OT-I Mice Research Articles

Dendritic Cells Exposed to Triiodothyronine Deliver Pro-Inflammatory Signals and Amplify IL-17-Driven Immune Responses.

Although a cross-talk between immune and endocrine systems has been well established, the precise pathways by which these signals co-regulate pro- and antiinflammatory responses on antigen-presenting cells remain poorly understood. In this work we investigated the mechanisms by which triiodothyronine (T3) controls T cell activity via dendritic cell (DC) modulation. DCs from wild-type (WT) and IL-6-deficient mice were pulsed with T3. Cytokine production and programmed death protein ligands (PD-L) 1 and 2 expression were assayed by flow cytometry and ELISA. Interferon-regulatory factor-4 (IRF4) expression was evaluated by RT-qPCR and flow cytometry. The ability of DCs to stimulate allogenic splenocytes was assessed in a mixed lymphocyte reaction and the different profile markers were analyzed by flow cytometry and ELISA. For in vivo experiments, DCs treated with ovalbumin and T3 were injected into OTII mice. Proliferation, cytokine production, frequency of FoxP3+ regulatory T (Treg) cells and PD-1+ cells were determined by MTT assay, ELISA and flow cytometry, respectively. T3 endows DCs with pro-inflammatory potential capable of generating IL-17-dominant responses and down-modulating expression of PD-L1 and 2. T3-stimulated WT-DCs increased the proportion of IL-17-producing splenocytes, an effect which was eliminated when splenocytes were incubated with T3-treated DCs derived from IL-6-deficient mice. Enhanced IL-17 expression was recorded in both, CD4- and CD4+ populations and involved the IRF-4 pathway. Particularly, γδ-T cells but not natural killer (NK), NKT, B lymphocytes nor CD8+ T cells were the major source of IL-17-production from CD4- cells. Moreover, T3-conditioned DCs promoted a decrease of the FoxP3+ Treg population. Furthermore, T3 down-modulated PD-1 expression on CD4- cells thereby limiting inhibitory signals driven by this co-inhibitory pathway. Thus, T3 acts at the DC level to drive proinflammatory responses in vitro. Accordingly, we found that T3 induces IL-17 and IFNγ-dominant antigen-specific responses in vivo. These results emphasize the relevance of T3 as an additional immune-endocrine checkpoint and a novel therapeutic target to modulate IL-17-mediated pro-inflammatory responses.

A Dual-Color Bioluminescence Reporter Mouse for Simultaneous in vivo Imaging of T Cell Localization and Function.

Non-invasive imaging technologies to visualize the location and functionality of T cells are of great value in immunology. Here, we describe the design and generation of a transgenic mouse in which all T cells constitutively express green-emitting click-beetle luciferase (CBG99) while expression of the red-emitting firefly luciferase (PpyRE9) is induced by Nuclear Factor of Activated T cells (NFAT) such as during T cell activation, which allows multicolor bioluminescence imaging of T cell location and function. This dual-luciferase mouse, which we named TbiLuc, showed high constitutive luciferase expression in lymphoid organs such as lymph nodes and the spleen. Ex vivo purified CD8+ and CD4+ T cells both constitutively expressed luciferase, whereas B cells showed no detectable signal. We cross-bred TbiLuc mice to T cell receptor-transgenic OT-I mice to obtain luciferase-expressing naïve CD8+ T cells with defined antigen-specificity. TbiLuc*OT-I T cells showed a fully antigen-specific induction of the T cell activation-dependent luciferase. In vaccinated mice, we visualized T cell localization and activation in vaccine-draining lymph nodes with high sensitivity using two distinct luciferase substrates, D-luciferin and CycLuc1, of which the latter specifically reacts with the PpyRE9 enzyme. This dual-luciferase T cell reporter mouse can be applied in many experimental models studying the location and functional state of T cells.

