Antibody-Mediated Depletion of Autoreactive T Lymphocytes through PD-1 Improves Disease Outcomes and Visualizes T Cell Activation in Experimental Autoimmune Encephalomyelitis.

Long-term therapeutic outcomes of multiple sclerosis (MS) remain hindered by the chronic nature of immune cell stimulation toward self-antigens. Development of novel methods to target and deplete autoreactive T lymphocytes remains an attractive target for therapeutics for MS. We developed a programmed cell death 1 (PD-1)-targeted radiolabeled mAb and assessed its ability to deplete activated PD-1+ T lymphocytes in vitro and its ability to reduce disease burden of the myelin oligodendrocyte glycoprotein 35-55 experimental autoimmune encephalomyelitis (EAE) model in C57BL/6 mice. We also investigated the upregulation of PD-1 on infiltrating lymphocytes in an animal model of MS. Finally, we demonstrate the (to our knowledge) first reported positron-emission tomography/computed tomography imaging of activated PD-1+ cells in the EAE animal model of MS. We found that the 177Lu radioisotope-labeled anti-PD-1 mAb demonstrated significant in vitro cytotoxicity toward activated CD4+PD-1+ T lymphocytes and led to significant reduction in overall disease progression in the EAE animal model. Our results show high expression of PD-1 on infiltrating lymphocytes in the spinal cords of EAE diseased animals. Positron-emission tomography/computed tomography imaging of the anti-PD-1 mAb demonstrated significant uptake in the cervical draining lymph nodes highlighting accumulation of activated lymphocytes. Targeted depletion of T lymphocytes using T cell activation markers such as PD-1 may present a novel method to reduce autoimmune attack and inflammation in autoimmune diseases such as MS. Development of multimodal nuclear theranostic agents may present the opportunity to monitor T cell activation via imaging radioisotopes and simultaneously treat MS using therapeutic radioisotopes.

Deficiency of leukocyte-specific protein 1 (LSP1) alleviates asthmatic inflammation in a mouse model.

BACKGROUND: Asthma is a major cause of morbidity and mortality in humans. The mechanisms of asthma are still not fully understood. Leukocyte-specific protein-1 (LSP-1) regulates neutrophil migration during acute lung inflammation. However, its role in asthma remains unknown. METHODS: An OVA-induced mouse asthma model in LSP1-deficient (Lsp1-/-) and wild-type (WT) 129/SvJ mice were used to test the hypothesis that the absence of LSP1 would inhibit airway hyperresponsiveness and lung inflammation. RESULTS: Light and electron microscopic immunocytochemistry and Western blotting showed that, compared with normal healthy lungs, the levels of LSP1 were increased in lungs of OVA-asthmatic mice. Compared to Lsp1-/- OVA mice, WT OVA mice had higher levels of leukocytes in broncho-alveolar lavage fluid and in the lung tissues (P < 0.05). The levels of OVA-specific IgE but not IgA and IgG1 in the serum of WT OVA mice was higher than that of Lsp1-/- OVA mice (P < 0.05). Deficiency of LSP1 significantly reduced the levels of IL-4, IL-5, IL-6, IL-13, and CXCL1 (P < 0.05) but not total proteins in broncho-alveolar lavage fluid in asthmatic mice. The airway hyper-responsiveness to methacholine in Lsp1-/- OVA mice was improved compared to WT OVA mice (P < 0.05). Histology revealed more inflammation (inflammatory cells, and airway and blood vessel wall thickening) in the lungs of WT OVA mice than in those of Lsp1-/- OVA mice. Finally, immunohistology showed localization of LSP1 protein in normal and asthmatic human lungs especially associated with the vascular endothelium and neutrophils. CONCLUSION: These data show that LSP1 deficiency reduces airway hyper-responsiveness and lung inflammation, including leukocyte recruitment and cytokine expression, in a mouse model of asthma.

CD40 signaling augments IL-10 expression and the tolerogenicity of IL-10-induced regulatory dendritic cells.

