 weeks). Additionally, we examined the fertilizing and developmental ability of the oocytes through in-vitro fertilization using frozen-thawed sperm, embryo culture and embryo transfer. The results showed that NOG mice produced the highest number of oocytes at 12 weeks old following the co-administration of eCG and IAS (collectively IASe) (70 oocytes/female). IASe was more effective in increasing the number of oocytes v. eCG at all ages. The IASe-derived oocytes demonstrated the ability to fertilize and develop into blastocysts and pups. Finally, we demonstrated that three strains of genetically modified NOG mice were efficiently produced through the optimized reproductive techniques. In summary, we developed an efficient system for the production of immunodeficient mice using 12-week-old, IASe-treated female NOG mice.

CAR T Cells Generated Using Sleeping Beauty Transposon Vectors and Expanded with an EBV-Transformed Lymphoblastoid Cell Line Display Antitumor Activity In Vitro and In Vivo.

Chimeric antigen receptor (CAR) T cell immunotherapy for the treatment of cancer is now an approved treatment for B cell malignancies. However, the use of viral vectors to provide long-term CAR expression is associated with high production costs and cumbersome quality controls, impacting the final cost of CAR T cell therapies. Nonviral integrative vectors, such as Sleeping Beauty (SB) transposons, provide an alternative to modify primary T cells. Therefore, we developed a protocol to expand SB-transfected 19BBζ CAR T cells using a lymphoblastoid cell line, and evaluated T cell phenotype as well as function along the T cell expansion. Electroporation of PBMCs with transposon plasmid decreased cell viability on day 1 but had a minor impact on the frequency of memory subpopulations when compared to mock condition. CAR+ lymphocytes showed increased proliferation compared to mock control and high cytotoxic activity towards CD19+ cells without significant differences in exhaustion markers expression. Moreover, CAR+ lymphocytes showed an increased frequency by the end of the stimulation cycle compared with day 1, suggesting that CAR expression confers a selective proliferation advantage. Immunodeficient NOD scid gamma chain knockout (NSG) mice engrafted with the human pre-B leukemic cell line RS4;11 and treated with 19BBζ CAR T cells showed improved overall survival when compared to mock T cells treated animals. The results showed that electroporation using the SB system is a simple and affordable method for inducing long-term CAR expression in T lymphocytes. Expansion of gene-modified T cells with the lymphoblastoid cell line provided up to 2 cycles of stimulations, generating effective T cells against leukemia in vitro and in vivo.

HIV‐1 Infected NSG‐BLT Humanized Mice as a Model for HIV‐Associated Lung Complications

Human immunodeficiency virus (HIV) is an incurable and life‐threatening disease impacting over 36 million people world‐wide. Antiretroviral treatment (ART) suppresses the virus and allows many patients to live longer lives. However, even patients undergoing ART are susceptible to complications in many organ systems including the lung. HIV has been successfully linked to pulmonary complications including: tuberculosis, cytomegalovirus, Hodgkin's lymphoma, chronic obstructive pulmonary disease (COPD), and pulmonary arterial hypertension (PAH). Some of these diseases have a higher prevalence amongst patients infected with HIV. For example, in HIV patients, the incidence of PAH is 1:200, which is approximately 300‐fold higher than idiopathic PAH in the general population. Due to the limited host specificity of HIV, there are not many well‐developed animal models to study these complications. However, previous research in NOD‐Scid IL‐2 receptor γ chain knockout mice engrafted with human hematopoietic stem cells (hCD34+) and surgically implanted with human fetal thymic and liver tissue (NSG‐BLT mice) has shown disseminated HIV infection, making NSG‐BLT mice a promising in vivo model. In this study, we aimed to confirm HIV dissemination to the lungs in these mice and to examine pulmonary pathologies after an acute infection period. We infected NSG‐BLT mice with HIV for 5, 10, or 15 weeks and looked for viral RNA and pro‐viral DNA in the lungs using qPCR and nested PCR, respectively. To begin investigating possible lung pathology, we measured cytokines from lung tissue and cell‐free bronchoalveolar (BAL) fluid using an ELISA‐based protein array. Of the many proteins that were dysregulated in HIV infected mice, RAGE and IL‐25 (also known as IL‐17) are of particular interest because they have been implicated in endothelial dysfunction or vascular remodeling. This suggests a role of these proteins in HIV‐directed lung pathology in these mice. We are currently confirming the dysregulation of these genes by qPCR. In conclusion, NSG‐BLT mice constitute a useful model for studying pulmonary dissemination and pathologies during HIV infection.Support or Funding InformationNational Institutes of Health (NIH) 1R01HL125050‐01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity.

