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Supplementary table from Mutant IDH Inhibits IFNγ–TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma

Supplementary Table 1

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II–restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo. In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II–restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II–restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell–mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Supplementary Figure from Mutant IDH Inhibits IFNγ–TET2 Signaling to Promote Immunoevasion and Tumor Maintenance in Cholangiocarcinoma

Supplementary Figures and Legends

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate–dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate–mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell–autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell–specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ–TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy.Significance:Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies.See related commentary by Zhu and Kwong, p. 604.This article is highlighted in the In This Issue feature, p. 587

Various monoclonal antibodies (mAb) target immune system molecules to enhance immunity by costimulating T cells (i.e., CD137, OX40, CD40, GITR) or interfering in coinhibitory signals (i.e., CTLA-4, PD-1). These powerful agents can be guided by cancer vaccines to enhance immunity against tumor but not self tissues. Clinically powerful therapeutic synergies are at hand.

Supplemental Information. Supplemental Materials and Methods Supplemental Fig. 1 : Chimeric mice selectively lacking MHCII expression on pDCs. Supplemental Fig. 2 : Similar T cell proliferation in µMT:WT and µMT pIII+IV-/-:WT chimeric mice. Supplemental Fig. 3 : Selective MHCII abrogation on pDCs in μMT pIII+IV-/-:WT mice does not affect MHCII expression and upregulation in other cell subtypes. Supplemental Fig. 4 : MHCII deficient pDCs exhibit normal TLR responses in vitro. Supplemental Fig. 5 : Vaccination using OVA protein + CpG-B significantly inhibits tumor growth after Ag presentation by pDCs. Supplemental Fig. 6 : Characterisation of in vitro generated Th0 and Th17 OT-II T cells. Supplemental Fig. 7 : Impaired cell migration toward tumor supernatant in mice lacking MHCII on pDCs. Supplemental Fig. 8 : CD8+ T cell depletion in EG7 bearing mice.

Supplementary Figures S1-S6. The IFN-I response innduced by SSFV-IL-12 is not affeccted by the IL-12 transgene but is impaired in the absence of IFN-I signaling (S1); IFN-I receptor function is critical for the therapeutic effect of SFV-IL-12 (S2); IFN-I receptor function on the hematopoietic cell compartment is critical for the therapeutic effect of SFV-IL-12 (S3); Gating strategy used to identify TAMs and the different MDSC and DC subsets in the tumor microenvironment (S4); The endogenous IFN-I system lies beneath the effects of SFV-IL-12 on the infiltrates of myeloid cells in the tumor microenvironment (S5); The immune gene expression signature in tumors provoked by SFV-IL-12 is largely dependent on the endogenous IFN-I system (S6).

Supplementary Figures 1-5 from Agonist Anti-CD137 mAb Act on Tumor Endothelial Cells to Enhance Recruitment of Activated T Lymphocytes

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research. Cancer Res; 75(3); 497–507. ©2014 AACR.

Isocitrate dehydrogenase 1 mutations (mIDH1) are common in cholangiocarcinoma. (R)-2-hydroxyglutarate generated by the mIDH1 enzyme inhibits multiple α-ketoglutarate–dependent enzymes, altering epigenetics and metabolism. Here, by developing mIDH1-driven genetically engineered mouse models, we show that mIDH1 supports cholangiocarcinoma tumor maintenance through an immunoevasion program centered on dual (R)-2-hydroxyglutarate–mediated mechanisms: suppression of CD8+ T-cell activity and tumor cell–autonomous inactivation of TET2 DNA demethylase. Pharmacologic mIDH1 inhibition stimulates CD8+ T-cell recruitment and interferon γ (IFNγ) expression and promotes TET2-dependent induction of IFNγ response genes in tumor cells. CD8+ T-cell depletion or tumor cell–specific ablation of TET2 or IFNγ receptor 1 causes treatment resistance. Whereas immune-checkpoint activation limits mIDH1 inhibitor efficacy, CTLA4 blockade overcomes immunosuppression, providing therapeutic synergy. The findings in this mouse model of cholangiocarcinoma demonstrate that immune function and the IFNγ–TET2 axis are essential for response to mIDH1 inhibition and suggest a novel strategy for potentiating efficacy. Significance: Mutant IDH1 inhibition stimulates cytotoxic T-cell function and derepression of the DNA demethylating enzyme TET2, which is required for tumor cells to respond to IFNγ. The discovery of mechanisms of treatment efficacy and the identification of synergy by combined CTLA4 blockade provide the foundation for new therapeutic strategies. See related commentary by Zhu and Kwong, p. 604. This article is highlighted in the In This Issue feature, p. 587

