Treg-deficient Mice Research Articles

Regulatory T-cell deficiency leads to features of autoimmune liver disease overlap syndrome in scurfy mice.

Scurfy mice have a complete deficiency of functional regulatory T cells (Treg) due to a frameshift mutation in the Foxp3 gene. The impaired immune homeostasis results in a lethal lymphoproliferative disorder affecting multiple organs, including the liver. The autoimmune pathology in scurfy mice is in part accompanied by autoantibodies such as antinuclear antibodies (ANA). ANA are serological hallmarks of several autoimmune disorders including autoimmune liver diseases (AILD). However, the underlying pathogenesis and the role of Treg in AILD remain to be elucidated. The present study therefore aimed to characterize the liver disease in scurfy mice. Sera from scurfy mice were screened for ANA by indirect immunofluorescence assay (IFA) and tested for a wide range of AILD-associated autoantibodies by enzyme-linked immunosorbent assay, line immunoassay, and addressable laser bead immunoassay. CD4+ T cells of scurfy mice were transferred into T cell-deficient B6/nude mice. Monoclonal autoantibodies from scurfy mice and recipient B6/nude mice were tested for ANA by IFA. Liver tissue of scurfy mice was analyzed by conventional histology. Collagen deposition in scurfy liver was quantified via hepatic hydroxyproline content. Real-time quantitative PCR was used to determine fibrosis-related hepatic gene expression. Hepatic immune cells were differentiated by flow cytometry. All scurfy mice produced ANA. AILD-associated autoantibodies, predominantly antimitochondrial antibodies, were detected at significantly higher levels in scurfy sera. CD4+ T cells from scurfy mice were sufficient to induce anti-dsDNA autoantibodies and ANA with an AILD-related nuclear envelope staining pattern. Liver histology revealed portal inflammation with bile duct damage and proliferation, as in primary biliary cholangitis (PBC), and interface hepatitis with portal-parenchymal necroinflammation, as found in autoimmune hepatitis (AIH). In scurfy liver, TNFα and fibrosis-related transcripts including Col1a1, Timp1, Acta2, Mmp2, and Mmp9 were upregulated. The level of proinflammatory monocytic macrophages (Ly-6Chi) was increased, while M2-type macrophages (CD206+) were downregulated compared to wildtype controls. Despite severe hepatic inflammation, fibrosis did not develop within 25 days, which is close to the lifespan of scurfy mice. Our findings suggest that Treg-deficient scurfy mice spontaneously develop clinical, serological, and immunopathological characteristics of AILD with overlapping features of PBC and AIH.

Gut regulatory T cells mediate immunological tolerance in Salmonella-infected super-spreader hosts.

Abstract Salmonella Typhimurium is an enteric pathogen that causes gastroenteritis in humans and Typhoid fever-like symptoms in mice. Our lab has shown that oral infection of 129X1/SvJ mice with Salmonella Typhimurium results in ~25% of mice becoming persistent high-shedders (>10 8CFU/g feces). Because these mice can transmit infection to a naïve host, we refer to them as super-spreaders (SS). Unexpectedly, SS mice are asymptomatic despite a persistent high-pathogen burden in the gut. We hypothesized that SS mice are disease resistant due to a gut-specific immune state that facilitates homeostasis between host and pathogen. To examine this, we performed extensive immuno-phenotyping of SS mice, which revealed a distinct type-1 immune state in the gut. We found that IFNγ producing effector CD4 T cells were the dominant population to accumulate in SS guts. Additionally, a population of Tbet+ Tregs were found only in the gut of SS mice, suggesting these cells may enable the SS asymptomatic state. Accordingly, specific ablation of Tregs resulted in a dramatic weight loss, gut inflammation, and a compromised epithelial barrier in SS mice. Bacterial burden was unchanged in Treg depleted mice, demonstrating that morbidity was not caused by Salmonella burden but likely due to immune pathology. Indeed, depletion of effector CD4+ T cells in Treg-deficient SS mice reversed this morbidity, confirming the disease state was directly caused by immune pathology. These results demonstrate that in SS mice Tregs play an essential tolerogenic role maintaining intestinal homeostasis. We are currently investigating the immunological mechanisms triggering this effect and whether this new immune-adapted state may impact infections with other enteric pathogens.

