T-cell Deficient Mice Research Articles

The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally

The host-microbiota relationship has evolved to shape mammalian physiology, including immunity, metabolism, and development. Germ-free models are widely used to study microbial effects on host processes such as immunity. Here, we find that both germ-free and Tcell-deficient mice exhibit a robust sebum secretion defect persisting across multiple generations despite microbial colonization and Tcell repletion. These phenotypes are inherited by progeny conceived during invitro fertilization using germ-free sperm and eggs, demonstrating that non-genetic information in the gametes is required for microbial-dependent phenotypic transmission. Accordingly, gene expression in early embryos derived from gametes from germ-free or Tcell-deficient mice is strikingly and similarly altered. Our findings demonstrate that microbial- and immune-dependent regulation of non-genetic information in the gametes can transmit inherited phenotypes transgenerationally in mice. This mechanism could rapidly generate phenotypic diversity to enhance host adaptation to environmental perturbations.

Deletion of MyD88 in T Cells Improves Antitumor Activity in Melanoma

Cytotoxic CD8+ T cells are central to the antitumor immune response by releasing cytotoxic granules that kill tumor cells. They are activated by antigen-presenting cells, which become activated by DAMPs (damage associated molecular patterns) through MyD88. However, the suppressive tumor microenvironment promotes T-cell tolerance to tumor antigens, in part by enhancing the activity of immune checkpoint molecules that prevent CD8+ T-cell activation and cytotoxicity. MyD88 limits CD4+ T-cell activation during cardiac adaptation to stress. A similar mechanism is hypothesized to exist in CD8+ T cells that could be modulated to improve antitumor immunity. Herein, adoptive transfer of MyD88-/- CD8+ T cells in melanoma-bearing T-cell-deficient mice resulted in slower tumor growth, greater intratumoral T-cell accumulation, and higher melanoma cell death compared with transfer of wild-type CD8+ T cells. These findings were also observed in T-cell-specific MyD88-/- mice compared with wild-type littermates implanted with melanoma. Mechanistically, deletion of MyD88 enhanced CD8+ T-cell activation and survival, and T-cell receptor induced degranulation of cytotoxic molecules, overall improving the killing of melanoma cells. This enhanced cytotoxicity was retained in mice bearing tumors expressing the specific antigen for which cytotoxic T-cells were restricted. This study's results demonstrate a conserved mechanism for MyD88 in modulating CD8+ T-cell activation and represent a novel target in improving cancer immunotherapy.

Abstract P3064: Cytotoxic T-cells Drive Doxorubicin Induced Cardiac Fibrosis And Systolic Dysfunction

Background: Doxorubicin (DR), the most commonly prescribed chemotherapy, results in dose-dependent cardiotoxicity and heart failure (HF), and patients respond less frequently to standard HF treatments. DR causes oxidative stress and myocyte death, both potent inducers of a T-cell immune response and cardiac inflammation in other causes of HF. However, mechanistic depth on the immune response elicited by DR is limited. We hypothesized that an aberrant T-cell immune response secondary to cardiac damage by DR contributes to DR-induced HF and cardiac remodeling. Methods and Results: We treated wild type (WT) and T-cell deficient ( Tcra -/- ) mice with 5.0 mg/kg DR or PBS weekly by intraperitoneal injection for 4 or 8 weeks. We found at both time points Tcra -/- mice had preserved ejection fraction (EF) by echocardiography and decreased cardiac fibrosis by picrosirius red staining, compared to WT. At 8 weeks, Tcra -/- mice had fewer TUNEL + cardiac cells than WT mice. DR treated mice had increased frequency of activated effector CD8 + CD62L lo CD44 hi cells in the cardiac draining lymph nodes, increased circulating CD8 + T-cells, and cardiac CD8 + T-cell infiltration at both time points. Histologic analysis demonstrated localization of CD8 + T-cells adjacent to cardiac fibroblasts (CFB) in the hearts of DR treated mice. Targeted antibody depletion of CD8 + but not CD4 + cells prior to 4 week DR treatment resulted in improved EF and decreased cardiac fibrosis. Moreover, cardiac CD8 + T-cells expressed higher IFNγ in vivo in DR compared to PBS treated mice, and DR hearts had increased gene expression of the IFNγ inducible chemokines Cxcl9 and Cxcl10 . Ex vivo DR (0.1 μg/mL) treatment of CD8 + T-cells during TCR activation with αCD3/CD28 increased IFNγ expression at the protein and transcript level. The same dose of DR also increased surface expression of ICAM-1 on CFB, and CD8 + T-cells adhered to DR treated CFB in vitro in greater numbers than untreated CFB, and induced more αSMA expression. Lastly, in patients treated with DR, plasma levels of CXCL9 and CXCL10 correlated with decreases in EF after 3 months of treatment. Conclusion: These data outline a novel CD8 T-cell-driven inflammatory mechanism in DR cardiotoxicity, driven by IFNγ-inducible chemokines in animal models and patients.