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

The authors show that autophagy in lymphatic endothelial cells (LECs) dampens collagen-induced arthritis by regulating two distinct pathways: the promotion of T reg cells in LNs and the inhibition of S1P-dependent migration of pathogenic Th17 cells in inflamed organs., Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II-restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II-restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II-restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell-mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Extracellular ATP is a central signaling molecule in inflammatory responses. Pannexin1 (Panx1) channels release ATP in a controlled manner and have been implicated in various inflammatory pathologies, but their role in atherogenesis remains elusive. Using atherosclerosis-susceptible mouse models with ubiquitous deletion of Panx1 (Panx1 −/− Apoe −/− ) or with Cre recombinase-mediated deletion of Panx1 in endothelial cells and monocytes (Tie2-Cre Tg Panx1 fl/fl Apoe −/− ; Panx1 del Apoe −/− ), we identified a novel role for Panx1 in the lymphatic vasculature. Atherosclerotic lesion development in response to high-cholesterol diet was enhanced in Panx1 del Apoe −/− mice, pointing to an atheroprotective role for Panx1 in endothelial and/or monocytic cells. Unexpectedly, atherogenesis was not changed in mice with ubiquitous Panx1 deletion, but Panx1 −/− Apoe −/− mice displayed reduced body weight, serum cholesterol, triglycerides and free fatty acids, suggesting altered lipid metabolism in these Panx1-deficient mice. Mechanistically, Panx1 −/− Apoe −/− mice showed impairment of lymphatic vessel function with decreased drainage of interstitial fluids and reduced dietary fat absorption. Thus, the detrimental effect of Panx1 deletion in endothelial and/or monocytic cells during atherogenesis is counterbalanced by an opposite effect resulting from impaired lymphatic function in ubiquitous Panx1-deficient mice. Collectively, our findings unveil a pivotal role of Panx1 in linking lymphatic function to lipid metabolism and atherosclerotic plaque development.

Plasmacytoid dendritic cells (pDCs) have been shown to both mediate and prevent autoimmunity, and the regulation of their immunogenic versus tolerogenic functions remains incompletely understood. Here we demonstrate that, compared to other cells, pDCs are the major expressors of Indoleamine-2,3-dioxygenase (IDO) in steady-state lymph nodes (LNs). IDO expression by LN pDCs was closely dependent on MHCII-mediated, antigen-dependent, interactions with Treg. We further established that IDO production by pDCs was necessary to confer suppressive function to Tregs. During EAE development, IDO expression by pDCs was required for the generation of Tregs capable of dampening the priming of encephalitogenic T cell and disease severity. Thus, we describe a novel crosstalk between pDCs and Tregs: Tregs shape tolerogenic functions of pDCs prior to inflammation, such that pDCs in turn, promote Treg suppressive functions during autoimmunity., Graphical abstract, Highlights • IDO expression by LN pDCs is closely dependent on MHCII-mediated, antigen-dependent, interactions with Tregs. • pDCs are the predominant source of IDO in both steady-state and inflamed lymph nodes. • IDO production by pDCs is necessary to confer suppressive function to Tregs in EAE.

Summary Inflammation triggers the differentiation of Ly6Chi monocytes into microbicidal macrophages or monocyte-derived dendritic cells (moDCs). Yet, it is unclear whether environmental inflammatory cues control the polarization of monocytes toward each of these fates or whether specialized monocyte progenitor subsets exist before inflammation. Here, we have shown that naive monocytes are phenotypically heterogeneous and contain an NR4A1- and Flt3L-independent, CCR2-dependent, Flt3+CD11c−MHCII+PU.1hi subset. This subset acted as a precursor for FcγRIII+PD-L2+CD209a+, GM-CSF-dependent moDCs but was distal from the DC lineage, as shown by fate-mapping experiments using Zbtb46. By contrast, Flt3−CD11c−MHCII−PU.1lo monocytes differentiated into FcγRIII+PD-L2−CD209a−iNOS+ macrophages upon microbial stimulation. Importantly, Sfpi1 haploinsufficiency genetically distinguished the precursor activities of monocytes toward moDCs or microbicidal macrophages. Indeed, Sfpi1+/− mice had reduced Flt3+CD11c−MHCII+ monocytes and GM-CSF-dependent FcγRIII+PD-L2+CD209a+ moDCs but generated iNOS+ macrophages more efficiently. Therefore, intercellular disparities of PU.1 expression within naive monocytes segregate progenitor activity for inflammatory iNOS+ macrophages or moDCs., Graphical Abstract, Highlights • Murine Ly6ChiCD115+ monocytes are heterogeneous • DC-related genes (Cd209a and MHCII) are expressed in a subset of FcγRIII+ monocytes • GM-CSF-dependent CD209a+ moDCs are generated by FcγRIII+CD209a+MHCII+ monocytes • iNOS+ macrophages are generated by FcγRIII+CD209a−MHCII− monocytes, Monocytes can differentiate into multiple progenies during inflammation. Here, Menezes et al. show that monocytes from naive mice are heterogeneous and contain distinct precursor subsets giving rise to iNOS+ inflammatory macrophages or GM-CSF-induced CD209a+ monocyte-derived dendritic cells.