Our data implicate type I IFN signaling as a necessary component of the sex bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. (Hepatology 2018;67:1408-1419).

Clinical responses to adoptive T-cell transfer can be modeled in an autologous immune-humanized mouse model

Immune checkpoint inhibitors and adoptive cell transfer (ACT) of autologous tumor-infiltrating T cells have shown durable responses in patients with melanoma. To study ACT and immunotherapies in a humanized model, we have developed PDXv2.0 — a melanoma PDX model where tumor cells and tumor-infiltrating T cells from the same patient are transplanted sequentially in non-obese diabetic/severe combined immune-deficient/common gamma chain (NOG/NSG) knockout mouse. Key to T-cell survival/effect in this model is the continuous presence of interleukin-2 (IL-2). Tumors that grow in PDXv2.0 are eradicated if the autologous tumor cells and T cells come from a patient that exhibited an objective response to ACT in the clinic. However, T cells from patients that are non-responders to ACT cannot kill tumor cells in PDXv2.0. Taken together, PDXv2.0 provides the potential framework to further model genetically diverse human cancers for assessing the efficacy of immunotherapies as well as combination therapies.

Incidence of spontaneous lymphomas in non-experimental NOD/Shi-scid, IL-2Rγnull (NOG) mice.

Severely immunodeficient NOD/Shi-scid, IL-2Rγnull (NOG) mice provide an in vivo model for human cell/tissue transplantation studies. NOG mice were established by combining interleukin-2 receptor-γ chain knockout mice and NOD/Shi-scid mice. They exhibit a high incidence of thymic lymphomas and immunoglobulin (Ig) leakiness. In this study, we assessed the incidence of malignant lymphomas and the occurrence of leakiness in 2,184 non-experimental NOG retired breeder mice aged 16–40 weeks. We established that the total incidence of lymphomas was only 0.60% (13/2,184). Most lymphomas (10/13) occurred in female mice by the age of around 25 weeks. No mice developed Ig leakiness. All lymphomas were derived from the thymus, and consisted mainly of CD3-positive and CD45R-negative lymphoblastic-like cells. Therefore, based on the absence of Ig leakiness and a very low incidence of lymphomas, including thymic lymphomas, NOG mice may be useful in regeneration medicine for xenotransplantation of human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, and in transplantation experiments involving tumor cells.

Anti-CD27 Enhances Lymphoma Immunotherapy through Profound Myeloid Cell Recruitment