CD40 expressed on stimulatory dendritic cells (DC) provides an important accessory signal for induction of effector T cell responses. It is also expressed at lower levels on regulatory DC (DCreg), but there is little evidence that CD40 signaling contributes to the tolerogenic activity of these cells. Indeed, CD40 silencing within DCreg has been reported to induce T cell tolerance in multiple disease models, suggesting that CD40 is superfluous to DC-induced tolerance. We critically assessed whether CD40 does have a role in tolerance induced by IL-10-differentiated DC (DC10) by using DC10 generating from the bone marrow of wild-type (w.t.) or CD40-/- donor mice, or IL-10-complemented CD40-/- DC10 to treat asthmatic mice. Wild-type DC10 ablated the OVA-asthma phenotype via induction of Foxp3+ Treg responses, but CD40-/- DC10 had no discernible effects on primary facets of the phenotype (e.g., IL-5, IL-9, IL-13 levels, IgE & IgG1 antibodies; p>0.05) and were ≤40% effective in reversal of others. Foxp3+ T cells from the lungs of CD40-/- DC10-treated mice expressed reduced levels of a panel of six Treg-specific activation markers relative to Treg from w.t. DC10-treated mice. Coculture with effector T cells from asthmatic mice induced a marked upregulation of cell surface CD40 on w.t. DC10. While untreated CD40-/- and w.t. DC10 secreted equally low levels of IL-10, stimulation of w.t. DC10 with anti-CD40 for 72 h increased their expression of IL-10 by ≈250%, with no parallel induction of IL-12. Complementing IL-10 expression in CD40-/- DC10 by IL-10 mRNA transfection fully restored the cells' abilities to suppress the asthma phenotype. In summary, CD40 signaling in DC10 contributes importantly to their expression of IL-10 and to a robust induction of tolerance, including activation of induced Treg.

225Ac-labeled CD33-targeting antibody reverses resistance to Bcl-2 inhibitor venetoclax in acute myeloid leukemia models.

PURPOSE: Despite the availability of new drugs, many patients with acute myeloid leukemia (AML) do not achieve remission and outcomes remain poor. Venetoclax is a promising new therapy approved for use in combination with a hypomethylating agent or with low-dose cytarabine for the treatment of newly diagnosed older AML patients or those ineligible for intensive chemotherapy. 225 Actinium-lintuzumab (225 Ac-lintuzumab) is a clinical stage radioimmunotherapy targeting CD33 that has shown evidence of single-agent activity in relapsed/refractory AML. Increased expression of MCL-1 is a mediator of resistance to venetoclax in cancer. EXPERIMENTAL DESIGN: Here we investigated the potential for 225 Ac-lintuzumab-directed DNA damage to suppress MCL-1 levels as a possible mechanism of reversing resistance to venetoclax in two preclinical in vivo models of AML. RESULTS: We demonstrated that 225 Ac-lintuzumab in combination with venetoclax induced a synergistic increase in tumor cell killing compared to treatment with either drug alone in venetoclax-resistant AML cell lines through both an induction of double-stranded DNA breaks (DSBs) and depletion of MCL-1 protein levels. Further, this combination led to significant tumor growth control and prolonged survival benefit in venetoclax-resistant in vivo AML models. CONCLUSIONS: There results suggest that the combination of 225 Ac-lintuzumab with venetoclax is a promising therapeutic strategy for the treatment of patients with venetoclax-resistant AML. Clinical trial of this combination therapy (NCT03867682) is currently ongoing.

Targeted lymphodepletion with a CD45-directed antibody radioconjugate as a novel conditioning regimen prior to adoptive cell therapy.