Summary CRISPR-Cas9 genome engineering has increased the pace of discovery for immunology and cancer biology, revealing potential therapeutic targets and providing insight into mechanisms underlying resistance to immunotherapy. However, endogenous immune recognition of Cas9 has limited the applicability of CRISPR technologies in vivo. Here, we characterized immune responses against Cas9 and other expressed CRISPR vector components that cause antigen-specific tumor rejection in several mouse cancer models. To avoid unwanted immune recognition, we designed a lentiviral vector system that allowed selective CRISPR antigen removal (SCAR) from tumor cells. The SCAR system reversed immune-mediated rejection of CRISPR-modified tumor cells in vivo and enabled high-throughput genetic screens in previously intractable models. A pooled in vivo screen using SCAR in a CRISPR-antigen-sensitive renal cell carcinoma revealed resistance pathways associated with autophagy and major histocompatibility complex class I (MHC class I) expression. Thus, SCAR presents a resource that enables CRISPR-based studies of tumor-immune interactions and prevents unwanted immune recognition of genetically engineered cells, with implications for clinical applications.

Intrahepatic cholangiocarcinoma (ICC) is a cancer of the liver bile ducts, which has been rising in incidence and carries a 5-year survival rate of less than 10%. Mutations in isocitrate dehydrogenase 1 and 2 (mIDH1/2) are among the most common genetic alterations in ICC, present in 20% patients. The hot-spot point mutations of IDH result in aberrant enzymatic function, reducing alpha-ketoglutarate (alphaKG) to the oncometabolite, 2-hydroxyglutarate (2-HG), which accumulates to millimolar concentrations within the cell. 2-HG competitively inhibits alphaKG-dependent histone and DNA demethylating enzymes, thereby altering chromatin regulation. The first-in-class small molecule selective IDH1m inhibitor, ivosidenib, has shown benefit in phase I and III trials for patients with IDH1m ICC. In order to gain insight into the mechanisms underlying the response to IDH1m inhibition, we developed the first genetically engineered mouse model (GEMM) of IDH1m ICC. We found that ivosidenib reduced the overall tumor burden and increased overall survival of syngeneic allografts of ICCs derived from the GEMM. The positive response to ivosidenib was associated with an induction of mature hepatocyte lineage markers and a reduction of tumor produced 2-HG >85%, supporting observations from the ivosidenib phase I data in ICC patients that mIDH1 inhibition may promote ICC tumor cell differentiation. In addition, there was pronounced induction of immune stimulatory interferon signaling, increased recruitment of CD8+ T cells to the tumors and strong PD-L1 upregulation. Thus, ivosidenib treatment leads to a sustained inhibition of 2-HG production, which correlated with an increased immune stimulation compared to vehicle treatment. We will present these data as well as ongoing studies interrogating the immune and transcriptomic changes with further resolution via fluorescence-activated cell sorting and single-cell RNA-sequencing analysis, direct functional testing of contributions of specific immune populations to the therapeutic efficacy of ivosidenib, and testing ivosidenib in combination with checkpoint inhibition. These studies highlight the impact of ivosidenib on hepatocyte differentiation and immune stimulation in IDH1m ICC and support the key role of IDHm in ICC tumor maintenance. Citation Format: Meng-Ju Wu, Lei Shi, Juan Dubrot, Christine Hudson, Joshua Merritt, Ramzi Adil, Qibiao Wu, Sebastien Ronseaux, Rohini Narayanaswamy, Robert Manguso, Brandon Nicolay, Nabeel Bardeesy. The mutant IDH1 inhibitor ivosidenib promotes tumor cell differentiation and anti-tumor immunity in intrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1810.

The ability to make rapid genetic alterations to tumor cells with CRISPR for screens and single-gene studies has been transformative for studying cancer biology. Recently, we and others have used CRISPR in murine tumor models in vivo to identify targets and resistance mechanisms for immunotherapy. However, the use of these techniques in immunocompetent mice is limited by endogenous immune recognition of CRISPR components such as Cas9. Here we report the design of a screening-compatible lentiviral vector system which allows selective CRISPR antigen removal (SCAR) from tumor cells before they are implanted in vivo. The SCAR system enables highly efficient genome engineering and reverses CD8+ T cell-mediated rejection of CRISPR modified tumor cells in vivo. Using this technology, we conducted a pooled, in vivo screen designed to probe tumor-immune interactions in the highly immunogenic CT26 colon carcinoma model and discovered that these tumors require intact interferon signaling to evade immune destruction. The SCAR system enables the study of tumor-immune interactions in any cancer model and can be more broadly applied to generate non-immunogenic lentivirally-engineered cells. Citation Format: Juan Dubrot, Sarah Kate Lane-Reticker, Emily Kessler, Clara Wolfe, Animesh Mahapatra, Peter Du, Robert T. Manguso. SCAR (Selective CRISPR Antigen Removal) vector system expands cancer immunology discovery with in vivo functional genomics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5866.