Direct-targeting monoclonal antibodies (mAb) such as anti-CD20 mAb are thought to elicit their anti-tumor function through antibody-dependent cellular phagocytosis (ADCP) mediated by myeloid cells (monocytes and macrophages), with little involvement of T cells. In contrast, immunomodulatory mAbs to TNFR superfamily members, CD27, OX40 and CD137, function by augmenting T-cell responses.We examined the therapeutic potential of combining anti-CD20 mAb with a panel of immunomodulatory mAbs (OX40, CD137, CD27, TIGIT, GITR, CTLA4, PD-1). In the syngeneic BCL1 B-cell lymphoma mouse model only an agonistic mAb to CD27, provided a synergistic effect when combined with anti-CD20. Anti-CD20 and anti-CD27 mAb individually provided modest therapeutic benefit (median survival 33 days and 62 days, respectively), but mice treated with the combination survived beyond 100 days. Similar synergistic survival benefit was observed in another B-cell lymphoma model, A31, and in BCL1-bearing human CD27 transgenic mice, when anti-CD20 was combined with varlilumab, an anti-human CD27 mAb currently under clinical investigation.We observed that in mice treated with anti-CD27, there was an early and substantial increase in intra-tumoral monocyte, neutrophil and macrophage infiltration. CD27 is expressed constitutively on T and NK cells but not myeloid cells or the tumor itself. To investigate whether CD27 agonism promotes intra-tumoral myeloid cell infiltration through T cells, we depleted T cells in the BCL1model. Surprisingly, CD4 or CD8 T-cell depletion had no effect on the survival of anti-CD20 and anti-CD27-treated mice, suggesting that the remaining CD27+ immune effector cells, NK cells, are required. To further probe the relative importance of these two sub-sets, we performed experiments in γ chain knockout mice, where activatory FcγR are not expressed. Here, anti-CD27 mediated T-cell activation can still occur via crosslinking from the inhibitory FcγRII, but effector function through NK cells, mediated through activatory FcγR, is abrogated. In this model, the therapeutic benefit of anti-CD27 was completely abolished, thereby supporting the requirement for NK cells.We hypothesize that anti-CD27 stimulates CD27+ NK cells to release chemokines that draw myeloid cells into the tumor, where they subsequently perform augmented anti-CD20 mediated ADCP.These data demonstrate the clear therapeutic potential of combining direct targeting and immunomodulatory mAb but that the therapeutic mechanism of action may differ to that expected; here involving a previously unheralded effect of anti-CD27 on myeloid infiltration. Based upon these data, we have implemented a phase II clinical trial examining rituximab and varlilumab in B-cell lymphoma, which will commence recruitment shortly. DisclosuresKeler:Celldex Therapeutics: Employment, Equity Ownership. Johnson:Celldex Therapeutics: Research Funding. Al-Shamkhani:Celldex Therapeutics: Patents & Royalties: On therapeutic use of antibodies targeting anti-CD27 and another applied for anti-CD20/anti-CD27 use, Research Funding. Glennie:Celldex Therapeutics: Patents & Royalties: Patent on therapeutics use of antibodies targeting human CD27 and patent for anti-CD20+anti-CD27 applied. Cragg:Baxalta: Consultancy; Gilead Sciences: Research Funding; GSK: Research Funding; Roche: Consultancy, Research Funding; Bioinvent International: Consultancy, Research Funding. Lim:Celldex Therapeutics: Patents & Royalties: Patent for anti-CD20+anti-CD27 applied, Research Funding.

C75 Fatty Acid Synthesis (FAS) Inhibitor Has Potent Immunosuppressive Activity

Fatty acid synthesis (FAS) or oxidation (FAO) are important regulatory pathways in immune response. In fact, FAS plays a pivotal role in antigen presentation and T cells activation and FAO leads to fatty acid degradation which has been previously shown to regulate hematopoietic stem cell maintenance. Here we hypothesized that FAS can be a new target to suppress T cell alloimmune responses in solid organ or stem cell transplantations. Therefore, we tested if the FAS inhibitor C75 could suppress T cell alloreactivity without impairing normal hematopoiesis.The immuno-suppressive (IS) effect of moderate FAS inhibition was demonstrated in mixed leukocyte cultures (MLC) where C75 at 10 mkg/ml significantly reduced T cell proliferation and prevented the expansion of CD3+CD25+ and CD3+CD69+ T cells. In T cells stimulated by alloantigen, C75 also induced the downregulation of NF-kB gene expression and the upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene involved in ubiquitination and degradation of NF-kB protein.When compared to other standard IS agents, such as anti-thymocyte globulin (ATG), Cyclosporine A, Rapamycin or inhibitor of FAO Etomoxir, C75 showed similar anti-T cell activity.The same dose of C75 (10 mkg/ml) did not cause apoptotic death of human CD34+ cells in vitro, nor affected CD34+ cell clonogenicity in vitro. In fact, C75 increased the number of BFU-E and CFU-GM colonies (P < 0.05). We observed that the expression of de novo DNA methyltrasferases DNMT3A and DNMT3B, which are important regulators of stem cell renewal, was strongly reduced in CD34+ cells co-cultured for 3 days with allogeneic T cells. On the contrary, in the presence of C75 the expression of DNMT3A and DNMT3B was not different from baseline control. To test the in-vivo effect of C75 we utilized a xenograft model of stem cell rejection where 2 x 105 human CD34+ cells and HLA incompatible T lymphocytes were injected in immunodeficient nonobese diabetic/ltsz-scid/scid - IL2 receptor gamma chain knockout (NSG) mice at 1:1 ratio. Four weeks after transplantation, control NSG mice showed complete rejection of huCD45+CD34+ cells and the expansion of T cells in the marrow and spleen. NSG mice treated with intra-peritoneum injections of C75 every 3 days for 2 weeks, instead, showed 10-15% human CD45+ myeloid cells in the marrow and spleen at week 4 after transplant, suggesting at least a partial effect on preventing rejection of incompatible stem cells.We showed here that moderate FAS inhibition may represent a novel immunosuppressive strategy and our findings will prompt preclinical investigations exploiting the effect of FAS inhibitors alone or in combination with standard IS agents in models of allogeneic transplantation or bone marrow failure. DisclosuresNo relevant conflicts of interest to declare.