CD8+-T Cells With Specificity for a Model Antigen in Cardiomyocytes Can Become Activated After Transverse Aortic Constriction but Do Not Accelerate Progression to Heart Failure

Heart failure due to pressure overload is frequently associated with inflammation. In addition to inflammatory responses of the innate immune system, autoimmune reactions of the adaptive immune system appear to be triggered in subgroups of patients with heart failure as demonstrated by the presence of autoantibodies against myocardial antigens. Moreover, T cell-deficient and T cell-depleted mice have been reported to be protected from heart failure induced by transverse aortic constriction (TAC) and we have shown recently that CD4+-helper T cells with specificity for an antigen in cardiomyocytes accelerate TAC-induced heart failure. In this study, we set out to investigate the potential contribution of CD8+-cytotoxic T cells with specificity to a model antigen (ovalbumin, OVA) in cardiomyocytes to pressure overload-induced heart failure. In 78% of cMy-mOVA mice with cardiomyocyte-specific OVA expression, a low-grade OVA-specific cellular cytotoxicity was detected after TAC. Adoptive transfer of OVA-specific CD8+-T cells from T cell receptor transgenic OT-I mice before TAC did not increase the risk of OVA-specific autoimmunity in cMy-mOVA mice. After TAC, again 78% of the mice displayed an OVA-specific cytotoxicity with on average only a three-fold higher killing of OVA-expressing target cells. More CD8+ cells were present after TAC in the myocardium of cMy-mOVA mice with OT-I T cells (on average 17.5/mm2) than in mice that did not receive OVA-specific CD8+-T cells (3.6/mm2). However, the extent of fibrosis was similar in both groups. Functionally, as determined by echocardiography, the adoptive transfer of OVA-specific CD8+-T cells did not significantly accelerate the progression from hypertrophy to heart failure in cMy-mOVA mice. These findings argue therefore against a major impact of cytotoxic T cells with specificity for autoantigens of cardiomyocytes in pressure overload-induced heart failure.

Production of Extracellular Adenosine by CD73+ Dendritic Cells Is Crucial for Induction of Tolerance in Contact Hypersensitivity Reactions

Dendritic cells (DCs) express the ecto-5'-nucleotidase CD73 that generates immunosuppressive adenosine (Ado) by dephosphorylation of extracellular Ado monophosphate and diphosphate. To investigate whether CD73-derived Ado has immune-suppressive activity, 2,4-dinitrothiocyanobenzene (DNTB) was applied to skin of wild-type (WT) or CD73-deficient (CD73-/-) mice, followed by sensitization and challenge with 2,4-dinitrofluorobenzene. In this model, we show the induction of tolerance by DNTB against 2,4-dinitrofluorobenzene only in WT but not in CD73-/- mice. Analysis of skin DCs showed increased expression of CD73 after application of DNTB in WT mice. That was accompanied by elevated concentrations of extracellular Ado in the lymph node. Moreover, T cells expressed markers for anergy, namely EGR2 and NDRG1 in DNTB-treated WT mice and they exhibited impaired proliferation upon exvivo re-stimulation. Similarly, invitro we observed that Ado-producing WT DCs, but not CD73-/- DCs, rendered transgenic T cells from OTII mice (OTII T cells) hyporeactive, decreased their T-cell costimulatory signaling, and induced up-regulation of EGR2 and NDRG1. Thus, these data show that expression of CD73 by DCs, which triggers elevated levels of extracellular Ado, is a crucial mechanism for the induction of anergic T cells and tolerance.

Platelet Gene Therapy Promotes Targeted Peripheral Tolerance by Clonal Deletion and Induction of Antigen-Specific Regulatory T Cells.

Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery. We used ovalbumin (OVA) as a surrogate non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the platelet-specific αIIb promoter. Platelet-specific OVA expression was introduced by bone marrow transduction and transplantation. Greater than 95% of OVA was stored in platelet α-granules. Control mice immunized with OVA generated OVA-specific IgG antibodies; however, mice expressing OVA in platelets did not. Furthermore, OVA expression in platelets was sufficient to prevent the rejection of skin grafts from CAG-OVA mice, demonstrating that immune tolerance developed in platelet-specific OVA-transduced recipients. To assess the mechanism(s) involved in this tolerance we used OTII mice that express CD4+ effector T cells specific for an OVA-derived peptide. After platelet-specific OVA gene transfer, these mice showed normal thymic maturation of the T cells ruling against central tolerance. In the periphery, tolerance involved elimination of OVA-specific CD4+ effector T cells by apoptosis and expansion of an OVA-specific regulatory T cell population. These experiments reveal the existence of natural peripheral tolerance processes to platelet granule contents which can be co-opted to deliver therapeutically important products.

Interleukin-6 signalling mediates Galectin-8 co-stimulatory activity of antigen-specific CD4 T-cell response.