During the last decade, there has been notable progress in cancer immunotherapy, including promising clinical success of immune checkpoint inhibitors. Moreover, there is an increasing number of new potential targets for cancer immunotherapy that are currently being developed both as monotherapy or in combination. However, the lack of durable clinical responses due in part to the resistance mechanisms that tumors exhibit in a significant proportion of patients urges novel approaches to find the right therapeutic strategies. The understanding of such tumor evasion mechanisms and the complex interactions that take place in the tumor microenvironment is essential to obtain successful treatments. Functional genomics has emerged as a powerful tool that can help to reveal some of these unknown processes, with CRISPR-Cas9 technology the most prominent example. However, in vivo genetic screens require stable expression of several components that in most cases are not derived from murine cells, which represents a major obstacle in the context of cancer immunology. The inoculation of engineered tumor cell lines in immunocompetent mice results in either tumor rejection or an aberrant response to immunotherapy, making the experiments technically impossible in the first case or providing inconsistent results in the latter. We have designed new vector strategies to overcome the altered immunogenicity of Cas9-expressing tumor cell lines using the Cre-lox system. Expression and effective removal of Cas9 and vector components are monitored using fluorescent reporter proteins. Both genome-editing capacity and normal in vivo behavior of all tested cell lines remain largely unaltered using this novel strategy. This technology represents a major advance for preclinical functional genomics, allowing studies in fully competent animals looking for better combination treatments and resistance mechanism in particular cancer models, and ultimately will help in the design of clinical trials. Citation Format: Juan Dubrot, Robert T. Manguso, Sarah Kate Lane-Reticker, Austin Ayer, Emily Kessler, Clara Wolfe, Animesh Mahapatra. Novel CRISPR/Cas9 vectors for in vivo tumor functional genomics [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B10.

MHCII-restricted antigen presentation by lymph node stromal cells is essential for regulatory T-cell proliferation and functions, and for the regulation of autoimmunity., How lymph node stromal cells (LNSCs) shape peripheral T-cell responses remains unclear. We have previously demonstrated that murine LNSCs, lymphatic endothelial cells (LECs), blood endothelial cells (BECs), and fibroblastic reticular cells (FRCs) use the IFN-γ–inducible promoter IV (pIV) of the MHC class II (MHCII) transactivator CIITA to express MHCII. Here, we show that aging mice (>1 yr old) in which MHCII is abrogated in LNSCs by the selective deletion of pIV exhibit a significant T-cell dysregulation in LNs, including defective Treg and increased effector CD4+ and CD8+ T-cell frequencies, resulting in enhanced peripheral organ T-cell infiltration and autoantibody production. The proliferation of LN-Tregs interacting with LECs increases following MHCII up-regulation by LECs upon aging or after exposure to IFN-γ, this effect being abolished in mice in which LECs lack MHCII. Overall, our work underpins the importance of LNSCs, particularly LECs, in supporting Tregs and T-cell tolerance.

Dendritic cells (DCs) travel through lymphatic vessels to transport antigens and present them to T cells in lymph nodes. DCs move directionally toward lymphatics by virtue of their CCR7 and a CCL21 chemotactic gradient. We evaluated in vivo and in bone marrow-derived dendritic cells (BMDCs) whether the gap junction protein Cx43 contributes to CCL21/CCR7-dependent DC migration in wild-type (WT) mice, heterozygous (Cx43+/-) mice and mice expressing a truncated form of Cx43 lacking its regulatory C-terminus (Cx43K258/-). In a model of flank skin inflammation, we found that the recruitment of myeloid DCs (mDCs) to skin draining lymph nodes was reduced in Cx43K258/- mice as compared to WT and Cx43+/- mice. In addition, the migration of Cx43K258/- BMDCs toward CCL21 was abolished in an in vitro chemotactic assay while it was only reduced in Cx43+/- cells. Both mutant genotypes showed defects in the directionality of BMDC migration as compared to WT BMDCs. No difference was found between the three populations of BMDCs in terms of expression of surface markers (CD11c, CD86, CD80, CD40, MHC-II, and CCR7) after differentiation and TLR activation. Finally, examination of the CCR7-induced signaling pathways in BMDCs revealed normal receptor-induced mobilization of intracellular Ca2+. These results demonstrate that full expression of an intact Cx43 is critical to the directionality and rate of DC migration, which may be amenable to regulation of the immune response.

Innate lymphoid cells keep gut T cells in check Trillions of bacteria inhabit our guts. So do many types of immune cells, including T cells, which might be expected to attack these bacteria. How, then, do our bodies manage to keep the peace? Working in mice, Hepworth et al. report one such mechanism. A population of immune cells, called innate lymphoid cells, directly killed CD4 + T cells that react to commensal gut microbes. Some of the specifics of this process parallel how the immune system keeps developing self-reactive T cells in check in the thymus. Furthermore, this peacekeeping process may be disrupted in children with inflammatory bowel disease. Science , this issue p. 1031

Host responses are increasingly considered important for the efficacious response to experimental cancer therapies that employ viral vectors, but little is known about the specific nature of host responses required. In this study, we investigated the role of host type I interferons (IFN-I) in the efficacy of virally delivered therapeutic genes. Specifically, we used a Semliki Forest virus encoding IL12 (SFV-IL12) based on its promise as an RNA viral vector for cancer treatment. Intratumoral injection of SFV-IL12 induced production of IFN-I as detected in serum. IFN-I production was abolished in mice deficient for the IFNβ transcriptional regulator IPS-1 and partially attenuated in mice deficient for the IFNβ signaling protein TRIF. Use of bone marrow chimeric hosts established that both hematopoietic and stromal cells were involved in IFN-I production. Macrophages, plasmacytoid, and conventional dendritic cells were each implicated based on cell depletion experiments. Further, mice deficient in the IFN-I receptor (IFNAR) abolished the therapeutic activity of SFV-IL12, as did a specific antibody-mediated blockade of IFNAR signaling. Reduced efficacy was not caused by an impairment in IL12 expression, because IFNAR-deficient mice expressed the viral IL12 transgene even more strongly than wild-type (WT) hosts. Chimeric host analysis for the IFNAR involvement established a strict requirement in hematopoietic cells. Notably, although tumor-specific CD8 T lymphocytes expanded robustly after intratumoral injection of WT mice with SFV-IL12, this did not occur in mice where IFNAR was inactivated genetically or pharmacologically. Overall, our results argued that the antitumor efficacy of a virally based transgene therapeutic relied strongly on a vector-induced IFN-I response, revealing an unexpected mechanism of action that is relevant to a broad array of current translational products in cancer research. Cancer Res; 75(3); 497–507. ©2014 AACR.