006 Pathogenic autoantibody derived from Treg-deficient scurfy mice targets Type VII Collagen and induces Epidermolysis bullosa acquisita-like blistering disease

Missing regulatory T cell (Treg) control contributes to the development of different autoimmune diseases. Scurfy mice have a missense mutation in the transcription factor Foxp3 which leads to dysfunctional Tregs. Previously, we have shown that scurfy mice develop high titers of autoantibodies with reactivity to structural proteins in the skin. Furthermore, the development of a pathogenic autoantibody that targets BP230 and induces a bullous pemphigoid-like phenotype indicates the progression of autoimmune blistering diseases (AIBD) in the absence of functional Tregs.

Tregs facilitate obesity and insulin resistance via a Blimp-1-IL-10 axis

Abstract Adipose-resident Tregs protect against systemic inflammation and metabolic disease by limiting expansion of pro-inflammatory cells, preserving insulin sensitivity and maintaining glucose tolerance. Although their basic markers and roles have been studied, less is known about the transcriptional machinery regulating their differentiation and function. B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional regulator known to be involved in development, polarization, and maintenance of various immune cells including CD4+ T cells. Using Blimp-1 reporter mice, we discovered that Blimp-1 is constitutively expressed in a subset of visceral adipose tissue (VAT) Tregs, and that Blimp-1+ VAT Tregs are phenotypically distinct from their Blimp-1-counterparts. We also found that Treg-specific Blimp-1 deletion led to altered differentiation and function of VAT and inguinal adipose tissue Tregs. Surprisingly, during diet-induced obesity, Blimp-1 Treg deficient mice gained less weight, had reduced body fat percentage, and exhibited improved insulin sensitivity compared to wild type mice. Furthermore, this was accompanied by upregulation of thermogenic genes such as Ucp1, Prdm16 and Dio2 in inguinal adipose tissue, and increased overall fatty acid oxidation. It has previously been shown that IL-10 can induce thermogenesis. Therefore, we repeated these experiments utilizing mice with Treg-specific deletion of IL-10 and found that they phenocopied the Blimp-1 Treg deficient mice. Based on these results, we hypothesize that cross-talk between Tregs and adipocytes via a Blimp-1-IL-10 axis suppresses thermogenesis, and that absence of Blimp-1+ Tregs is metabolically protective during diet-induced obesity.

FoxP3 deficiency causes no inflammation or neurodegeneration in the murine brain

Regulatory T cells (Treg) maintain immunological self-tolerance and their functional or numerical deficits are associated with progression of several neurological diseases. We examined the effects of Treg absence on the structure and integrity of the unchallenged murine brain. When compared to control, Treg-deficient FoxP3sf mutant mice showed no differences in brain size, myelin amount and oligodendrocyte numbers. FoxP3sf strain displayed no variations in quantity of neurons and astrocytes, whereas microglia numbers were slightly reduced. We demonstrate lack of neuroinflammation and parenchymal responses in the brains of Treg-deficient mice, suggesting a minor Treg role in absence of blood-brain barrier breakdown.

The Lymphoma Tumor Microenvironment Influences Toxicity after CD19 CAR T Cell Therapy