Hyperthermic Intrathoracic Chemotherapy (HITOC) Improves Malignant Pleural Mesothelioma Control Through a Tumor Specific Cytotoxic Immune Response

Abstract Background The management of malignant pleural mesothelioma (MPM) remains challenging with poor patient survival. Local therapies such as hyperthermic intrathoracic cisplatin (HITOC) have shown good tumor control in selected patients. HITOC was shown to increase MPM drug exposure while limiting systemic side effects but alternative mechanisms for HITOC are still lacking. Aims We hypothesized that HITOC induces an immune response directed against MPM which decreases cancer related mortality. Methods A perfusion circuit was downsized to administer cisplatin in the thoracic cavity of MPM bearing mice at normo (37°C, ITOC) or hyperthermic (39°C, HITOC) conditions for 30 minutes. Tumor platinum content and distribution were determined by inductively coupled plasma mass spectrometry (ICP-MS) and by laser ablation ICP-MS respectively. The impact of (H)ITOC on the MPM immune microenvironment was questioned using flow cytometry and immunohistochemistry. Tumor growth and survival was assessed in immunocompetent and T-cell deficient mice. Finally, the combination of HITOC with anti-CTLA-4 and anti-PD-1 antibodies was evaluated in immunocompetent animals. Results MPM tumor control and mouse survival were significantly improved by HITOC compared to controls (ITOC, saline 37 and 39°C). This correlated with higher tumor platinum content that was mainly located at the surface of tumors. Furthermore, HITOC enhanced MPM infiltration by cytotoxic T-cells and decreased the level of protumor (M2-like) macrophages at day 7. The beneficial impact of HITOC on tumor control and mice survival was lost in immunocompromised animals. Interestingly, HITOC significantly induced PD-1 and CTLA-4 immune checkpoint molecules expression and administration of dual immune checkpoint blockade after HITOC increased of almost two fold the median survival of the animals. Conclusions HITOC improves MPM control through a T lymphocyte immune mediated response. The potentiation of the immune checkpoint blockade response by HITOC opens new perspectives for the combination of immune checkpoint inhibitors with HITOC in clinic.