Abstract A164: Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy

Abstract Pre-emptive immunotherapy after HSC transplantation based on minimal residual disease (MRD) status with donor lymphocyte infusions (DLI) using cytokine-induced killer (CIK) cells may be beneficial to prevent relapse with a reduced risk of causing graft-versus-host-disease (GvHD) compared to conventional T cell infusion. CIK cells are a heterogeneous effector cell population including T cells (CD3+CD56-), natural killer (NK) cells (CD3-CD56+) and natural killer T (T-NK) cells (CD3+CD56+), that exhibit non-MHC-restricted cytotoxic activity and are generated by ex vivo expansion of peripheral blood mononuclear cells (PBMC) through the addition of interferon (IFN)-γ, anti-CD3 antibody, IL-2 and IL-15. While CIK cells have shown potent in vivo activity against various cancer types such as lymphomas or colorectal cancer, their cytotoxicity against B-ALL, characterized by the expression of CD19, has been limited. Hence, retargeting of CIK cells using chimeric antigen receptors (CARs) to facilitate selective target cell recognition and enhance specific cytotoxicity represents a promising approach. CAR comprise an extracellular scFv antibody fragment as an antigen-binding domain, linked via a flexible hinge region and a transmembrane domain to an intracellular signaling moiety such as CD3 zeta chain (first generation CAR), or zeta chain fused to a co-stimulatory protein domain such as CD28 (second generation CAR). We established an optimized protocol for transduction of CIK cells with CD19-specific lentiviral CAR constructs, and characterized cells for expression of an EGFP marker gene and CAR surface expression. Effects of exposure to lentiviral vector particles on the development of CIK cell subpopulations were monitored. In in vitro cytotoxicity assays we could demonstrate potent and selective cytotoxicity of retargeted CIK cells towards established cancer cell lines expressing CD19 and primary pre-B-ALL blasts. In addition, we observed significantly enhanced degranulation of CAR-CIK cells upon target cell contact and showed increased secretion of pro-inflammatory cytokines, while no secretion of immunosuppressive IL-10 could be detected. Cytotoxity towards non-malignant allo-PBMCs remained low. In addition, we investigated anti-leukemic activity of retargeted CIK cells in vivo in NOD/SCID common gamma chain knockout (NSG) mouse models using bioluminescence and fluorescence imaging. We observed potent reduction of tumor load after only two CIK cell applications in mice with an engrafted primary pre-B-ALL yielding long-term survivors with a negative MRD status (4/6). In a separate model, we demonstrated migration of CD19-specific CIK cells into local, subcutaneous tumor sites. In conclusion, CAR-CIK cells represent a promising alternative to CAR-T cell therapy, as relatively high numbers of conventional CIK cells have already been infused in clinical trials, which demonstrated a low risk of causing severe side effects. Citation Format: Sarah Oelsner, Juliane Wagner, Miriam E. Friede, Verena Pfirrmann, Eva Rettinger, Ralf Schubert, Heike Pfeifer, Evelyn Ullrich, Peter Bader, Winfried S. Wels. Genetically modified cytokine-induced killer (CIK) cells for targeted cancer therapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A164.

F4/80+ Host Macrophages Are a Barrier to Murine Embryonic Stem Cell-Derived Hematopoietic Progenitor Engraftment In Vivo.