Lymph node stromal cells (LNSCs) have newly been promoted to the rank of new modulators of T cell responses. The different non-hematopoietic cell subsets in lymph node (LN) were considered for years as a simple scaffold, forming routes and proper environment for antigen (Ag)-lymphocyte encountering. Deeper characterization of those cells has recently clearly shown their impact on both dendritic cell and T cell functions. In particular, lymphatic endothelial cells (LECs) control lymphocyte trafficking and homeostasis in LNs and limit adaptive immune responses. Therefore, the new role of LECs in shaping immune responses has drawn the attention of immunologists. Striking is the discovery that LECs, among other LNSCs, ectopically express a large range of peripheral tissue-restricted Ags (PTAs), and further present PTA-derived peptides through major histocompatibility class I molecules to induce self-reactive CD8(+) T cell deletional tolerance. In addition, both steady-state and tumor-associated LECs were described to be capable of exogenous Ag cross-presentation. Whether LECs can similarly impact CD4(+) T cell responses through major histocompatibility class II restricted Ag presentation is still a matter of debate. Here, we review and discuss our current knowledge on the contribution of Ag-presenting LECs as regulators of peripheral T cell responses in different immunological contexts, including autoimmunity and cancer.

Mutations in the gap junction protein connexin47 (Cx47) are associated with lymphedema. However, the role of Cx47 in lymphatic pathophysiology is unknown. We demonstrate that Cx47 is expressed in lymphatic endothelial cells by whole-mount immunostaining and qPCR. To determine if Cx47 plays a role in lymphatic vessel function we analysed Cx47-/- mice. Cx47-deficiency did not affect lymphatic contractility (contractile amplitude or frequency) or lymphatic morphology (vessel diameter or number of valves). Interstitial fluid drainage or dendritic cell migration through lymphatic vessels was also not affected by Cx47-deficiency. Cx47 is dispensable for long-chain fatty acid absorption from the gut but rather promotes serum lipid handling as prolonged elevated triglyceride levels were observed in Cx47-deficient mice after oral lipid tolerance tests. When crossed with Apolipoprotein E-deficient (Apoe-/-) mice, LDL-cholesterol was decreased in young Cx47-/-Apoe-/- adults as compared to Apoe-/- mice, which was inverted later in life. Finally, advanced atherosclerotic plaques in thoracic-abdominal aortas of 15 months-old mice tended to be larger in Cx47-/-Apoe-/- mice. These plaques contained fewer macrophages but similar amounts of T lymphocytes, collagen and lipids than plaques of Apoe-/- mice. In conclusion, Cx47 is expressed in lymphatic endothelium and seems modestly implicated in multiple aspects of lymphatic pathophysiology.

Plasmacytoid dendritic cells (pDC) rapidly and massively produce type I IFN and other inflammatory cytokines in response to foreign nucleic acids, thereby indirectly influencing T-cell responses. Moreover, antigen (Ag)-presenting pDCs directly regulate T-cell differentiation. Depending on the immune environment, pDCs exhibit either tolerogenic or immunogenic properties. Here, we show that CpG-activated pDCs promote efficient Th17 differentiation. Indeed, Th17 responses are defective in mice selectively lacking MHCII on pDCs upon antigenic challenge. Importantly, in those mice, the frequency of Th17 cells infiltrating solid tumors is impaired. As a result, the recruitment of infiltrating leukocytes in tumors, including tumor-specific cytotoxic T lymphocytes (CTL), is altered and results in increased tumor growth. Importantly, following immunization with tumor Ag and CpG-B, MHCII-restricted Ag presentation by pDCs promotes the differentiation of antitumor Th17 cells that induce intratumor CTL recruitment and subsequent regression of established tumors. Our results highlight a new role for Ag presenting activated pDCs in promoting the development of Th17 cells and impacting on antitumor immunity. Cancer Res; 74(22); 6430–40. ©2014 AACR.