Galectin-8 (Gal-8) is a mammalian lectin endowed with the ability to co-stimulate antigen-specific immune responses. We have previously demonstrated that bone-marrow-derived dendritic cells produce high levels of interleukin-6 (IL-6) in response to Gal-8 stimulation. As IL-6 is a pleiotropic cytokine that has a broad effect on cells of the immune system, we aimed to elucidate whether IL-6 was involved in Gal-8-dependent co-stimulatory signals during antigen recognition by specific CD4 T cells. With this aim, splenocytes from DO11.10 mice were incubated with a low dose of the cognate ovalbumin peptide in combination with Gal-8. Interleukin-6 was found significantly increased in cultures stimulated with Gal-8 alone or Gal-8 plus cognate peptide. Moreover, IL-6 signalling was triggered during Gal-8-induced co-stimulation, as determined by phosphorylation of signal transducer and activator of transcription 3. Interleukin-6 blockade by neutralizing monoclonal antibody precluded Gal-8 co-stimulatory activity but did not affect the antigen-specific T-cell receptor activation. Different subsets of dendritic cells, as well as macrophages and B cells, were identified as the cellular source of IL-6 during Gal-8-induced co-stimulation. To confirm that IL-6 mediated the Gal-8 co-stimulatory effect, antigen-presenting cells from IL-6-deficient or wild-type mice were co-cultured with purified CD4 T cells from OTII mice in the presence of cognate peptide and Gal-8. Notably, Gal-8-induced co-stimulation, but not the antigen-specific response, was significantly impaired in the presence of IL-6-deficient antigen-presenting cells. In addition, exogenous IL-6 fully restored Gal-8-induced co-stimulation. Taken together, our results demonstrate that IL-6 signalling mediates the Gal-8 immune-stimulatory effect.

CD28 Costimulation of T Helper 1 Cells Enhances Cytokine Release In Vivo.

Compared to naive T cells, differentiated T cells are thought to be less dependent on CD28 costimulation for full activation. To revisit the role of CD28 costimulation in mouse T cell recall responses, we adoptively transferred in vitro generated OT-II T helper (Th) 1 cells into C57BL/6 mice (Thy1.2+) and then either blocked CD28–ligand interactions with Fab fragments of the anti-CD28 monoclonal antibody (mAb) E18 or deleted CD28 expression using inducible CD28 knock-out OT-II mice as T cell donors. After injection of ovalbumin protein in adjuvant into the recipient mice we observed that systemic interferon (IFN)γ release strongly depended on CD28 costimulation of the Th1 cells, while secondary clonal expansion was not reduced in the absence of CD28 costimulation. For human memory CD4+ T cell responses we also noted that cytokine release was reduced upon inhibition of CD28 costimulation. Together, our data highlight the so far underestimated role of CD28 costimulation for the reactivation of fully differentiated CD4+ T cells.

Ibrutinib has a novel immunomodulatory effect by enhancing DC maturation both in vivo and in a model of inflammation

Abstract Myeloid derived suppressor cells (MDSCs) can inhibit T cell activation by altering the characteristics and activation of dendritic cells (DCs). We had previously reported that Ibrutinib enhances the expression of co-stimulatory molecules on DCs and promotes DC-mediated T cell activation. In the present study we further determined the mechanisms of DC immunomodulation by Ibrutinib. We evaluated the effect of Ibrutinib on (i) DC maturation and migration from the injection site to draining lymph nodes, (ii) T cell proliferation in vivo, and (iii) monocyte maturation to DCs using a mouse inflammation model. DCs were generated from mouse bone marrow-derived cells in the presence of vehicle or 10μM lbrutinib. CFES-labelled DCs were injected subcutaneously into the footpads of naive mice, and their migration to the regional lymph nodes and maturation profile was analyzed by flow cytometry. To evaluate T cell priming, CFSE-labelled OVA-specific T cells isolated from OT-II mice were injected into the tail vein of syngeneic mice that were prior injected with mature DCs loaded with OVA peptide. After four days, T cell proliferation was evaluated by CFSE dilution. In the inflammation model, C57BL6 mice were treated with vehicle or 6mg/kg/day Ibrutinib orally for a week. A sub-lethal dose of LPS was injected in the dorsal skin and the frequency of inflammatory monocytes and mature DCs was determined. We found that Ibrutinib significantly improves DC maturation and migration, inducing T cell activation and proliferation in vivo. In addition, Ibrutinib enhances monocyte maturation to DCs in the skin. The results suggest a novel effect of Ibrutinib, which may have therapeutic use for cancers characterized by a high MDSC infiltration.