Understanding how embryonic stem cells and their derivatives interact with the adult host immune system is critical to developing their therapeutic potential. Murine embryonic stem cell-derived hematopoietic progenitors (ESHPs) were generated via coculture with the bone marrow stromal cell line, OP9, and then transplanted into NOD.SCID.Common Gamma Chain (NSG) knockout mice, which lack B, T, and natural killer cells. Compared to control mice transplanted with adult lineage-negative bone marrow (Lin− BM) progenitors, ESHP-transplanted mice attained a low but significant level of donor hematopoietic chimerism. Based on our previous studies, we hypothesized that macrophages might contribute to the low engraftment of ESHPs in vivo. Enlarged spleens were observed in ESHP-transplanted mice and found to contain higher numbers of host F4/80+ macrophages compared to BM-transplanted controls. In vivo depletion of host macrophages using clodronate-loaded liposomes improved the ESHP-derived hematopoietic chimerism in the spleen but not in the BM. F4/80+ macrophages demonstrated a striking propensity to phagocytose ESHP targets in vitro. Taken together, these results suggest that macrophages are a barrier to both syngeneic and allogeneic ESHP engraftment in vivo.

Preclinical Study for the Use of Abatacept to Prevent Rejection of Allogeneic CD34+ Cells in a Xenograft Model

The aims of this study were to analyze the role of T cells on the engraftment of allogeneic CD34+ cells after transplantation in immunodeficient nonobese diabetic/ltsz-scid/scid (NOD/SCID) IL2 receptor gamma chain knockout (NSG) mice and to test the in-vivo ability of abatacept (CTLA4-Ig) in preventing graft failure. Human CD34+ cells (0.2x106 cells/animal) were co- transplanted with allogeneic CD3+ T cells into sublethally irradiated NSG mice at ratios ranging from 1:50 to 1:0.5, or without T cells as a control. The engraftment of huCD45+ cell subsets in the bone marrow and spleen was measured by flow cytometry after 4-8 weeks. An expansion of T cells without engraftment of CD34+ cells was detected in each group of mice transplanted with CD34:T cells at ratios ≥ 1:0.5. To test whether T cells prevented any engraftment of CD34+ cells, or caused rejection after initial CD34+ cell homing in the bone marrow, kinetics experiments were performed by analyzing the marrow and spleen of mice at 1,2 or 4 weeks after transplant of CD34+ and T cells at 1:1 ratio. These experiments showed that at two weeks after transplant, CD34+ cells had repopulated the bone marrow but not the spleen, while T cells were found primarily in the spleen. Instead, in mice sacrificed at 4 weeks after transplant the marrow and the spleen contained only T cells and the CD34+ cells had been rejected. Based on our previous in-vitro studies showing that CD34+ cell immunogenicity is mainly dependent on B7:CD28 costimulatory signaling, we then tested whether costimulatory blockade with abatacept (CTLA4-Ig, Bristol Myers Squibb) would block stem cell rejection. Three groups of mice were transplanted with CD34+ and allo-T cells at 1:1 ratio and injected with Abatacept at 250 ug i.p. every other day from: a) day -1 to +28, b) day -1 to day +14 or c) day +14 to +28, then the animals were sacrificed at day +56 (8 weeks) after transplant to assess the engraftment. In Group a) the overall engraftment of huCD45+ cells was only <10%, but Abatacept completely prevented T cell-mediated stem cell rejection with >98% huCD45+ cells of myeloid or B cell lineage and <1% T cells in the marrow and spleen. In Group b) 70% of huCD45+ cells both in the marrow and spleen were T cells, and the remaining fraction of myeloid or B cells were derived from CD34+ cells. In Group c), instead, 100% of huCD45+ cells were T cells, with complete rejection of CD34+ cells. T cells recovered from the spleen of mice in groups b) and c) were also tested as responders in MLC stimulated with the original CD34+ cells and showed a brisk proliferation, consistent with lack of tolerance. Finally, another group of mice that received Abatacept from day -1 to + 28 was rechallenged with a boost of CD34+ cells on day +28 to test whether the low CD34+ cell engraftment was secondary to a latent rejection or partial stem cell exhaustion. The CD34+ cell boost resulted in a full hematopoietic recovery with 37% huCD45+CD3- cells, including myeloid and B cells, as well as CD34+ cells in the bone marrow and spleen.In this preclinical xenograft model we demonstrated that costimulatory blockade with Abatacept at the time of allogeneic transplant of human CD34+ cells can prevent T cell mediated rejection provide the basis for the future non-myeloablative protocols for incompatible stem cell transplantation. DisclosuresNo relevant conflicts of interest to declare.