Chimeric antigen receptor (CAR) T cell therapies, and adoptive cell therapy (ACT) in general, represent one of the most promising anti-cancer strategies. Conditioning has been shown to improve the immune homeostatic environment to enable successful ACT or CAR-T engraftment and expansion in vivo following infusion, and represents potential point of intervention to decrease serious toxicities following CAR-T treatment. In contrast to relatively non-specific chemotherapy-derived lymphodepletion, targeted lymphodepletion with radioimmunotherapy (RIT) directed to CD45 may be a safer and more effective alternative to target and deplete immune cells. Here we describe the results of preclinical studies with an anti-mouse CD45 antibody 30F11, labeled with two different beta-emitters 131Iodine (131I) and 177Lutetium (177Lu), to investigate the effect of anti-CD45 RIT lymphodepletion on immune cell types and on tumor control in a model of adoptive cell therapy. Treatment of mice with 3.7 MBq 131I-30F11 or 1.48 MBq 177Lu-30F11 safely depleted immune cells such as spleen CD4+ and CD8+ T Cells, B and NK cells as well as Tregs in OT I tumor model while sparing RBC and platelets and enabled E. G7 tumor control. Our results support the application of CD45-targeted RIT lymphodepletion with a non-myeloablative dose of 131I-30F11 or 177Lu-30F11 antibody prior to adoptive cell therapy.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors.

Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

Detection and targeting insulin growth factor receptor type 2 (IGF2R) in osteosarcoma PDX in mouse models and in canine osteosarcoma tumors.

Osteosarcoma (OS) represents 3.4% of all childhood cancers with overall survival of 70% not improving in 30 years. The consistent surface overexpression of insulin-like growth factor-2 receptor (IGF2R) has been reported in commercial and patient-derived xenograft (PDX) OS cell lines. We aimed to assess efficacy and safety of treating PDX and commercial OS tumors in mice with radiolabeled antibody to IGF2R and to investigate IGF2R expression on canine OS tumors. IGF2R expression on human commercial lines 143B and SaOS2 and PDX lines OS-17, OS-33 and OS-31 was evaluated by FACS. The biodistribution and microSPECT/CT imaging with 111Indium-2G11 mAb was performed in 143B and OS-17 tumor-bearing SCID mice and followed by radioimmunotherapy (RIT) with 177Lutetium-2G11 and safety evaluation. IGF2R expression in randomly selected canine OS tumors was measured by immunohistochemistry. All OS cell lines expressed IGF2R. Biodistribution and microSPECT/CT revealed selective uptake of 2G11 mAb in 143B and OS-17 xenografts. RIT significantly slowed down the growth of OS-17 and 143B tumors without local and systemic toxicity. Canine OS tumors expressed IGF2R. This study demonstrates the feasibility of targeting IGF2R on OS in PDX and spontaneous canine tumors and sets the stage for further development of RIT of OS using comparative oncology.

Prospective Analysis of the Effects of Maternal Immune Activation on Rat Cytokines during Pregnancy and Behavior of the Male Offspring Relevant to Schizophrenia.

Influenza during pregnancy is associated with the development of psychopathology in the offspring. We sought to determine whether maternal cytokines produced following administration of viral mimetic polyinosinic-polycytidylic acid (polyI:C) to pregnant rats were predictive of behavioral abnormalities in the adult offspring. Timed-pregnant Sprague Dawley rats received a single intravenous injection of 4-mg/kg polyI:C or saline on gestational day (GD)15. Blood was collected 3 h later for serum analysis of cytokine levels with ELISA. Male offspring were tested in a battery of behavioral tests during adulthood and behavior was correlated with maternal cytokine levels. Maternal serum levels of CXCL1 and interleukin (IL)-6, but not tumor necrosis factor (TNF)-α or CXCL2, were elevated in polyI:C-treated dams. PolyI:C-treated dams experienced post-treatment weight loss and polyI:C pups were smaller than controls at postnatal day (PND)1. Various behavior alterations were seen in the polyI:C-treated offspring. Male polyI:C offspring had enhanced MK-801-induced locomotion, and reduced sociability. PolyI:C offspring failed to display crossmodal and visual memory, and oddity preference was also impaired. Set-shifting, assessed with a lever-based operant conditioning task, was facilitated while touchscreen-based reversal learning was impaired. Correlations were found between maternal serum concentrations of CXCL1, acute maternal temperature and body weight changes, neonatal pup mass, and odd object discrimination and social behavior. Overall, while the offspring of polyI:C-treated rats displayed behavior abnormalities, maternal serum cytokines were not related to the long-term behavior changes in the offspring. Maternal sickness effects and neonatal pup size may be better indicators of later effects of maternal inflammation in the offspring.