Diarylheptanoid from rhizomes of Curcuma kwangsiensis (DCK) inhibited imiquimod-induced dendritic cells activation and Th1/Th17 differentiation

Background and objectDendritic cells (DCs) are critical for initiating the activation and differentiation of T cells in inflammatory diseases including psoriasis. Curcuma kwangsiensis S.G. Lee & C.F. Liang is a herb for treating psoriasis and we previously found Diarylheptanoid from rhizomes of Curcuma kwangsiensis (DCK) inhibited keratinocytes proliferation. However, it is unknown whether DCK influences DC functions. Thus we aimed to explore whether DCK affect the major immunological functions of DCs. Materials and methodsPrimary DCs derived from mouse bone marrow cells and spleen were used for examining their general immunological functions, and OVA-specific T cells from OT-II mice were used for examining the DC-mediated T-helper (Th) 1 and Th17 cells differentiation and effect. ResultsWe demonstrated DCK suppressed DC uptake of FITC-labeled ovalbumin (OVA) and DC maturation characterized by decreased MHCII, CD80 and CD86 following imiquimod (IMQ) stimulation. DCK also reduced DC expression of the lymphoid-homing chemokine receptor CCR7, and DC migration towards CCL21, the ligand for CCR7. Importantly, DCK significantly reduced the production of proinflammatory cytokines including IL-12, IL-6 and IL-1β by IMQ-stimulated DCs. Moreover, in the coculture of OVA323–339 peptide-pulsed DCs and OVA-specific T cells from OT-II mice, DCK significantly inhibited T cell proliferation and the differentiation of Th1 and Th17 cells. Furthermore, DCK treatment greatly reduced phosphorylation of p65-associated cell signaling pathway in IMQ-stimulated DCs. ConclusionThese data together demonstrate a potential role of DCK in suppressing the biological function of DCs, and provide a possible mechanism for understanding the effects of herb Curcuma kwangsiensis in treating psoriasis.

Protein tyrosine phosphatase PTPN22 regulates IL-1β dependent Th17 responses by modulating dectin-1 signaling in mice.

A single nucleotide polymorphism within the PTPN22 gene is a strong genetic risk factor predisposing to the development of multiple autoimmune diseases. PTPN22 regulates Syk and Src family kinases downstream of immuno‐receptors. Fungal β‐glucan receptor dectin‐1 signals via Syk, and dectin‐1 stimulation induces arthritis in mouse models. We investigated whether PTPN22 regulates dectin‐1 dependent immune responses. Bone marrow derived dendritic cells (BMDCs) generated from C57BL/6 wild type (WT) and Ptpn22−/− mutant mice, were pulsed with OVA323‐339 and the dectin‐1 agonist curdlan and co‐cultured in vitro with OT‐II T‐cells or adoptively transferred into OT‐II mice, and T‐cell responses were determined by immunoassay. Dectin‐1 activated Ptpn22−/− BMDCs enhanced T‐cell secretion of IL‐17 in vitro and in vivo in an IL‐1β dependent manner. Immunoblotting revealed that compared to WT, dectin‐1 activated Ptpn22−/− BMDCs displayed enhanced Syk and Erk phosphorylation. Dectin‐1 activation of BMDCs expressing Ptpn22R619W (the mouse orthologue of human PTPN22R620W) also resulted in increased IL‐1β secretion and T‐cell dependent IL‐17 responses, indicating that in the context of dectin‐1 Ptpn22R619W operates as a loss‐of‐function variant. These findings highlight PTPN22 as a novel regulator of dectin‐1 signals, providing a link between genetically conferred perturbations of innate receptor signaling and the risk of autoimmune disease.