Detection of HTLV-1 replication and CD4+ proliferation in the humanized BLT mouse model

Human T-lymphotropic virus type-1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL) and a number of lymphocyte-mediated inflammatory conditions including HTLV-1–associated myelopathy/tropical spastic paraparesis. Development of animal models to study the pathogenesis of HTLV-1–associated diseases has been problematic and the mechanisms driving disease remain poorly understood. Here we report our findings of HTLV-1 infection in humanized nonobese diabetic (NOD) severe combined immunodeficiency (SCID) common gamma chain knockout (NSG) mice that have been implanted with fetal bone marrow-liver-thymus tissue (BLT). BLT mice were injected with lethally irradiated CD4+ HTLV-1 producing cells. Blood samples were collected every four weeks to monitor CD4 and CD8 phenotypes and DNA isolation. Concomitant with an increase in the number of CD4+CD25+ T-lymphocytes in exposed animals compared to controls, we observed a significant increase in the HTLV-1 viral DNA load. Surprisingly, unlike what we observed in macaques, HTLV-1 virus mutated to knockout Orf-I protein expression was able to infect BLT mice and high viral DNA loads were detected. However, after sequencing the orf-I region of proviral DNA from infected animals, we found reversion of the point mutation to wild type. This was observed as early as four weeks after exposure, indicating in vivo viral replication and selection for Orf-I expression. Thus, the BLT mouse model successfully recapitulates HTLV-1 infection and may serve as an important tool for investigating in vivo mechanisms of HTLV-1 disease pathology and in evaluating drugs and treatment strategies.

­Impact of Porcine-Human Mixed Hematopoietic Chimerism on Human NK Cell Response to Porcine Cells

Mixed hematopoietic chimerism permits durable tolerance of T, B and NK cells to xenoantigens in a rat to mouse bone marrow transplant model. However, it is unclear whether tolerance of human NK cells to pig xenoantigens can be induced by mixed hematopoietic chimerism. We assessed the tolerance of human NK cells towards pig cells in a humanized mouse model with established pig and human mixed xenogeneic chimerism. Pig and human mixed chimeras (MCs) were generated by injection of pig bone marrow cells to irradiated pig cytokine (IL3, GMCSF and SCF) transgenic NOD-scid common gamma chain knockout (NSG) mice followed by injection of human fetal liver CD34+ cells 3 day later. In the control group, only human CD34+ cells were transplanted. 12 weeks post-transplant, hydrodynamic injection of plasmid encoding human Flt3L followed by injection of three rounds of recombinant IL15/IL-15 receptor alpha Fc complex was given to promote human NK cell reconstitution. The control non–mixed chimeric group (Non-MC) received the same treatment without pig cells. 12 days following induction of human NK cell reconstitution, human NK cells from both MC and Non-MC mice were isolated from the spleen and their cytotoxic responses in vitro to pig cells were determined in a chromium release assay. In addition, the presence of pig cells in various tissues of the chimeric mice was studied.While human NK cells were usually undetectable in peripheral blood prior to the injection of human Flt3L plasmid and IL-15/IL-15 receptor alpha-Fc complex, they were detected by 5 days post-injection of IL-15 protein. 12 days post-induction of human NK cell reconstitution, pig cells remained detectable in peripheral blood, spleen, bone marrow and liver in the chimeric mice together with human NK cells. The co-existence of human NK cells and pig cells suggested that human NK cells in MCs might be tolerant to pig cells. Consistent with this notion, cytotoxicity assays showed that human NK cells from MCs demonstrated decreased killing of pig PBMC blasts compared to NK cells from Non-MC mice. Importantly, killing of K562 cells by NK cells from MCs mice was also decreased compared to that of Non-MC mice, suggesting that human NK cell tolerance to pig cells induced by mixed chimerism was associated with global hyporesponsiveness, as we have previously observed in a rat-to-mouse bone marrow transplantation model. Moreover, higher percentages of CD56highCD16low and lower percentages of CD56lowCD16high human NK cell subsets were observed in bone marrow of chimeric mice than in non-chimeric mice, indicating that the development of human NK cells in bone marrow might be altered by the presence of pig cells. In summary, our data suggest that mixed xenogeneic chimerism may induce tolerance of human NK cells towards porcine cells, but the tolerance may be associated with global hyporesponsiveness. DisclosuresNo relevant conflicts of interest to declare.