Abstract 5166: Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models

Abstract Advances in immunotherapy have revolutionized cancer treatment, with patients with otherwise incurable disease having long-lasting responses. Unfortunately, deep remissions only occur in a select subset of patients. Cancer progression is common as many current immunotherapies only target one or two pathways, which can quickly lead to tumor resistance. Novel approaches that target several pathways concurrently are therefore needed to promote stronger responses in a wider patient population. Our group has recently discovered that the protein Ubiquitin-specific protease 6 (USP6) upregulates multiple, distinct immunostimulatory pathways. Tumor cell-specific expression of USP6 significantly increases CXCL9, CXCL10, and CCL5 chemokine production that enhances cytotoxic immune cell recruitment. Simultaneously, USP6 also upregulates several ligands and receptors that promote immune cell activation and killing of tumor cells such as CD112, MICA/B, ICAM1, and MHC Class I. In addition, USP6 increases the tumor cell’s sensitivity to immune-derived factors such as Type I and Type II interferons and TRAIL by raising the surface expression of their respective receptors. USP6 is hominid-specific and therefore requires xenografting human tumor cells into athymic, T-cell deficient nude mice. Despite the immunocompromised model, USP6 expression was found to increase immune cell recruitment to the tumor while repolarizing the immune microenvironment toward a more activated, tumor-suppressive state. Strikingly, immune cells in the peripheral blood were also affected with an increase in natural killer cell (NK) activation and a skewing of the myeloid population away from an immunosuppressive phenotype. Our group subsequently demonstrated that a USP6+ tumor was able to inhibit the growth of a USP6− distal tumor, with a concordant increase in NK, macrophage, and dendritic cell activation in the distal USP6− tumor. Given our data, we sought to translate USP6 into a novel immunotherapy. Delivery of mRNA encoding the USP6 protein recapitulates the immunostimulatory effects of USP6 in vitro. Intratumoral (I.T) injection of a lipid nanoparticle (LNP) encapsulating USP6 mRNA significantly inhibited Ewing sarcoma and acute myeloid leukemia growth in athymic mice, despite the model lacking a fully functional immune system. Moreover, I.T injection of USP6 mRNA LNPs inhibited the growth of a non-injected, distal tumor. Altogether, these data support that USP6 is a potentially powerful target and continued development of I.T USP6 mRNA LNPs as a novel immunotherapy. Citation Format: Ian Henrich, Margaret Billingsley, Kanika Jain, Laura Quick, Rob Young, Margaret Chou, Michael Mitchell. Intratumoral delivery of mRNA encoding USP6 activates multiple immuno-stimulatory pathways simultaneously and inhibits local and distal tumor growth in murine models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5166.

Microbe–Immune–Stress Interactions Impact Behaviour during Postnatal Development

Decades of research have established the role of microbiota-brain communication in behaviour and brain function. Studies have shown that microbiota composition and diversity are influenced by a variety of factors including host genetics, diet, and other environmental exposures, with implications for the immunological and neurobiological development of the host organism. To further understand early-life interactions between environment, genetic factors, the microbiome and the central nervous system, we investigated the impact of postnatal stress in C57Bl/6 wild type and T-cell deficient mice on microbe-brain interactions and behaviour. Mice were exposed to immune challenge with lipopolysaccharide (LPS) at postnatal day (P) 3 and maternal separation at P9 (16 h overnight). Behavioural assessment of growth and development as well as behaviour (righting reflex, ultrasonic vocalizations in response to brief maternal separation, open field, sociability, and grooming) was conducted. Microbiota diversity and composition of fecal samples collected at P24 revealed reduced alpha diversity in T-cell-deficient mice as well as genotype- and stress-related taxa. Notably, integrated analyses of microbiota and behaviour in the context of immunocompromise revealed key behavioural related taxa that may be important to brain development. These findings are important to determining the influence of genetic and environmental factors on gut microbiota and advances our understanding microbiome-brain signaling pathways on neurodevelopment and behaviour.

Abstract P3062: T-cell Myd88 Regulates Cardiac Fibrosis In Heart Failure By Modulating T-cell Survival And Pro-inflammatory Signaling