Plasmacytoid dendritic cells (pDCs) exhibit both innate and adaptive functions. In particular they are the main source of type I IFNs and directly impact T cell responses through antigen presentation. We have previously demonstrated that during experimental autoimmune encephalomyelitis (EAE) initiation, myelin-antigen presentation by pDCs is associated with suppressive Treg development and results in attenuated EAE. Here, we show that pDCs transferred during acute disease phase confer recovery from EAE. Clinical improvement is associated with migration of injected pDCs into inflamed CNS and is dependent on the subsequent and selective chemerin-mediated recruitment of endogenous pDCs to the CNS. The protective effect requires pDC pre-loading with myelin antigen, and is associated with the modulation of CNS-infiltrating pDC phenotype and inhibition of CNS encephalitogenic T cells. This study may pave the way for novel pDC-based cell therapies in autoimmune diseases, aiming at specifically modulating pathogenic cells that induce and sustain autoimmune inflammation., Highlights • pDC therapy ameliorates established EAE. • CNS inflammation is locally modulated after pDC transfer. • Upon pDC transfer, resting endogenous pDCs are selectively recruited to the CNS via chemerin/CMKLR1 axis. • Therapeutic pDC injection promotes a tolerogenic environment and inhibits encephalitogenic T cells in the CNS.

IFN-α/β link innate and adaptive immune responses by directly acting on naïve CD8(+) T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8(+) CD45RO(-) cells were stimulated with beads coated with anti-CD3 and anti-CD28 mAb, mimicking Ag (type-1) and co-stimulatory (type-2) signals, in the presence or absence of IFN-α and their transcriptional profiles were defined by cDNA-microarrays. We show that IFN-α provides a strong third signal directly to human CD8(+) T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8(+) T-cell population. Thus, whereas IFN-α increases the proliferative capacity of naïve CD8(+) T cells, it inhibits or does not affect the proliferation of Ag-experienced cells, such as memory and effector CTL, including CMV-specific lymphocytes. Cytolysis and IFN-γ-secretion of all these populations are enhanced by IFN-α-derived signals, which are critical in naïve CD8(+) T cells for acquisition of effector functions. Our findings in human CD8(+) T cells are informative to understand and improve IFN-α-based therapies for viral and malignant diseases.

Anti-CTLA-4 monoclonal antibodies (mAb) that block the interaction of CTLA-4 with CD80 and CD86 such as tremelimumab and ipilimumab are currently being tested in the clinic for cancer treatment exploiting their properties to de-repress tumor-specific cellular immunity. Addition of the fully human anti-CTLA-4 (tremelimumab) to cultures of human T cells with allogenic dendritic cells (DCs) did not increase proliferation. Magnetic bead-mediated elimination of CD4(+) CD25(+) regulatory T cells (T(reg)) before setting up those alloreactive cultures also largely failed to increase primary proliferation. In contrast, predepletion of CD4(+) CD25(+) T(reg) and culture in the presence of tremelimumab synergistically resulted in increased proliferation and DC:T-cell aggregation. These effects were much more prominent in CD4 than in CD8 T cells. The synergy mechanism can be traced to enhanced CTLA-4 expression in effector cells as a result of T(reg) elimination, thereby offering more targets to the blocking antibody. Human T cells and allogenic DCs (derived both from healthy donors and advanced cancer patients) were coinjected in the peritoneum of Rag2(-/-) IL-2Rγ(-/-) mice. In these conditions, tremelimumab injected intravenously did not significantly enhance alloreactive proliferation unless T(reg) cells had been predepleted. Synergistic effects in vivo were again largely restricted to the CD4 T-cell compartment. In addition, T(reg) depletion and CTLA-4 blockade synergistically enhanced specific cytotoxicity raised in culture against autologous EBV-transformed cell lines. Taken together, these experiments indicate that tremelimumab therapy may benefit from previous or concomitant T(reg) depletion.

Repeated administration of gene therapies is hampered by host immunity toward vectors and transgenes. Attempts to circumvent antivector immunity include pharmacological immunosuppression or alternating different vectors and vector serotypes with the same transgene. Our studies show that B-cell depletion with anti-CD20 monoclonal antibody and concomitant T-cell inhibition with clinically available drugs permits repeated liver gene transfer to a limited number of nonhuman primates with recombinant adenovirus. Adenoviral vector–mediated transfer of the herpes simplex virus type 1 thymidine kinase (HSV1-tk) reporter gene was visualized in vivo with a semiquantitative transgene-specific positron emission tomography (PET) technique, liver immunohistochemistry, and immunoblot for the reporter transgene in needle biopsies. Neutralizing antibody and T cell–mediated responses toward the viral capsids were sequentially monitored and found to be repressed by the drug combinations tested. Repeated liver transfer of the HSV1-tk reporter gene with the same recombinant adenoviral vector was achieved in macaques undergoing a clinically feasible immunosuppressive treatment that ablated humoral and cellular immune responses. This strategy allows measurable gene retransfer to the liver as late as 15 months following the first adenoviral exposure in a macaque, which has undergone a total of four treatments with the same adenoviral vector.