Introduction: Of patients receiving CD19 CAR T cell therapy for large B cell lymphoma (LBCL), approximately 1 in 10 experience severe cytokine release syndrome (CRS) and 1 in 3 experience severe neurotoxicity. While CAR T cells trigger the onset of these toxicities, CRS and neurotoxicity are thought to occur as a consequence of activated myeloid cells amplifying cytokine and catecholamine release, thereby stimulating inflammation both systemically and at the blood-brain barrier. However, patient and tumor-related factors that account for differences in the amount of toxicity remain poorly understood. Methods: Serum cytokine levels were measured on an ELLA point of care device prior to lymphodepleting chemotherapy and throughout inpatient treatment with CD19 CAR T cell therapy (axicabtagene ciloleucel) for LBCL. Catecholamine levels were measured as we have previously reported. Tumor biopsies were taken within 1 month prior to infusion of CAR T cells. RNA expression was measured by RNAseq and/or a Nanostring IO360 panel consisting of 770 genes found in the tumor microenvironment (TME) in cancer. Analysis used nSolver to identify cell types, GSEA and differential gene expression between groups. Mouse CAR T cell studies utilized mouse CD19-targeted CAR T cells derived from C57BL/6 splenocytes and cultured in vitro with myeloid cells and target cells to evaluate cytotoxicity and/or cytokine secretion. Elicited mouse macrophages were collected from peritoneal fluid 4 days after IP injection of 3% Brewer's thioglycollate medium. In vivo studies with mouse CD19-targeted CAR T cells were performed in IL2Ra-/- mice given cyclophosphamide as a pre-conditioning chemotherapy followed by adoptive transfer and analyses for CAR T cell and B cell persistence, as well as cytokines. Results: Of 58 patients undergoing CD19 CAR T cell therapy for LBCL, 8 (14%) had severe (grade 3 or higher) CRS and 16 (28%) had severe (grade 3 or higher) neurotoxicity. At baseline, peripheral blood levels of IL-6, IFN-γ, IL-15 and ferritin were significantly higher in patients who would subsequently experience severe CRS and severe neurotoxicity. Confirming our recent animal model of CRS we determined that peak serum catecholamine levels were higher in patients experiencing severe CRS. To identify if myeloid cells potentiate cytokine release we co-cultured CAR T cells with CD19 target and macrophages obtained from elicited mouse peritoneum. When these macrophages were added, IL-6 release from CAR T cells significantly increased compared to when macrophages were absent. Next, we studied the baseline TME in LBCL CAR T patients. Of 36 patients, 10 (27%) experienced severe neurotoxicity following CAR T cell therapy. By cell type score, the severe neurotoxicity group had a lower expression of genes associated with T cells overall and specifically Tregs. Also significantly lower in the severe neurotoxicity group were T cell genes including multiple subunits of CD3, CD3ζ, FOXP3, ICOS, CD62L and others. Association of increased T cell infiltration in the TME with low neurotoxicity raised the possibility that suppressive T cell subsets play a role in limiting toxicity post-CAR T cell therapy. To test this hypothesis, we injected CD19-targeted CAR T cells into an immune competent mouse model of Treg depletion (IL2Ra-/-) with established CD19+ leukemia. Treg deficient mice experienced a massive cytokine release after CAR T infusion and died prematurely due to CAR T toxicity compared to control mice with Tregs intact. Conclusions: Our observations suggest that the incidence of severe toxicity following CD19 CAR T cell therapy is influenced by baseline characteristics that are present prior to the infusion of CAR T cells. These include systemic inflammation characterized by high cytokine levels and a TME notable for a lack of infiltrating T cells. We posit a model whereby inflammation primes myeloid cells that are further activated upon CAR T cell infusion to release toxic amounts of cytokines and catecholamines. T cell subsets in the TME may modulate CAR T cells at the site of antigen encounter and prevent excessive CAR T activation. Reducing systemic inflammation or encouraging T cell infiltration into tumor prior to CAR T infusion are potential strategies for lowering the toxicity associated with CAR T therapy. Disclosures Jain: Kite/Gilead: Consultancy. Chavez:Kite Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Genentech: Speakers Bureau; Janssen Pharmaceuticals, Inc.: Speakers Bureau. Shah:Novartis: Honoraria; Spectrum/Astrotech: Honoraria; Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; AstraZeneca: Honoraria. Bachmeier:Kite/Gilead: Speakers Bureau. Mullinax:Iovance: Research Funding. Locke:Novartis: Other: Scientific Advisor; Cellular BioMedicine Group Inc.: Consultancy; Kite: Other: Scientific Advisor. Davila:Anixa: Consultancy; Precision Biosciences: Consultancy; Novartis: Research Funding; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Celgene: Research Funding; Atara: Research Funding; Bellicum: Consultancy.