Background: Despite the association of heart failure (HF) with cardiac and systemic inflammation, anti-inflammatory therapies have shown limited success. Cardiac inflammation is characterized by the presence of damage associated molecular patterns (DAMPs), myeloid and T-helper cell infiltration, and the activation of cardiac fibroblasts (CFB). The adaptor “Myeloid differentiation response 88” (MyD88) is central to DAMP sensing in myeloid cells, but its role in T-cell function and cardiac inflammation is largely unknown. We hypothesized that T-cell signaling through MyD88 modulates cardiac inflammation and fibrosis in HF. Methods and Results: We reconstituted T-cell deficient ( Tcra -/- ) mice, normally protected from cardiac fibrosis, with WT or Myd88 -/- Type 1 helper T-cells (Th1) in the onset of transaortic constriction (TAC). We found mice given Myd88 -/- Th1 cells exhibited significantly higher levels of cardiac T-cell infiltration, increased cardiac fibrosis, and lower fractional shortening than mice given WT Th1 cells. T-cell specific MyD88 deficient mice ( Myd88 fl/fl CD4 Cre ) also developed more fibrosis and increased cardiac T-cells compared to Cre- littermate controls in response to TAC. RNA-seq analysis of WT and Myd88 -/- Th1 cells revealed enrichment of pro-inflammatory signaling and pro-survival genes in Myd88 -/- Th1 cells, which we validated by immunoblotting for phospho-ERK, NF-kB, and AKT. In contrast, IFNγ, the Th1 signature cytokine, was similar by qPCR and flow cytometry in WT and Myd88 -/- cells. Moreover, we discovered that Myd88 -/- Th1 cells have increased survival in vitro, by monitoring propidium iodide incorporation in real time, and in vivo by adoptive transfer of equal numbers of WT and Myd88 -/- cells together into Tcra -/- mice and quantifying both cell types in lymphoid organs over time. Lastly, more Myd88 -/- Th1 cells adhered to immobilized VCAM-1 and CFB in vitro compared to WT, resulting in enhanced CFB transformation. Conclusion: T-cell MyD88 limits cardiac fibrosis in TAC through modulation of T-cell inflammatory signaling and cell survival. Disruption of this pathway in T-cells results in enhanced transformation of fibroblasts and worsened systolic function.

Anti-GD2 Antibodies Conjugated to IL15 and IL21 Mediate Potent Antitumor Cytotoxicity against Neuroblastoma.

Half of the patients with high-risk neuroblastoma who receive GD2-targeted mAb do not achieve long-term remissions. Recently, the antibody hu14.18 has been linked to IL2 (hu14.18-IL2) to enhance its efficacy and shown promising preclinical and clinical activity. We developed two new immunocytokines (IC) by linking two other γc cytokines, IL15 and IL21, to hu14.18. The purpose of this study was to compare hu14.18-IL15 and -IL21 with hu14.18-IL2 in their ability to induce antibody-dependent cell-mediated cytotoxicity (ADCC) against neuroblastoma. We assessed ADCC of hu14.18-IL15 and -IL2 (human cytokines, cross-reactive to mouse) against GD2low and GD2high neuroblastoma cell lines in vitro. T-cell-deficient mice with orthotopic patient-derived xenografts (PDX) and immunocompetent mice with transplantable orthotopic neuroblastoma were used to test all three ICs, including hu14.18-IL21 (murine IL21, not cross-reactive to human). Mechanistic studies were performed using single-cell RNA-sequencing (scRNA-seq). hu14.18-IL15 and hu14.18-IL2 mediated equivalent in vitro ADCC by human NK cells. When combined with chemotherapy, all three ICs similarly controlled the growth of PDXs in nude mice with murine NK effector cells. However, hu14.18-IL15 and -IL21 outperformed hu14.18-IL2 in immunocompetent mice with syngeneic neuroblastoma, inducing complete tumor regressions and extending survival. scRNA-seq data revealed an increase in CD8+ T cells and M1 tumor-associated macrophages and decreased regulatory T cells and myeloid-derived suppressor cells in the tumor microenvironment. Hu14.18-IL15 and Hu14.18-IL21 exhibit robust preclinical activity, warranting further consideration for clinical testing in patients with GD2-expressing neuroblastoma.