Contributions of direct versus indirect mechanisms for regulatory dendritic cell suppression of asthmatic allergen-specific IgG1 antibody responses.

IL-10-differentiated dendritic cells (DC10) can reverse the asthma phenotype in mice, but how they suppress the asthmatic B cell response is unclear. Herein we assessed the mechanism(s) by which DC10 and DC10-induced Treg affect IgG1 production in asthma. We observed a rapid decline in lung-resident OVA-specific IgG1-secreting B cells on cessation of airway allergen challenge, and intraperitoneal DC10 therapy did not amplify that (p>0.05). It did however increase the loss of IgG1-B cells from the bone marrow (by 45+/-7.2%; p≤0.01) and spleen (by 65+/-17.8%; p≤0.05) over 2 wk. Delivery of OVA-loaded DC10 directly into the airways of asthmatic mice decreased the lung IgG1 B cell response assessed 2 dy later by 33+/-9.7% (p≤0.01), while their co-culture with asthmatic lung cell suspensions reduced the numbers of IgG1-secreting cells by 56.5+/-9.7% (p≤0.01). This effect was dependent on the DC10 carrying intact allergen on their cell surface; DC10 that had phagocytosed and fully processed their allergen were unable to suppress B cell responses, although they did suppress asthmatic Th2 cell responses. We had shown that therapeutic delivery of DC10-induced Treg can effectively suppress asthmatic T and B cell (IgE and IgG1) responses; herein CD4+ cells or Treg from the lungs of DC10-treated OVA-asthmatic mice suppressed in vitro B cell IgG1 production by 52.2+/-8.7% (p≤0.001) or 44.6+/-12.2% (p≤0.05), respectively, but delivery of DC10-induced Treg directly into the airways of asthmatic mice had no discernible impact over 2 dy on the numbers of lung IgG1-secreting cells (p≥0.05). In summary, DC10 treatment down-regulates OVA-specific B cell responses of asthmatic mice. While DC10 that carry intact allergen on their cell surface can dampen this response, DC10-induced Treg are critical for full realization of this outcome. This suggests that infectious tolerance is an essential element in regulatory DC control of the B cell response in allergic asthma.

Metformin inhibits the development, and promotes the resensitization, of treatment-resistant breast cancer.

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin's potential clinical use against MDR cancers.

Therapeutic reversal of food allergen sensitivity by mature retinoic acid-differentiated dendritic cell induction of LAG3+CD49b-Foxp3- regulatory T cells.

BACKGROUND: Anaphylaxis is a life-threatening condition for which we have limited therapeutic options. Although specific immunotherapy for food allergies is becoming more effective, it is still laborious and carries substantial risk of adverse events. On the other hand, regulatory dendritic cell (DC) therapy is effective in mouse models of allergic disease and has been shown to work with TH2 cells from atopic asthmatic patients. OBJECTIVE: We assessed whether DC immunotherapy could reverse food allergen sensitivity in mouse models to provide proof of concept relating to their use in the clinic. METHODS: We generated and characterized mature retinoic acid-skewed dendritic cells (DC-RAs) and assessed their abilities to reverse ovalbumin or peanut allergies in mouse models, as well as their operative mechanisms. RESULTS: DC-RAs displayed a mature yet tolerogenic phenotype, expressing IL-10, TGF-ß, IL-27, and aldehyde dehydrogenase 1A2 but not IL-12 or IL-35; IL-10 and TGF-ß together drove their suppression of TH2 cell proliferation. Delivery of specific allergen-presenting DC-RAs to half-maximally sensitized mice with ovalbumin or peanut allergy reduced anaphylactic responses to oral allergen challenge by 84% to 90%, as well as diarrhea, mast cell activation, and TH2 cytokine responses and serum allergen-specific IgE/IgG1 levels. DC-RA expression of IL-27 was important to their induction of CD25+ lymphocyte activation gene 3 (LAG3)+, CD49b-, forkhead box P3 (Foxp3)- regulatory T cells in vitro, such that ß subunit of IL-27 (Ebi)-/- (ie, IL-27-incompetent) DC-RAs were ineffective in inducing food allergen tolerance. CONCLUSION: Our data indicate that regulatory DC immunotherapy can be effective for food allergies and suggest that induction of Foxp3- regulatory T cells might be a useful strategy for tolerance induction in this context.