Abstract 006: Hypertension is Accompanied With Over Activity of the Adaptive Immune System

Adaptive immune response plays an important role in the pathogenesis of hypertension, but how does hypertension influence on the immune system is still poorly understood. To understand this, we studied antigen presenting cells (APCs) isolated from normotensive and experimental hypertensive mice induced by angiotensin II (490 ng/kg/min, 2 weeks). The APCs were loaded with ovalbumin(OVA) or its MHC class I epitope SIINFEKL (SKL) for 3h. Then, splenocytes from OT-I mice, containing OVA-specific, CD8 + T cells (OT-I cells), were added for 4h. Significantly more activated T cells expressing surface marker CD69 and cytokine IFN-γ were detected by flow cytometry when stimulated with APCs from hypertensive mice than equivalent cells from normotensive mice (P<0.05). Thus, in vitro, APCs derived from hypertensive mice more effectively present antigen to T cells. To study antigen presentation in vivo , we immunized AngII and sham treated mice with OVA and adjuvant. Consistently more OVA-specific CD8 + T cells were induced in the blood (2.7 ± 0.23% vs. 1.2 ± 0.47%, P<0.05) and the spleen (1.51 ± 0.17% vs. 2.62 ± 0.31%, P<0.05) of AngII mice vs sham when measured by flow using an H-2k b -SKL tetramer. We also evaluated the APC and T cell activation signals before and after hypertension. Flow analysis showed that splenic DCs divided from AngII treated mice resulted in a substantially increased surface expression of co-stimulatory factor CD86 compare with DCs from normotensive controls (MFI: 131±7.8 vs. 97±5.3). RIP-mOVA mice were used to study if hypertension affects APC cross-presentation of self-antigens. This transgenic mouse line expresses membrane-bound OVA in pancreatic islet β cells. When OT-I cells (5x10 6 ) are injected into RIP-mOVA mice, they activate by cross-presentation of OVA and cause insulitis and diabetes. RIP-mOVA mice were made hypertensive with either AngII or L-NAME (1.5 mg/ml, 4 weeks). When OT-I cells were i.v. infused into RIP-mOVA mice, the hypertensive mice had more OT-I cells infiltration into the pancreas (AngII 75%, L-NAME 73%, control 13%) and developed severer diabetes (Blood glucose: AngII 331, L-NAME 315, control 168 mg/dl). In conclusion, high blood pressure itself is associated with over activity of the adaptive immune system.

Immunological Properties of Murine Parthenogenetic Stem Cells and Their Differentiation Products.

The perspective to transplant grafts derived from pluripotent stem cells has gained much attention in recent years. Parthenogenetic stem cells (PSCs) are an alternative pluripotent stem cell type that is attractive as source of grafts for allogeneic transplantations because most PSCs are haploidentical for the major histocompatibility complex (MHC). This reduced immunogenetic complexity of PSCs could tremendously simplify the search for MHC-matched allogeneic stem cells. In this study, we have characterized immunological properties of the MHC haploidentical PSC line A3 (H2d/d) and the heterologous PSC line A6 (H2b/d). Both PSC lines largely lack MHC class I molecules, which present peptides to cytotoxic T lymphocytes (CTLs) and serve as ligands for inhibitory natural killer (NK) receptors. They express ligands for activating NK receptors, including the NKG2D ligand RAE-1, and the DNAM-1 ligands CD112 and CD155. Consequently, both PSC lines are highly susceptible to killing by IL-2-activated NK cells. In vitro-differentiated cells acquire resistance and downregulate ligands for activating NK receptors but fail to upregulate MHC class I molecules. The PSC line A6 and differentiated A6 cells are largely resistant to CTLs derived from T cell receptor transgenic OT-I mice after pulsing of the targets with the appropriate peptide. The high susceptibility to killing by activated NK cells may constitute a general feature of pluripotent stem cells as it has been also found with other pluripotent stem cell types. This activity potentially increases the safety of transplantations, if grafts contain traces of undifferentiated cells that could be tumorigenic in the recipient.

180 The aorta can act as site of t cell priming and promotes a local cd4+ adaptive immune response in early stage atherosclerosis in apolipoprotein-e-/- mice

Artery tertiary lymphoid organs (ATLOs) in the adventitia adjacent to intimal plaque in aortas from aged apolipoprotein-E (apoE)-/- mice have recently been shown by us to orchestrate the aortic immune...

CD8+ memory T-cell inflation renders compromised CD4+ T-cell-dependent CD8+ T-cell immunity via naïve T-cell anergy.