Abstract 3529: T-cell trafficking and extravasation is suppressed in distal tumors during gastrointestinal tract dysbiosis

Abstract The crosstalk between the microbiota and the host is crucial to maintaining immune homeostasis. A growing body of evidence shows that microbial imbalance affects cancer susceptibility and progression. A common significant influence on the microbiota in the gastrointestinal (GI) tract is the use of antibiotics. Oral antibiotics severely alter the bacteria in the GI tract by destroying beneficial bacteria alongside pathogenic ones — producing a state of microbial imbalance called dysbiosis. The overall use of antibiotics has increased by more than 30% in recent years, and moreover, many cancer patients are prescribed antibiotics during treatment, as infection is frequently a complication. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby causing stromal immune surveillance suppression. We found that antibiotics as well as tumorigenesis caused changes in the bacterial abundance, composition, or diversity in the GI tract. On the host side, we found that antibiotic-induced dysbiosis caused the small intestine and cecum to enlarge and elongate, yet the colon was not affected. Moreover, B16-F10 melanoma and Lewis Lung carcinoma progressed more quickly during dysbiosis. Mechanistically, tumor progression was mediated by suppressed TNF-α levels, both locally and systemically. In contrast, IFN-γ and VEGF did not change significantly during dysbiosis. The suppressed levels of TNF-α, a pleiotropic regulator of intercellular adhesion molecule-1 (ICAM-1), resulted in reduced expression of tumor endothelial cell associated ICAM-1, and a subsequent decrease in the number of activated and effector CD8+ T-cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1 (LFA-1), was not seen in established vasculature during dysbiosis as assessed in the spleen or the thymus. While dysbiosis enhanced tumor growth in ICAM-1+/+ and immunocompetent mice, it did not affect tumor growth in ICAM-1-/- or T-cell deficient mice, highlighting the influence and impact of dysbiosis on T-cell trafficking and extravasation. TNF-α supplementation in dysbiotic mice was able to overcome the dysbiosis-induced suppression as evident by increased ICAM-1 expression, leukocyte trafficking and tumor growth inhibition. Overall, these results demonstrate the importance of commensal bacteria in supporting immune surveillance and expand our understanding of the crosstalk between the gut microbiome and the stroma in distal non-GI tract tumors. Moreover, this knowledge has the potential to influence clinical decision-making when treating patients for solid cancers with dysbiosis-inducing antibiotics and may further inform concurrent and (neo-)adjuvant treatments to overcome the adverse effects of the antibiotics. Citation Format: Samir V. Jenkins, Michael S. Robeson, Eric R. Siegel, Charles M. Quick, Robert J. Griffin, Martin J. Cannon, Kieng B. Vang, Ruud P. Dings. T-cell trafficking and extravasation is suppressed in distal tumors during gastrointestinal tract dysbiosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3529.

Abstract CT526: A Phase 1/2a Study of a PI3Kδγ dual inhibitor ZX-101A in patients with advanced solid tumors

Abstract ZX-101A is a next-generation PI3Kδ/γ dual inhibitor, exerting synergistic effects on PI3Kδ and PI3Kγ-mediated processes through multifaceted mechanisms of actions. Phase 1/2a study, ZX-101A-101 (NCT04504708) is ongoing to assess safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy in patients with advanced hematologic malignancies. Furthermore, recent preclinical studies of ZX-101A showed that dual inhibition of PI3Kδ and PI3Kγ restored the immune surveillance to inhibit solid tumor growth. ZX-101A-201 is a phase 1/2a open-label, multiple-dose ascending study to investigate the safety, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of once daily oral ZX-101A in 28-day cycles in patients with advanced solid tumors.ZX-101A had minimal effects on solid tumor-derived CT26, 4T1 and A20 cells in in vitro cell proliferation assay. In contrast, ZX-101A inhibited tumor growth when CT26 cells were grafted to wild-type syngeneic mice, while only showed a limited tumor growth inhibition when CT26 tumors grew on T-cell deficient nude mice. Thus, in vivo tumor growth inhibition in wild-type mice was attributed to the enhanced anti-tumor immunity by ZX-101A treatment. Flow cytometry analysis on ex vivo tumor tissues from syngeneic mouse demonstrated that ZX-101A decreased regulatory T cells and increased M1/M2 macrophage polarization in the tumor microenvironment. In A20 syngeneic model, ZX-101A in combination of anti-PD1 antibody achieved 99.7% TGI after 18 days of treatment, while anti-PD1 antibody alone only had TGI at 11.7%. Synergistic inhibitory effect of ZX-101A and anti-PD1 antibody was also observed in LLC, CT26 and humanized MC38 syngeneic mouse models. Safety assessment on the mouse spleen, thymus and colon organs indicated minimal side effects by the treatment of the ZX-101A alone and in combination with anti-PD1. These results suggested ZX-101A as a potential immunotherapeutic agent to treat appropriate solid tumor types. ZX-101A-201 study consists of 2 parts, Dose Escalation and Dose Expansion. Part 1 dose escalation is designed to determine the safety, tolerability, and recommended phase 2 dose (RP2D) of ZX-101A monotherapy in patient with advanced solid tumors. Part 2 dose expansion will evaluate ZX-101A activity in up to three disease-specific cohorts. Inclusion of combination therapy is planned after obtaining the initial clinical assessment of ZX-101A on its safety, tolerability, pharmacokinetics, and pharmacodynamics in patients. Citation Format: Xiaoli Qin, Lori Luo, Li Ying-ying, Yin Wang, Shuaijun Sun, Xiaolin Hao, Jinfu Yang. A Phase 1/2a Study of a PI3Kδγ dual inhibitor ZX-101A in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT526.