Division of labour among cells of the immune system is a prevailing theme in the orchestration of immune responses. Contrary to this paradigm, a particular leukocyte population in the mouse seems to be equipped with the ability to kill transformed and virally infected cells and with the capability to mediate antigen processing and presentation to T cells. Those two functions are classically ascribed separately to Natural Killer (NK) cells and Dendritic cells (DC). IKDC (interferon-producing killer dendritic cell) were defined in mice as cells expressing CD11c, although to a lesser extent than conventional DCs (cDCs), while coexpressing B220, NK1.1, CD49b (VLA-2) and MHC Class II molecules. Absence of CD3, CD19 and Gr1(Ly49c) expression also featured this minor subset upon multicolour FACS characterization. These cells kill a variety tumor cell lines in a TRAIL-dependent fashion. Furthermore, IKDC produce high amounts of Interferon (IFN) gamma (IFN-γ) and type I IFN upon activation by combined interleukins (IL-18, IL-2, IL-12 or IL-15), tumor cells, or TLR-9 nucleotide agonists. Importantly, these cells may present antigen to prime CD4 and CD8 T cells. The discovery of this subset in mice was reported in two back to back articles published in Nature Medicine in 2006, along with a comment that highlighted how such a cell may provide the ability to kill cells in an innate fashion while starting T-cell adaptive immunity by means of cross-presentation of antigens collected from the apoptotic remains of their victim cells. However, two years later three articles in a single issue of the Journal of Experimental Medicine contended that such a population did not exist and that IKDC were actually nothing more than activated NK cells. The arguments were based on the inability of IKDC to present antigen and prime T cells, and on their dependency on IL-15 for differentiation and survival. Later on, the groups that first described IKDC have provided evidence that such a subpopulation can mediate antigen presentation and have also stressed several characteristics that discriminate activated NK cells and IKDC. The reasons for some of these experimental discrepancies are obscure. Yet, the core of the controversy is to establish wether the hybrid abilities proposed for such minor leukocyte population are relevant to physiology, pathology or therapy. To date, no equivalent population has been described in human lymphoid tissues.

Anti-CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti-tumor effect involves co-stimulation of tumor-specific CD8(+) T cells. Whether antigen cross-presenting DC are required for the efficacy of anti-CD137 mAb treatment has never been examined. Here we show that the administration of anti-CD137 mAb eradicates EG7-OVA tumors by a strictly CD8beta(+) T-cell-dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross-presentation revealed that CD11c(+) cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor-draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti-OVA CTL induction. Using CD11c diphtheria toxin receptor-green fluorescent protein-->C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross-presentation in CTL induction against OVA(257-264) epitope and in the antitumor efficacy induced by anti-CD137 mAb.

Vascular endothelial growth factor (VEGF) inhibits differentiation and maturation of dendritic cells (DC), suggesting a potential immunosuppressive role for this proangiogenic factor. Bevacizumab, sorafenib and sunitinib target VEGF-mediated angiogenesis and are active against several types of cancer, but their effects on the immune system are poorly understood. In this study, VEGF and supernatants of renal carcinoma cell lines cultured under hypoxia were found to alter the differentiation of human monocytes to DC. Resulting DC showed impaired activity, as assessed by the alloreactive mixed T-lymphocyte reaction. Bevacizumab and sorafenib, but not sunitinib, reversed the inhibitory effects of VEGF, but not of those mediated by tumour supernatants. Dendritic cells matured under the influence of VEGF expressed less human leukocyte antigen-DR (HLA-DR) and CD86, and this effect was restored by bevacizumab and sorafenib. Finally, tumour-cell supernatants decreased interleukin-12 (IL-12) production by mature DC, and such inhibition was not restored by any of the tested drugs, delivered either as single agents or in combination. The deleterious effects of tumour-cell supernatants were mainly mediated by thermostable molecules distinct from VEGF. These results indicate that inhibition of the differentiation of monocytes to DC is a multifactorial effect, and that they support the development of combinations of angiogenesis inhibitors with immunological modulators.

The “danger theory” is a set of postulates formally proposed by Polly Matzinger fifteen years ago. As a theory, it explains how it is possible that immune responses take place without infection. It provokingly proposed that the immune systems have not evolved for self/non-self discrimination, but to respond against what is causing tissue damage. The proposal of the “danger model” coincided in time with the discovery of the immune potentiating effects of microbial molecular patterns. Charles Janeway et al. proposed that infection would be detected by innate receptors for microbiological biomolecules that are either absent or different in mammals. These agents were found to stimulate antigen presenting cells in such a way that would provide T lymphocytes with appropriate costimulatory molecules that critically complement the signals raised by antigen recognition. This was considered absolutely critical to ignite and sustain immune responses. The danger theory predicted the existence of endogenous molecules released or modified by danger that would act in a similar fashion to the microbial molecular patterns on dendritic cell costimulatory functions. Recent evidence points to various molecules capable of sounding the alarm in aseptic conditions. These include: the nuclear protein HBGM-1, uric acid, Interferon-α, chaperones of the heat shock protein family, alternatively spliced domains of fibronectin, and self nucleic acids. Some of these agents act through the same Toll like receptors involved in microbial pattern recognition. Identification of these mechanisms provides molecular support for the danger theory and has an extraordinary importance for tumor and transplantation immunology.