Rapamycin reduces fibroblast proliferation without causing quiescence and induces STAT5A/B-mediated cytokine production.

Rapamycin is a well-known inhibitor of the Target of Rapamycin (TOR) signaling cascade; however, the impact of this drug on global genome function and organization in normal primary cells is poorly understood. To explore this impact, we treated primary human foreskin fibroblasts with rapamycin and observed a decrease in cell proliferation without causing cell death. Upon rapamycin treatment chromosomes 18 and 10 were repositioned to a location similar to that of fibroblasts induced into quiescence by serum reduction. Although similar changes in positioning occurred, comparative transcriptome analyses demonstrated significant divergence in gene expression patterns between rapamycin-treated and quiescence-induced fibroblasts. Rapamycin treatment induced the upregulation of cytokine genes, including those from the Interleukin (IL)-6 signaling network, such as IL-8 and the Leukemia Inhibitory Factor (LIF), while quiescent fibroblasts demonstrated up-regulation of genes involved in the complement and coagulation cascade. In addition, genes significantly up-regulated by rapamycin treatment demonstrated increased promoter occupancy of the transcription factor Signal Transducer and Activator of Transcription 5A/B (STAT5A/B). In summary, we demonstrated that the treatment of fibroblasts with rapamycin decreased proliferation, caused chromosome territory repositioning and induced STAT5A/B-mediated changes in gene expression enriched for cytokines.

Behavioral alterations in rat offspring following maternal immune activation and ELR-CXC chemokine receptor antagonism during pregnancy: implications for neurodevelopmental psychiatric disorders.

Research suggests that maternal immune activation (MIA) during pregnancy increases the risk of neurodevelopmental disorders including schizophrenia and autism in the offspring. Current theories suggest that inflammatory mediators including cytokines and chemokines may underlie the increased risk of these disorders in humans. For example, elevated maternal interleukin-8 (IL-8) during pregnancy is associated with increased risk of schizophrenia in the offspring. Given this association, the present experiments examined ELR-CXC chemokines CXCL1 and CXCL2, rodent homologues of human IL-8, and activation of their receptors (CXCR1 and CXCR2) in an established rodent model of MIA. Pregnant Long Evans rats were treated with the viral mimetic polyinosinic-polycytidylic acid (polyI:C; 4 mg/kg, i.v.) on gestational day 15. Protein analysis using multiplex assays and ELISA showed that polyI:C significantly increased maternal serum concentrations of interleukin-1ß, tumor necrosis factor, and CXCL1 3h after administration. Subsequent experiments tested the role of elevated maternal CXCL1 on behavior of the offspring by administering a CXCR1/CXCR2 antagonist (G31P; 500 µg/kg, i.p.; 1h before, 48 and 96 h after polyI:C treatment). The male offspring of dams treated with polyI:C demonstrated subtle impairments in prepulse inhibition (PPI), impaired associative and crossmodal recognition memory, and altered behavioral flexibility in an operant test battery. While G31P did not completely reverse the behavioral impairments caused by polyI:C, it enhanced PPI during adolescence and strategy set-shifting and reversal learning during young adulthood. These results suggest that while polyI:C treatment significantly increases maternal CXCL1, elevations of this chemokine are not solely responsible for the effects of polyI:C on the behavior of the offspring.

Regulatory dendritic cells for immunotherapy in immunologic diseases.