Microbe-Immune Crosstalk: Evidence That T Cells Influence the Development of the Brain Metabolome.

Cross-talk between the immune system and the brain is essential to neuronal development, neuronal excitability, neuroplasticity, and neurotransmission. Gut microbiota are essential to immune system development and immune function; hence, it is essential to consider more broadly the microbiota-immune-brain axis in neurodevelopment. The gut, brain, and microbial metabolomes obtained from C57Bl/6 and T-cell-deficient mice across four developmental timepoints (postnatal day 17, 24, 28, and 84) were studied by 1H NMR spectroscopy. 16S rRNA gene sequencing was performed on cecal and fecal samples. In the absence of T-cells, the developmental trajectory of the gut microbiota and of the host’s metabolic profile was altered. The novel insights from this work include (1) the requirement of functional T-cells for the normal trajectory of microbiotal development and the metabolic maturation of the supra-organism, (2) the potential role for Muribaculaceae taxa in modulating the cecal availability of metabolites previously implicated with a role in the gut-brain axis in T-cell deficient mice, and (3) the impact of T-cell-deficiency on central levels of neuroactive metabolites.

Innate Lymphoid Cells Promote Recovery of Ventricular Function After Myocardial Infarction

BackgroundInnate lymphoid cells type 2 (ILC2s) play critical homeostatic functions in peripheral tissues. ILC2s reside in perivascular niches and limit atherosclerosis development. ObjectivesILC2s also reside in the pericardium but their role in postischemic injury is unknown. MethodsWe examined the role of ILC2 in a mouse model of myocardial infarction (MI), and compared mice with or without genetic deletion of ILC2. We determined infarct size using histology and heart function using echocardiography. We assessed cardiac ILC2 using flow cytometry and RNA sequencing. Based on these data, we devised a therapeutic strategy to activate ILC2 in mice with acute MI, using exogenous interleukin (IL)-2. We also assessed the ability of low-dose IL-2 to activate ILC2 in a double-blind randomized clinical trial of patients with acute coronary syndromes (ACS). ResultsWe found that ILC2 levels were increased in pericardial adipose tissue after experimental MI, and genetic ablation of ILC2 impeded the recovery of heart function. RNA sequencing revealed distinct transcript signatures in ILC2, and pointed to IL-2 axis as a major upstream regulator. Treatment of T-cell–deficient mice with IL-2 (to activate ILC2) significantly improved the recovery of heart function post-MI. Administration of low-dose IL-2 to patients with ACS led to activation of circulating ILC2, with significant increase in circulating IL-5, a prototypic ILC2-derived cytokine. ConclusionsILC2s promote cardiac healing and improve the recovery of heart function after MI in mice. Activation of ILC2 using low-dose IL-2 could be a novel therapeutic strategy to promote a reparative response after MI.