Whether inflation of CD8+ memory T (mT) cells, which is often derived from repeated prime-boost vaccinations or chronic viral infections in the elderly, would affect late CD8+ T-cell immunity is a long-standing paradox. We have previously established an animal model with mT-cell inflation by transferring ConA-stimulated monoclonal CD8+ T cells derived from Ova-specific T-cell-receptor transgenic OTI mice into irradiation-induced lymphopenic B6 mice. In this study, we also established another two animal models with mT-cell inflation by transferring, 1) ConA-stimulated monoclonal CD8+ T cells derived from lymphocytic choriomeningitis virus glycoprotein-specific T-cell-receptor transgenic P14 mice, and 2) ConA-stimulated polyclonal CD8+ T cells derived from B6.1 mice into B6 mice with irradiation-induced lymphopenia. We vaccinated these mice with recombinant Ova-expressing Listeria monocytogenes and Ova-pulsed dendritic cells, which stimulated CD4+ T cell-independent and CD4+ T-cell-dependent CD8+ T-cell responses, respectively, and assessed Ova-specific CD8+ T-cell responses by flow cytometry. We found that Ova-specific CD8+ T-cell responses derived from the latter but not the former vaccination were significantly reduced in mice with CD8+ mT-cell inflation compared to wild-type B6 mice. We determined that naïve CD8+ T cells purified from splenocytes of mice with mT-cell inflation had defects in cell proliferation upon stimulation in vitro and in vivo and upregulated T-cell anergy-associated Itch and GRAIL molecules. Taken together, our data reveal that CD8+ mT-cell inflation renders compromised CD4+ T-cell-dependent CD8+ T-cell immunity via naïve T-cell anergy, and thus show promise for the design of efficient vaccines for elderly patients with CD8+ mT-cell inflation.

Enhancement of antigen-specific CD4+ and CD8+ T cell responses using a self-assembled biologic nanolipoprotein particle vaccine

To address the need for vaccine platforms that induce robust cell-mediated immunity, we investigated the potential of utilizing self-assembling biologic nanolipoprotein particles (NLPs) as an antigen and adjuvant delivery system to induce antigen-specific murine T cell responses. We utilized OT-I and OT-II TCR-transgenic mice to investigate the effects of NLP-mediated delivery of the model antigen ovalbumin (OVA) on T cell activation. Delivery of OVA with the TLR4 agonist monophosphoryl lipid A (MPLA) in the context of NLPs significantly enhanced the activation of both CD4+ and CD8+ T cells in vitro compared to co-administration of free OVA and MPLA. Upon intranasal immunization of mice harboring TCR-transgenic cells, NLPs enhanced the adjuvant effects of MPLA and the in vivo delivery of OVA, leading to significantly increased expansion of CD4+ and CD8+ T cells in lung-draining lymph nodes. Therefore, NLPs are a promising vaccine platform for inducing T cell responses following intranasal administration.

Role of ROCK2 in CD4+ cells in allergic airways responses in mice.

Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration. To determine the role of ROCK2 acting within CD4+ cells in allergic airways responses. ROCK2-haploinsufficient (ROCK2+/- ) and wild-type mice were sensitized with ovalbumin (OVA). ROCK2+/- mice then received either CD4+ cells from ROCK2-sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 h before challenge with aerosolized OVA. Wild-type mice received saline before challenge. Allergic airways responses were measured 48 h after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4+ cells (ROCK2CD4Cre mice) vs. control (CD4-Cre and ROCK2flox/flox ) mice. OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2+/- vs. wild-type mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2+/- mice, adoptive transfer with CD4+ cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wild-type levels, whereas eotaxin and airway hyperresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2-insufficient mice, allergic inflammation was not different in ROCK2CD4Cre vs. control mice. ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non-lymphocyte cells involved in lymphocyte activation and migration into the airways.

Restricted CD4+ T cell receptor repertoire impairs cognitive function via alteration of Th2 cytokine levels.

ABSTRACTDespite the effects of CD4+ T cell dysfunction on cognitive and behavioral impairment are well established, the effects of Th2 cytokines on the adult hippocampal neurogenesis and cognitive function in restricted CD4+ T cell receptor (TCR) repertoire model have not been fully elucidate. We found that mice with restricted CD4+ repertoire TCR showed decreased adult hippocampal neurogenesis using OT-II mice. Moreover, we demonstrated that OT-II mice showed increased Th2 cytokine levels in peripheral organs and IL-4 levels in brain. Taken together, altered Th2 cytokine levels may impact learning and memory via impaired adult neurogenesis in restricted CD4+ repertoire TCR mice.