We recognize well the abilities of dendritic cells to activate effector T cell (Teff cell) responses to an array of antigens and think of these cells in this context as pre-eminent antigen-presenting cells, but dendritic cells are also critical to the induction of immunologic tolerance. Herein, we review our knowledge on the different kinds of tolerogenic or regulatory dendritic cells that are present or can be induced in experimental settings and humans, how they operate, and the diseases in which they are effective, from allergic to autoimmune diseases and transplant tolerance. The primary conclusions that arise from these cumulative studies clearly indicate that the agent(s) used to induce the tolerogenic phenotype and the status of the dendritic cell at the time of induction influence not only the phenotype of the dendritic cell, but also that of the regulatory T cell responses that they in turn mobilize. For example, while many, if not most, types of induced regulatory dendritic cells lead CD4(+) naïve or Teff cells to adopt a CD25(+)Foxp3(+) Treg phenotype, exposure of Langerhans cells or dermal dendritic cells to vitamin D leads in one case to the downstream induction of CD25(+)Foxp3(+) regulatory T cell responses, while in the other to Foxp3(-) type 1 regulatory T cells (Tr1) responses. Similarly, exposure of human immature versus semi-mature dendritic cells to IL-10 leads to distinct regulatory T cell outcomes. Thus, it should be possible to shape our dendritic cell immunotherapy approaches for selective induction of different types of T cell tolerance or to simultaneously induce multiple types of regulatory T cell responses. This may prove to be an important option as we target diseases in different anatomic compartments or with divergent pathologies in the clinic. Finally, we provide an overview of the use and potential use of these cells clinically, highlighting their potential as tools in an array of settings.

Comparison of induced versus natural regulatory T cells of the same TCR specificity for induction of tolerance to an environmental antigen.

Recent evidence shows that natural CD25(+)Foxp3(+) regulatory T cells (nTreg) and induced CD25(+)Foxp3(+) regulatory T cells (iTreg) both contribute to tolerance in mouse models of colitis and asthma, but there is little evidence regarding their relative contributions to this tolerance. We compared the abilities of nTreg and iTreg, both from OVA-TCR-transgenic OTII mice, to mediate tolerance in OVA-asthmatic C57BL/6 mice. The iTreg were differentiated from Th2 effector T cells by exposure to IL-10-differentiated dendritic cells (DC10) in vitro or in vivo, whereas we purified nTreg from allergen-naive mice and exposed them to DC10 before use. Each Treg population was subsequently repurified and tested for its therapeutic efficacy in vitro and in vivo. DC10 engaged the nTreg in a cognate fashion in Forster (or fluorescence) resonance energy transfer assays, and these nTreg reduced in vitro OVA-asthmatic Th2 effector T cell responses by 41-56%, whereas the comparator iTreg reduced these responses by 72-86%. Neutralization of IL-10, but not TGF-ß, eliminated the suppressive activities of iTreg but not nTreg. Delivery of 5 × 10(5) purified nTreg reduced allergen challenge-induced airway IL-4 (p ≤ 0.03) and IL-5 (p ≤ 0.001) responses of asthmatic recipients by ≤ 23% but did not affect airway hyperresponsiveness or IgE levels, whereas equal numbers of iTreg of identical TCR specificity reduced all airway responses to allergen challenge by 82-96% (p ≤ 0.001) and fully normalized airway hyperresponsiveness. These data confirm that allergen-specific iTreg and nTreg have active roles in asthma tolerance and that iTreg are substantially more tolerogenic in this setting.

Induction of prolonged asthma tolerance by IL-10-differentiated dendritic cells: differential impact on airway hyperresponsiveness and the Th2 immunoinflammatory response.