Short communication: Efficacy of albendazole in Echinococcus multilocularis-infected mice depends on the functional immunity of the host

Alveolar echinococcosis (AE) is a deadly parasitic disease that requires lifelong treatment with albendazole. Development of host immunity is pivotal with regard to the clinical outcome of AE, but its influence on conventional albendazole treatment is unknown. Using T-cell deficient athymic nude mice, we demonstrated that functional immunity is required for albendazole to be efficacious against murine AE. These results call for attention given the increasing number of immunocompromised patients with AE.

Protein Tyrosine Phosphatase Non-Receptor Type 2 Function in Dendritic Cells Is Crucial to Maintain Tissue Tolerance.

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) plays a pivotal role in immune homeostasis and has been associated with human autoimmune and chronic inflammatory diseases. Though PTPN2 is well-characterized in lymphocytes, little is known about its function in innate immune cells. Our findings demonstrate that dendritic cell (DC)-intrinsic PTPN2 might be the key to explain the central role for PTPN2 in the immune system to maintain immune tolerance. Partial genetic PTPN2 ablation in DCs resulted in spontaneous inflammation, particularly in skin, liver, lung and kidney 22 weeks post-birth. DC-specific PTPN2 controls steady-state immune cell composition and even incomplete PTPN2 deficiency in DCs resulted in enhanced organ infiltration of conventional type 2 DCs, accompanied by expansion of IFNγ-producing effector T-cells. Consequently, the phenotypic effects of DC-specific PTPN2 deficiency were abolished in T-cell deficient Rag knock-out mice. Our data add substantial knowledge about the molecular mechanisms to prevent inflammation and maintain tissue tolerance.

CCR6-Positive γδ T Cells Provide Protection Against Intracorneal HSV-1 Infection

γδ T cells offer an important early immune defense against many different pathogens, both bacterial and viral. Herein, we examined the capacity of γδ T cell subsets to provide protection in the cornea against herpes simplex virus-1 (HSV-1). C57Bl/6 (wild-type [WT]), γδ T-cell deficient (TCRδ-/-) and CCR6-deficient (CCR6-/-) mice were infected intracorneally with HSV-1. At multiple time points following infection, corneas were excised, and cells were immunostained for surface markers, intracellular cytokines, and analyzed using flow cytometry. WT and CCR6-/- γδ T cells were adoptively transferred into TCRδ-/- mice and corneal scores and survival were measured. Intracorneal infection of mice lacking γδ T cells exhibited increased corneal opacity scores, elevated viral titers, and higher mortality compared with WT mice. Both CCR6+ and CCR6neg γδ T cell subsets were observed in corneas after virus infection. CCR6+ γδ T cells produced IL-17A and were predominantly CD44+CD62L+, consistent with natural IL-17+ γδ T cells. In contrast IL-17A production by CCR6neg γδ T cells was infrequent, and this subset was largely single positive for CD62L or CD44. The CCR6+ subset appeared to provide protection against HSV-1 as follows: (1) CCR6-/- mice had more severe corneal opacity compared with WT mice; and (2) adoptive transfer of γδ T cells from WT mice restored protection in TCRδ-/- mice whereas transfer of γδ T cells from CCR6-/- mice did not. γδ T cells in the cornea can be divided into CCR6+ and CCR6neg subsets with the former conferring protection early after intracorneal HSV-1 infection.

Mechanisms of fibronectin-binding protein A (FnBPA110–263) vaccine efficacy in Staphylococcus aureus sepsis versus skin infection

Increasing rates of life-threatening infections and decreasing susceptibility to antibiotics urge an effective vaccine targeting Staphylococcus aureus. Here we investigate the role of cellular immunity in FnBPA110–263 mediated protection in Staphylococcus aureus infection. This study revealed FnBPA110–263 broadly protected mice from seven FnBPA isotypes strains in the sepsis model. FnBPA110–263 immunized B-cell deficient mice were protected against lethal challenge, while T-cell deficient mice were not. Reconstituting mice with FnBPA110–263 specific CD4+ T-cells conferred antigen specific protection. In vitro assays indicated that isolated FnBPA110–263 specific splenocytes from immunized mice produced abundant IL-17A. IL-17A deficient mice were not protected from a lethal challenge by FnBPA110–263 vaccination. Moreover, neutralizing IL-17A, but not IFN-γ,reverses FnBPA110–263-induced protective efficacy in sepsis and skin infection model. These findings suggest that IL-17A producing Th17 cells play an essential role in FnBPA110–263 vaccine-mediated defense against S. aureus sepsis and skin infection in mice.