Platelet-Targeted Gene Transfer Prevents Graft Rejection and Induces Immune Tolerance Even in a Primed Model

Induction of antigen-specific immune tolerance is desirable in autoimmune diseases, transplantation, gene therapy, and some protein therapies in order to prevent or reverse adverse immune responses. Our previous studies have demonstrated that targeting FVIII or FIX expression to platelets under control of the platelet-specific aIIb promoter can restore hemostasis and induce immune tolerance in hemophilia mice. Here we explored if this approach can be applied to prevent graft rejection and induce immune tolerance to a non-coagulant protein even with pre-existing immunity. We used ovalbumin (OVA) as a non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the aIIb promoter (2bOVA). We designed another vector, 2bVpOVA, which includes the VWF propeptide (Vp) cassette to secure OVA storage in platelet granules. The third construct, 2bGFP, in which the GFP cassette is driven by the same promoter, was used as a control vector.We confirmed that 2bOVA or 2bVpOVA lentiviral gene delivery to hematopoietic stem cells (HSCs) can result in OVA expression with greater than 95% of OVA stored in platelet a-granules. Under a non-myeloablative conditioning 6.6Gy total body irradiation (TBI), platelet-OVA expression levels were 24.22±8.72 ng/108 platelets and 1.41±0.73 ng/108 platelets in 2bOVA and 2bVpOVA transduced wild type (WT) recipients, respectively. When recipients were immunized with OVA, anti-OVA antibody titers in both the 2bOVA (560±68, n=10) and the 2bVpOVA group (320±34, n=10) were significantly lower than in un-transduced controls (10424±2837, n=24), demonstrating that platelet-specific OVA gene delivery to HSCs can suppress the anti-OVA immune response. To explore whether platelet targeted gene transfer can be applied to prevent graft rejection, skin grafts from Act-mOVA transgenic mice, in which OVA is expressed on the cell surfaces of all organs, were transplanted onto transduced recipients. Full thickness tail skin successfully grafted onto OVA-immunized 2bOVA- or 2bVpOVA-transduced recipients and was sustained for the rest of the animals’ lives or during the study course for up to 6 months. In contrast, skin grafts were rejected in OVA-immunized untransduced WT and 2bGFP-transduced animals within 6 weeks.To explore how immune suppression is established after platelet-specific gene transfer, we transduced HSCs from OVA-specific TCR transgenic (OTII/CD45.2) mice with 2bOVA, 2bVpOVA, or 2bGFP and transplanted into CD45.1/B6 WT recipients preconditioned with 6.6Gy TBI. After BM reconstitution, the engraftments among the 3 groups were similar (85.7±4.3%, 84.9±3.9%, and 86.5±2.9%, respectively), but donor-derived CD45.2+CD4+ T cells in the 2bOVA (0.2±0.1%, n=10) and 2bVPOVA (0.8±0.3%, n=11) groups were significantly lower than in the 2bGFP group (2.6±0.5%, n=11) in peripheral blood. Similarly, donor-derived CD45.2+CD4+ T cells in both spleen and lymph nodes were significantly lower in the 2bOVA and the 2bVpOVA groups compared to the 2bGFP group. However, there were no differences in the thymus among the 3 groups, indicating that central tolerance may not play a role in platelet-targeted gene therapy.To investigate whether platelet-targeted gene transfer can still induce the immune tolerance when immune system is primed, Sca-1+ cells from OTII mice were transduced with 2bOVA lentivirus and transplanted into OVA-primed CD45.1/B6 WT animals preconditioned with 6.6Gy TBI. We found that the platelet-OVA expression level in the OVA-primed group after 2bOVA gene transfer was not significantly different from the unprimed group (26.47±4.47 vs. 29.56±6.29 ng/108 platelets). The engraftments were comparable among the primed, the unprimed, and the primed untransduced control groups. Similar to the unprimed model, OVA-specific CD4+ T cells in 2bOVA-transduced OVA-primed recipients were significantly lower than in the untransduced controls (0.23±0.07% vs. 3.98±0.61%). Importantly, the anti-OVA total IgG declined with time after 2bOVA gene transfer, even when the animals were rechallenged with OVA.In summary, our data demonstrate that platelet-targeted gene transfer can prevent graft rejection and induce immune tolerance even when the immune system is primed, suggesting that platelet gene therapy can be a promising approach to induce immune tolerance and prevent undesired immune responses. DisclosuresNo relevant conflicts of interest to declare.