IL-10-differentiated dendritic cells (DC10s) can prevent allergen sensitization and reverse the asthma phenotype in mice with established disease. However, little is known about the time-frames over which this tolerance is effective. We report that at 2 wk after i.p. or transtracheal delivery of 1 × 10(6) OVA-, but not house dust mite- presenting, DC10s to OVA-asthmatic mice, significant diminution of airway hyperresponsiveness (AHR) was first apparent, whereas AHR was abrogated between 3 and 10 wk posttreatment. At 13 wk, AHR returned to pretreatment levels but could again be reversed by DC10 retreatment. The impact of a single DC10 treatment on airway eosinophil and Th2 cytokine responses to recall OVA challenge, and on OVA-specific IgE/IgG1 responses, was substantial at 3 wk posttreatment, but progressively increased thereafter, such that at 8 mo, airway eosinophil and Th2 responses to recall allergen challenge remained ∼85-95% suppressed relative to saline-treated asthmatic mice. Four biweekly DC10 treatments, whether transtracheal or i.p., reduced all asthma parameters to near background by 8 wk, whereas s.c. DC10 treatments did not affect AHR but did reduce the airway Th2 responses (i.v. DC10 had no discernible effects). Repeated challenge of the DC10-treated mice with aerosolized OVA (100 µg/ml) did not reverse tolerance, but treatment with the indoleamine-2,3-dioxygenase antagonist 1-methyltryptophan or neutralizing anti-IL-10R from days 12 to 21 after DC10 therapy partially reversed tolerance (Th2 cytokine responses, but not AHR). These findings indicate that DC10-induced Th2 tolerance in asthmatic animals is long lived, but that DC10s employ distinct mechanisms to affect AHR versus Th2 immunoinflammatory parameters.

Mast cells and IgE activation do not alter the development of oral tolerance in a murine model.

BACKGROUND: In addition to their well-known role as potent effector cells in patients with allergic disease, mast cells have important immunomodulatory roles regulating tolerance in allograft rejection models. The roles of mast cells in oral tolerance development have not previously been examined. OBJECTIVE: We sought to evaluate the importance of mast cells, IgE-mediated mast cell activation, and histamine receptor 1 or 2 blockade on oral tolerance development in mice. METHODS: Oral tolerance was assessed in 2 mast cell-deficient murine strains (Kit(W-sh/W-sh) and Kit(W/W-v) mice) and control mice. Mice were fed ovalbumin (OVA) or peanut butter for 1 week and then immunized and boosted with relevant protein antigens. Antibody responses were assessed by using ELISA. The oral antihistamines pyrilamine and ranitidine were administered during tolerance induction to OVA. IgE-mediated mast cell activation was initiated during oral tolerance induction or OVA immunization. OVA-specific regulatory T cells were assessed in the Peyer patches, mesenteric lymph nodes, and spleens by using flow cytometry after adoptive transfer. RESULTS: Oral tolerance was successfully induced to OVA and peanut butter in mast cell-deficient mice. Kit(W-sh/W-sh) mice had higher proportions of antigen-specific regulatory T cells in the mesenteric lymph nodes than mast cell-containing control mice. However, mast cell reconstitution studies suggested this effect was mast cell independent. Oral antihistamine treatments with pyrilamine or ranitidine did not impair tolerance and neither did IgE-mediated activation. CONCLUSIONS: Mast cells are not necessary for the induction of oral tolerance, and allergic activation of mast cells does not impair tolerance to OVA. Oral antihistamine treatments do not disrupt the development of oral tolerance.

Tissue eosinophilia in a mouse model of colitis is highly dependent on TLR2 and independent of mast cells.

The mechanisms initiating eosinophil influx into sites of inflammation have been well studied in allergic disease but are poorly understood in other settings. This study examined the roles of TLR2 and mast cells in eosinophil accumulation during a nonallergic model of eosinophilia-associated colitis. TLR2-deficient mice (TLR2(-/-)) developed a more severe colitis than wild-type mice in the dextran sodium sulfate (DSS) model. However, they had significantly fewer eosinophils in the submucosa of the cecum (P < 0.01) and mid-colon (P < 0.01) than did wild-type mice after DSS treatment. Decreased eosinophilia in TLR2(-/-) mice was associated with lower levels of cecal CCL11 (P < 0.01). Peritoneal eosinophils did not express TLR2 protein, but TLR2 ligand injection into the peritoneal cavity induced local eosinophil recruitment, indicating that TLR2 activation of other cell types can mediate eosinophil recruitment. After DSS treatment, mast cell-deficient (Kit(W-sh/W-sh)) mice had similar levels of intestinal tissue eosinophilia were observed as those in wild-type mice. However, mast cell-deficient mice were partially protected from DSS-induced weight loss, an effect that was reversed by mast cell reconstitution. Overall, this study indicates a critical role for indirect TLR2-dependent pathways, but not mast cells, in the generation of eosinophilia in the large intestine during experimental colitis and has important implications for the regulation of eosinophils at mucosal inflammatory sites.