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes.

Tumor-associated macrophages can promote growth of cancers. In neuroblastoma, tumor-associated macrophages have greater frequency in metastatic versus loco-regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high-risk patients who have MYCN-non-amplified disease. The contribution of cytotoxic T-lymphocytes to anti-neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T-cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF-1R can improve the response to chemotherapy. In vitro, CSF-1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co-injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14+ and CD163+ cells and mouse F4/80+ cells after CSF-1R blockade. In subcutaneous or intra-renal models in immunodeficient NSG or NOD/SCID mice, CSF-1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF-1R inhibitor BLZ945, either without or with anti-human and anti-mouse CSF-1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor-associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T-lymphocytes, suggesting the possibility that combination of CSF-1R blockade with chemotherapy might be effective in patients who have limited anti-tumor T-cell responses.

Development of an in vivo infection model to study Mouse papillomavirus-1 (MmuPV1)

Preclinical model systems to study multiple features of the papillomavirus life cycle are extremely valuable tools to aid our understanding of Human Papillomavirus (HPV) biology, disease progression and treatments. Mouse papillomavirus (MmuPV1) is the first ever rodent papillomavirus that can infect the laboratory strain of mice and was discovered recently in 2011. This model is an attractive model to study papillomavirus pathogenesis due to the ubiquitous availability of lab mice and the fact that this mouse species is easily genetically modifiable. Several other groups, including ours, have reported that MmuPV1-induced papillomas are restricted to T-cell deficient immunosuppressed mice. In our lab we showed for the first time that MmuPV1 causes skin cancers in UVB-irradiated immunocompetent animals. In this report we describe in detail the MmuPV1-UV infection model that can be adapted to study MmuPV1 biology in immunocompetent animals.

Abstract IA21: Immunomodulatory therapy of multiple myeloma with IAP antagonists

Abstract IA21: Immunomodulatory therapy of multiple myeloma with IAP antagonists

Characterization of the distribution, retention, and efficacy of internal radiation of 188Re-lipid nanocapsules in an immunocompromised human glioblastoma model.

Internal radiation strategies hold great promise for glioblastoma (GB) therapy. We previously developed a nanovectorized radiotherapy that consists of lipid nanocapsules loaded with a lipophilic complex of Rhenium-188 (LNC188Re-SSS). This approach resulted in an 83 % cure rate in the 9L rat glioma model, showing great promise. The efficacy of LNC188Re-SSS treatment was optimized through the induction of a T-cell immune response in this model, as it is highly immunogenic. However, this is not representative of the human situation where T-cell suppression is usually encountered in GB patients. Thus, in this study, we investigated the efficacy of LNC188Re-SSS in a human GB model implanted in T-cell deficient nude mice. We also analyzed the distribution and tissue retention of LNC188Re-SSS. We observed that intratumoral infusion of LNCs by CED led to their complete distribution throughout the tumor and peritumoral space without leakage into the contralateral hemisphere except when large volumes were used. Seventy percent of the 188Re-SSS activity was present in the tumor region 24h after LNC188Re-SSS injection and no toxicity was observed in the healthy brain. Double fractionated internal radiotherapy with LNC188Re-SSS triggered survival responses in the immunocompromised human GB model with a cure rate of 50 %, which was not observed with external radiotherapy. In conclusion, LNC188Re-SSS can induce long-term survival in an immunosuppressive environment, highlighting its potential for GB therapy.