Antibiotic-modulated microbiome suppresses lethal inflammation and prolongs lifespan in Treg-deficient mice

BackgroundRegulatory T cell (Treg) deficiency leads to IPEX syndrome, a lethal autoimmune disease, in Human and mice. Dysbiosis of the gut microbiota in Treg-deficient scurfy (SF) mice has been described, but to date, the role of the gut microbiota remains to be determined.ResultsTo examine how antibiotic-modified microbiota can inhibit Treg deficiency-induced lethal inflammation in SF mice, Treg-deficient SF mice were treated with three different antibiotics. Different antibiotics resulted in distinct microbiota and metabolome changes and led to varied efficacy in prolonging lifespan and reducing inflammation in the liver and lung. Moreover, antibiotics altered plasma levels of several cytokines, especially IL-6. By analyzing gut microbiota and metabolome, we determined the microbial and metabolomic signatures which were associated with the antibiotics. Remarkably, antibiotic treatments restored the levels of several primary and secondary bile acids, which significantly reduced IL-6 expression in RAW macrophages in vitro. IL-6 blockade prolonged lifespan and inhibited inflammation in the liver and lung. By using IL-6 knockout mice, we further identified that IL-6 deletion provided a significant portion of the protection against inflammation induced by Treg dysfunction.ConclusionOur results show that three antibiotics differentially prolong survival and inhibit lethal inflammation in association with a microbiota—IL-6 axis. This pathway presents a potential avenue for treating Treg deficiency-mediated autoimmune disorders.

Dormant pathogenic CD4+ T cells are prevalent in the peripheral repertoire of healthy mice

Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3− T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3− cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including self-peptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage.

Noc4L-Mediated Ribosome Biogenesis Controls Activation of Regulatory and Conventional T Cells

Regulatory Tcell (Treg) activation is crucial for maintaining self-tolerance, but the translational regulation of this process is still poorly understood. Although ribosome biogenesis is considered a housekeeping process, emerging evidence supports the hypothesis that ribosome biogenesis can selectively regulate protein synthesis by tuning translation. Here, we focused on the ribosome biogenesis factor Noc4L, based on the observations that Noc4L is highly expressed in activated Tregs. Conditional Noc4L knockout in Tregs resulted in a lethal autoimmune phenotype resembling Treg-deficient scurfy mice. Interestingly, the Noc4L defect did not globally affect overall protein translation in Tregs but was selectively detrimental to the expression of mRNAs related to Treg activation. These results demonstrate the critical role of Noc4L-mediated ribosome biogenesis in controlling the activation of Tregs and maintaining immune tolerance.

Noc4L Dependent Ribosome Biogenesis Controls Treg Activation

Tregs activation is crucial for maintaining self-tolerance, but the translational regulation of this process is still poorly understood. Although ribosome biogenesis is considered a housekeeping process, emerging evidence supports a new hypothesis that different types of cells may actually contain distinct repertoires of ribosome biogenesis factors that generate specialized ribosomes and confer the translation of selected mRNAs. Here, we focused on the ribosome biogenesis factor Noc4L, based on the observations that Noc4L is highly expressed in activated Treg cells. Conditional Noc4L knockout in Tregs resulted in a lethal autoimmune phenotype, resembling Treg deficient scurfy mice. Interestingly, the Noc4L defect did not globally affect the overall protein translation in Tregs but rather was selectively detrimental to the expression of genes related to Treg activation. These results demonstrate the critical role of Noc4L dependent ribosome biogenesis in controlling the activation of Tregs and maintaining immune tolerance.