Resumen Las celulas mieloides supresoras (Myeloid-derived suppressor cells, MDSC) pertenecen a un subtipo de leucocitos causantes de inmunosupresion en individuos portadores de tumor. En ratones, estas celulas han sido definidas como CD11b+GR1+IL-4Rα+ y, debido a la presencia de tumores en modelos experimentales, se acumulan tanto en la lesion tumoral como en los organos linfoides. CD137 (4-1BB) es un receptor de coestimulo de la familia de receptores del factor de necrosis tumoral (TNF) principalmente expresado sobre la membrana de linfocitos T y de celulas NK (Natural Killer) activados, aunque tambien se encuentra en la superficie de otros leucocitos de estirpe mieloide como mastocitos, granulocitos, macrofagos, celulas dendriticas y endotelio. Anticuerpos agonistas frente a CD137 incrementan la respuesta inmune antitumoral potenciando los CTLs antitumorales. En este trabajo, celulas de carcinoma de colon C26 transfectadas para expresar GM-CSF se inocularon por via subcutanea a ratones singenicos debido a sus propiedades inductoras de un gran aumento en el numero de las MDSCs. Mediante citometria de flujo multicolor hemos confirmado un notable aumento en el numero de estas celulas CD11b+GR1+IL-4Rα+ tanto en el estroma tumoral como en el bazo de los ratones portadores de tumor. La expresion de CD137 en este subtipo celular sin embargo, mostro resultados negativos. Por tanto, se pueden excluir los efectos directos de los mAbs sobre MDSCs como mecanismo de accion de la inmunoterapia con anticuerpos anti-CD137. Segun esto las terapias dirigidas a disminuir el numero o funcion de MDSCs podrian sinergizar con anticuerpos inmunoterapeuticos anti-CD137 ya que actuan sobre dianas diferentes.

Better understanding of the mechanisms that mediate spontaneous immune rejections ought to be important in the quest for improvements in immunotherapy of cancer. A set of intraperitoneal tumors of mesenchymal origin that had been chemically induced in ubiquitously expressing EGFP transgenic mice provided a model in which both T and NK cells were absolutely required for tumor rejection. Tumor cells were traceable because of being fluorescent and readily grafted in RAG1(-/-) immunodeficient mice, whereas they were rejected in a majority of syngeneic C57BL/6 and EGFP-transgenic mice. Tumor-cell clones with the highest EGFP expression tended to be rejected, but a direct involvement of EGFP as the antigen recognized for the immune rejections was ruled out. Rejections were absolutely dependent on NK cells as well as on CD4(+) and CD8(+) T lymphocytes according to selective depletion studies. Furthermore, CD8(+) and CD4(+) T lymphocytes as well as NK cells were detected in the inflammatory infiltrate that mediates tumor rejection along with some DC. The effects of IFN gamma, produced at the tumor site by T and NK lymphocytes, were only required at the malignant cell level and were necessary for tumor eradication. NK recognition of tumor cells was mediated by the NKG2D-activating receptor and blocking its function in vivo partially interfered with rejection. Therefore, complete rejection of these mesenchymal tumors requires a concerted set of activities including direct tumor-cell destruction and IFN gamma production that are mediated by both NK and T cells.

Toxoplasma gondii possesses sets of dense granule proteins (GRAs) that either assemble at, or cross the parasitophorous vacuole membrane (PVM) and exhibit motifs resembling the HT/PEXEL previously identified in a repertoire of exported Plasmodium proteins. Within Plasmodium spp., cleavage of the HT/PEXEL motif by the endoplasmic reticulum-resident protease Plasmepsin V precedes trafficking to and export across the PVM of proteins involved in pathogenicity and host cell remodelling. Here, we have functionally characterized the T. gondii aspartyl protease 5 (ASP5), a Golgi-resident protease that is phylogenetically related to Plasmepsin V. We show that deletion of ASP5 causes a significant loss in parasite fitness in vitro and an altered virulence in vivo. Furthermore, we reveal that ASP5 is necessary for the cleavage of GRA16, GRA19 and GRA20 at the PEXEL-like motif. In the absence of ASP5, the intravacuolar nanotubular network disappears and several GRAs fail to localize to the PVM, while GRA16 and GRA24, both known to be targeted to the host cell nucleus, are retained within the vacuolar space. Additionally, hypermigration of dendritic cells and bradyzoite cyst wall formation are impaired, critically impacting on parasite dissemination and persistence. Overall, the absence of ASP5 dramatically compromises the parasite’s ability to modulate host signalling pathways and immune responses., Author Summary The opportunistic pathogen Toxoplasma gondii infects a large range of nucleated cells where it replicates intracellularly within a parasitophorous vacuole (PV) surrounded by a membrane (PVM). Parasites constitutively secrete dense-granule proteins (GRAs) both into and beyond the PV which participate in remodelling of the PVM, recruitment of host organelles, neutralization of the host cellular defences, and subversion of host cell functioning. In addition, the GRAs critically contribute to cyst wall formation, a process that critically ensures parasite persistence and transmission. To act as effector molecules, some of the GRAs must be translocated across the PVM. Within the related apicomplexan parasite P. falciparum, a repertoire of proteins exported beyond the PVM contain a motif cleaved by a specific protease, Plasmepsin V. Examination of the repertoire of GRAs in T. gondii revealed that some proteins exhibit such export-like motifs suggestive of protease involvement. In this study, we have functionally characterized the related aspartyl protease 5 (TgASP5) in both virulent and persistent T. gondii strains, and have investigated the phenotypic consequences of its deletion in the context of overall parasite biology, its intracellular niche, the infected host cells and the murine model. Our findings revealed fundamental roles of TgASP5 at the host-parasite interface.