Apurinic/Apyrimidinic Endonuclease 1/Redox Factor-1 (Ape1/Ref-1) Modulates Antigen Presenting Cell-mediated T Helper Cell Type 1 Responses

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ape1/Ref-1) is a multifunctional protein possessing DNA repair, redox control, and transcriptional regulatory activities. Although Ape1/Ref-1 plays multiple roles in the immune system, its functions in helper T (Th) cell activation and differentiation are largely unknown. In this study, the function of Ape1/Ref-1 in Th cell activation was analyzed using an Ape1/Ref-1 redox-specific inhibitor, E3330. When splenocytes from OT-II mice, which are ovalbumin (OVA)-specific T-cell receptor transgenic mice, were activated with OVA in the presence of E3330, the induction of IFN-γ-producing OT-II T cells was significantly increased. In contrast, E3330 did not enhance IFN-γ production from plate-bound anti-CD3 antibody-stimulated CD4+ T cells in the absence of antigen presenting cells (APCs). Furthermore, E3330-pretreated and OVA-pulsed APCs also enhanced the IFN-γ production from OT-II T cells. These results suggested that E3330 enhances Th1 responses by modifying APC function. E3330 did not alter the surface expression of MHC-II or the co-stimulatory molecules CD80 and CD86 on APCs. On the other hand, E3330 up-regulated the IL-12 p35 and p40 gene expression, and IL-12 surface retention, but decreased the IL-12 secretion from Toll-like receptor (TLR) ligand-stimulated APCs. These results were confirmed with Ape1/Ref-1 knockdown experiments. Taken together, our findings indicated that the suppression of Ape1/Ref-1 redox function leads to an increased cell surface retention of IL-12 and enhances Th1 responses. This is the first study to demonstrate that Ape1/Ref-1 modulates the IL-12 production and secretion from APCs and controls Th1 immune responses.

Abstract A070: The role of the centrosome in cytotoxic T cell function

Abstract Cytotoxic T lymphocytes (CTLs) are essential for productive immune responses and recent studies have demonstrated their strong clinical potential as immunotherapeutic agents against established tumors. Cytotoxic responses in CTLs are induced by the T cell receptor, which recognizes cognate peptide-major histocompatibility complex molecules on the surfaces of infected or transformed cells, which induces the formation the immunological synapse. Synapse formation is accompanied by a series of actin and microtubule remodeling events, the most striking of which is the dramatic reorientation of the centrosome to a position just beneath the center of the contact site. It has been proposed that lytic granules containing perforin and granzyme cluster around the centrosome in activated CTLs. This polarization toward the synapse promotes the selective fusion of these granules with the synaptic membrane, leading to directional release of their contents toward the target cell. In order to study more precisely how the centrosome influences secretory responses and how these contributions affect CTL activity, we have developed an approach using OT-I mice bearing a conditional allele of both p53 (p53flox) as well as SAS4 (SAS4flox), a scaffolding protein that is absolutely required for the formation and maintenance of the centrosome. OT-I SAS4−/−p53−/− cells were generated by transducing SAS4flox/floxp53flox/flox cells with a retrovirus expressing Cre. Using gamma-tubulin and pericentrin staining, we were able to show that SAS4 deletion effectively eliminates interphase centrosomes in CTLs. OT-I SAS4−/− p53−/− cells exhibited a marked defect in target cell killing after 24h, implying a crucial role of the centrosome in cytotoxic responses. This killing defect was accompanied by a decrease in both granzyme B and perforin production. IFN-gamma production and secretion were not altered by the loss of the centrosome. Importantly, TCR induced signaling was normal in these cells. In summary, we were able to show that centrosome deletion impairs target cell killing without altering their overall magnitude. Given the importance of centrosome polarization for lytic granules delivery, we can assume that the centrosome ablation selectively alters cytotoxic efficiency by disrupting the directional synaptic secretion. Citation Format: Fella Tamzalit, Ariella Kepecs, Hisham Bazzi, Kathryn Anderson, Morgan Huse. The role of the centrosome in cytotoxic T cell function [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A070.