A role for the nonclassical MHC molecule DO in Treg selection and function

Abstract Selection of CD4 T cells into the conventional and regulatory lineages is thought to be dependent on the strength of interaction of rearranged T cell receptors with MHCII-bound peptides displayed on APCs in the thymus. The nonclassical MHC molecule DO inhibits DM-mediated peptide exchange and has been shown to play a role in selection of peptides for presentation to CD4 T cells. In mice deficient in DO (H2-O), alterations in T cell responses and in antigen presentation as well as increased autoimmunity have been described. We show that DO−/− mice have increased numbers of thymic and peripheral Tregs, which also display a more activated phenotype. These DO−/− Tregs additionally exert greater suppressive capacity in vivo as compared to WT Tregs, as demonstrated by reduced T cell activation following Treg transfer into Treg-deficient mice. T cell receptor Vβ chain sequencing indicates the Treg population is less diverse and the conventional CD4 T cell population more diverse in DO−/− mice, suggesting skewing of thymic selection as a result of DO expression. To understand the mechanism behind altered T cell selection due to DO, we eluted peptides from thymic and B cell-derived I-Ab and characterized them by tandem mass spectrometry. Peptide diversity was increased in both cell types isolated from WT as compared to DO−/− mice. We speculate that through control of peptide diversity, DO tunes the number and specificity of thymocytes selected into the Treg lineage. These results highlight a previously unappreciated role for DO in controlling Treg selection and function.

Globule Leukocytes and Other Mast Cells in the Mouse Intestine.

Only 2 major mast cell (MC) subtypes are commonly recognized in the mouse: the large connective tissue mast cells (CTMCs) and the mucosal mast cells (MMCs). Interepithelial mucosal inflammatory cells, most commonly identified as globule leukocytes (GLs), represent a third MC subtype in mice, which we term interepithelial mucosal mast cells (ieMMCs). This term clearly distinguishes ieMMCs from lamina proprial MMCs (lpMMCs) while clearly communicating their common MC lineage. Both lpMMCs and ieMMCs are rare in normal mouse intestinal mucosa, but increased numbers of ieMMCs are seen as part of type 2 immune responses to intestinal helminth infections and in food allergies. Interestingly, we found that increased ieMMCs were consistently associated with decreased mucosal inflammation and damage, suggesting that they might have a role in controlling helminth-induced immunopathology. We also found that ieMMC hyperplasia can develop in the absence of helminth infections, for example, in Treg-deficient mice, Arf null mice, some nude mice, and certain graft-vs-host responses. Since tuft cell hyperplasia plays a critical role in type 2 immune responses to intestinal helminths, we looked for (but did not find) any direct relationship between ieMMC and tuft cell numbers in the intestinal mucosa. Much remains to be learned about the differing functions of ieMMCs and lpMMCs in the intestinal mucosa, but an essential step in deciphering their roles in mucosal immune responses will be to apply immunohistochemistry methods to consistently and accurately identify them in tissue sections.

Regulatory T cells promote myelin regeneration in the central nervous system.

Regeneration of central nervous system (CNS) myelin involves differentiation of oligodendrocytes from oligodendrocyte progenitor cells (OPC). In multiple sclerosis (MS), remyelination can fail despite abundant OPC, suggesting impairment of oligodendrocyte differentiation. T cells infiltrate the CNS during MS, yet little is known about T cell functions in remyelination. Here, we report that regulatory T cells (Treg) promote oligodendrocyte differentiation and (re)myelination. Treg-deficient mice exhibited significantly impaired remyelination and oligodendrocyte differentiation that was rescued by adoptive transfer of Treg. In brain slice cultures, Treg accelerated developmental myelination and remyelination, even in the absence of overt inflammation. Treg directly promoted OPC differentiation and myelination in vitro. We identified CCN3 as a novel Treg-derived mediator of oligodendrocyte differentiation and myelination in vitro. These findings reveal a new regenerative function of Treg in the CNS, distinct from immunomodulation. Although originally named ‘Treg’ to reflect immunoregulatory roles, this also captures emerging, regenerative Treg functions aptly.

Mouse immune thrombocytopenia is associated with Th1 bias and expression of activating Fcγ receptors.

Immune thrombocytopenia (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies. We recently established a mouse ITP model exhibiting regulatory T-cell (Treg) deficiency, although only one-third of the Treg-deficient mice developed ITP. To clarify mechanisms involved in the emergence of platelet-specific autoimmunity in this model, we examined the T helper (Th)-cell balance and macrophage Fcγ receptor (FcγR) expression profiles in Treg-deficient mice with and without ITP. Splenocytes from both populations of Treg-deficient mice and control BALB/c mice were subjected to flow cytometry-based analyses to evaluate Th cell subset proportions and the expression of activating and inhibitory FcγRs on macrophages. In addition, IgG subclass distribution of anti-platelet autoantibodies in splenocyte culture supernatants was determined by flow cytometry using IgG subclass-specific antibodies. Treg-deficient ITP mice exhibited a significantly higher proportion of Th1 cells than either Treg-deficient non-ITP or control mice. The predominant anti-platelet autoantibody subclasses in the ITP mice were Th1-associated IgG2a and IgG2b. Furthermore, the FcγRI/FcγRIIB expression ratio in splenic macrophages was higher in the Treg-deficient ITP than in the Treg-deficient non-ITP and control mice. In summary, Th1 polarization and macrophages' activating FcγR expression profile are associated with the development of ITP in Treg-deficient mice.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