Tolerogenic dendritic cells induce CD4+CD25hiFoxp3+ regulatory T cell differentiation from CD4+CD25-/loFoxp3- effector T cells.

IL-10-differentiated dendritic cells (DC10) induce allergen tolerance in asthmatic mice, during which their lung Th2 effector T cells (Teffs) are displaced by activated CD4(+)CD25(hi)Foxp3(+) T cells. Intestinal DCs promote oral tolerance by inducing Ag-naive T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), but whether DCs can induce Teffs to differentiate into Tregs remains uncertain. In this study, we addressed this question in OVA-asthmatic mice that were treated with DC10. OVA-presenting DC10 treatment maximally activated lung Tregs in these animals at 3 wk posttreatment, as determined by upregulation of activation markers (ICOS, programmed cell death-1, glucocorticoid-induced TNFR-related protein, LAG3, and CTLA-4) and in functional assays. This in vitro regulatory activity was ≥90% reduced by treatment with anti-IL-10 but not anti-TGF-ß Abs. In parallel cultures, OVA- but not house dust mite (HDM)-presenting DC10 induced ≈43% of CFSE-labeled CD25(-/lo)Foxp3(-) Teffs from asthmatic OVA-TCR transgenic mice to differentiate into tolerogenic CD25(hi)Foxp3(+) Tregs. We recapitulated this in vivo using OVA-asthmatic mice that were coinjected with OVA- or HDM-presenting DC10 (i.p.) and CFSE-labeled CD4(+)CD25(-/lo)Foxp3(-) Teffs (i.v.) from the lungs of asthmatic DO11.10 mice. From ≈7 to 21% of the activated (i.e., dividing) DO11.10 Teffs that were recovered from the lungs, lung-draining lymph nodes, or spleens of the OVA-DC10 recipients had differentiated into CD4(+)CD25(hi)Foxp3(+) Tregs, whereas no CFSE-positive Tregs were recovered from the HDM-DC10-treated animals. These data indicate that DC10 treatments induce tolerance at least in part by inducing Teffs to differentiate into CD4(+)CD25(hi)Foxp3(+) Tregs.

Mast cells, histamine, and IL-6 regulate the selective influx of dendritic cell subsets into an inflamed lymph node.

In response to bacterial stimuli, multiple dendritic cell (DC) populations accumulate within the draining lymph node, thus enhancing opportunities for effective T cell-DC interaction. DC subpopulations, such as plasmacytoid, CD8(+), and CD11b(+) subsets, have distinct roles in determining the nature of the immune response. The mechanisms whereby individual DC subpopulations are mobilized and the extent to which these processes are linked to increases in overall lymph node cellularity have not been determined. In the current study, the mechanisms of DC subset mobilization to the draining auricular lymph node were examined after intradermal injection of Staphylococcus aureus-derived peptidoglycan. Using mast cell-deficient mice and local mast cell reconstitution, plasmacytoid and CD8(+) DC responses were shown to be mast cell dependent, whereas the CD11b(+) DC response was not. A histamine H2 receptor-dependent, CXCL9-independent pathway controlled the selective influx of both plasmacytoid and CD11b(+) DC into the lymph node, but not lymph node cellularity. In contrast, IL-6 was important for the mobilization of CD8(+) and CD11b(+) DC. TNF and IL-1 receptor were dispensable for plasmacytoid, CD11b(+), and CD8(+) DC responses. These findings provide novel opportunities for the selective mobilization of specific DC subsets to lymph nodes and demonstrate critical roles for both histamine and IL-6 in this process.