Homeostasis and function of Tregs at the site of chronic inflammation (LYM2P.728)

Abstract This study is aimed at characterizing Treg dynamics at the site of chronic inflammation in the islets of NOD mice. Tregs expressing CD103 were present primarily in the inflamed islets and low in the pancreatic LN and spleen. Both CD103+ and CD103- Tregs from inflamed NOD islets could prevent diabetes when transferred to Treg-deficient CD28KO mice. CD103 was selectively induced and maintained in inflamed islets after transfer. However, fewer CD103+ Tregs were recovered after transfer compared with CD103- Tregs, suggesting that CD103+ Tregs have a survival defect. Indeed, CD103+ Tregs had lower Bcl2 expression despite higher CD25 expression and higher responsiveness to IL-2. CD103+ islet Tregs have reduced TCRb repertoire diversity and higher BrdU incorporation, indicating they clonally expand in the islets. Lastly, in NOD mice expressing a TCR signaling reporter, the Nur77-GFP transgene, CD103+ islet Tregs were found to be uniformly Nur77hi suggesting they were more recently activated by antigens. TCRb sequences used by high frequency clones of CD103+ Tregs could be found in Nur77hiCD103- but not Nur77loCD103- Tregs, consistent with sequential TCR activation and CD103 expression. Together, our results demonstrate that chronically inflamed islets in NOD mice contain functional Tregs, but fail to maintain the highly antigen-specific activated subset. Maintenance of this subset may have important implications for the treatment of chronic inflammatory disease.

Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation.

Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4(+)CD25(hi)Foxp3(+) Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3(DTR)) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 10(5) DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS.

40. Transgene Product-Specific Treg Are Initially Induced in the Celiac Lymph Node During Tolerance Induction By Hepatic AAV Gene Transfer

Previous work by us and several other laboratories has shown that liver-directed gene transfer can induce transgene product-specific immune tolerance. Induction of CD4+CD25+FoxP3+ regulatory T cells (Treg) is a critical component of the tolerance mechanism. Gaps in knowledge exist about the identity of the antigen-presenting cells (APCs) and the origin of peripherally induced Treg. To characterize the interaction of adeno-associated virus (AAV) antigen expression in the liver with immune cells, we used AAV8-EF1a vectors, which have a high tropism for murine liver, expressing either a secreted ovalbumin (AAV8-ova) or cytoplasmic ovalbumin (AAV8-Cytoova). In Treg-deficient mice transgenic for an ova-specific CD4+ T cell receptor (DO11.10-tg Rag-2-/- BALB/c), Treg were induced with either vector. For secreted ova, multiple organs (liver, spleen, lymph nodes [LN]) showed a similar level and time course of Treg induction, with greater than 1% of the CD4+ T cells being Treg starting at 4 weeks. Substantial T cell activation using the very early activation marker CD69 was also observed in these tissues starting at 3 weeks after AAV8-ova. However, Treg induction was observed in the celiac LN (one of the liver-draining LN) as early as 3 weeks after AAV8-Cyto-ova gene transfer, followed by induction of Treg in other tissues by 4 weeks. Interestingly, T cell activation was notably higher in liver and celiac LN after AAV8-Cyto-ova with over 70% activated CD4+ T cells after 4 weeks. In addition, T cell activation was observed earlier in all tissues after AAV8-Cyto-ova when compared to AAV8-ova. Literature data document a role for tolerogenic antigen presentation in the liver itself via sinusoidal endothelial cells and possibly hepatocytes. Our data suggest an important role of conventional mechanisms of adaptive immunity involving draining LNs that survey tissue antigens leading to early T cell activation and Treg induction of hepatic expressed non-systemic antigens. To start defining the critical APCs, BALBc mice were given AAV8-ova vector two weeks prior to adoptive transfer of CellTrace Violet-labeled CD4+ T cells from DO11.10 donor mice and their proliferation was measured using flow cytometry after 5 days. Half of these mice had their Kupffer cells and M1 macrophages inactivated with Gadolinium chloride for two days prior to DO11.10 adoptive transfer. DO11.10 cells proliferated in an antigen-specific manner, and proliferation was blocked by Gadolinium chloride, supporting a role for professional APCs in presentation of hepatic transgene product. Our data suggest and reinforce the importance of Kupffer cells as key APCs for hepatic antigens and further characterize the impact of the route of antigen expression on T cell activation and Treg induction.

Lung Angiogenesis Requires CD4(+) Forkhead Homeobox Protein-3(+) Regulatory T Cells.

Angiogenesis in ischemic organs is modulated by immune cells. Systemic neovascularization of the ischemic lung requires macrophages, with chemokines playing a central role in new vessel growth. Because regulatory T (Treg) cells modulate tumor-induced neovascularization, we questioned whether this CD4(+) lymphocyte subset impacts blood vessel growth during ischemia. In a model of left lung ischemia, an increase in CD4(+) CD25(+) forkhead homeobox protein-3 (Foxp3)(+) cells was observed 3-5 days after the onset of ischemia in wild-type C57Bl/6 mice. Using transgenic mice where Foxp3(+) Treg cells can be depleted with diphtheria toxin (DT; Foxp3(DTR)), we unexpectedly found that Foxp3(+) Treg depletion led to markedly reduced lung angiogenesis (90% reduction from Foxp3(gfp) controls). Adoptive transfer studies using CD4(+) CD25(+) splenocytes from congenic CD45.1 mice into Foxp3(+) Treg-depleted mice showed an almost complete recovery of the angiogenic phenotype (80% of Foxp3(gfp) controls). A survey of lung gene expression of angiogenic (lipopolysaccharide-induced CXC chemokine [LIX], IL-6, IL-17) and angiostatic (IFN-γ, transforming growth factor-β, IL-10) cytokines showed Treg-dependent differences only in LIX (CXCL5) and IL-6. Protein confirmation demonstrated a significant reduction in LIX in Treg-deficient mice compared with controls 5 days after the onset of ischemia. Phenotyping other inflammatory cells in the lung by multicolor flow cytometry demonstrated a significantly reduced number of macrophages (major histocombatibility complex class II [MHCII](int), CD11C(+)) in Treg-deficient lungs compared with Treg-sufficient lungs. Treg cells are essential for maximal systemic angiogenesis after pulmonary ischemia. One likely mechanism responsible for the decrease in angiogenesis in Treg-depleted mice was the decline in the essential CXC chemokine, LIX.

Regulatory T Cells Shape the Resident Memory T Cell Response to Virus Infection in the Tissues

Regulatory T cells (Tregs) are well known for their role in dampening the immune responses to self-Ags and, thereby, limiting autoimmunity. However, they also must permit immune responses to occur against foreign infectious agents. Using a mouse model of West Nile virus infection, we examined the role of Tregs in the generation of effector and memory T cell responses in the secondary lymphoid organs, as well as the infected tissues. We found that Treg numbers and activation increased in both the secondary lymphoid organs and CNS postinfection. Using Foxp3(DTR) knock-in mice, we found that Treg-deficient mice had increased Ag-driven production of IFN-γ from both CD4(+) and CD8(+) T cells in the spleen and CNS during the effector phase. In mice lacking Tregs, there were greater numbers of short-lived effector CD8(+) T cells in the spleen during the peak of the immune response, but the memory CD8(+) T cell response was impaired. Specifically, we demonstrate that Treg-dependent production of TGF-β results in increased expression of CD103 on CD8(+) T cells, thereby allowing for a large pool of resident memory T cells to be maintained in the brain postinfection.