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1.  Engineered Tregulatory cells co-expressing MHC class II:peptide complexes are efficient inhibitors of autoimmune T cell function and prevent the development of autoimmune arthritis 
Treg cells are critical homeostatic components in preventing the development of autoimmunity, and are a major focus for their therapeutic potential for autoimmune diseases. In order to enhance the efficacy of Treg cells in adoptive therapy, we developed a strategy for generating engineered Tregs that have the capacity to target autoimmune T cells in an antigen specific manner. Using a retroviral expression system encoding Foxp3 and HLA-DR1 covalently linked to the immunodominant peptide of the autoantigen type II collagen (DR1-CII), naïve T cells were engineered to become Treg cells that express DR1-CII complexes on their surface. When these cells were tested for their ability to prevent the development of collagen induced arthritis, both the engineered DR1-CII-Foxp3 and Foxp3 only Treg cells significantly reduced the severity and incidence of disease. However, the mechanism buy which these two populations of Treg cells inhibited disease differed significantly. Disease inhibition by the DR1-CII-Foxp3 Treg cells was accompanied by significantly lower numbers of autoimmune CII-specific T cells in vivo and lower levels of autoantibodies in comparison to engineered Tregs expressing Foxp3 alone. Additionally, the numbers of IFN-γ and IL-17 expressing T cells in mice treated with DR1-CII-Foxp3 Tregs were also significantly reduced in comparison to mice treated with Foxp3 engineered Treg cells or vector control cells. These data indicate that the co-expression of class II autoantigen-peptide complexes on Treg cells provides these cells with a distinct capacity to regulate autoimmune T cell responses that differs from that used by conventional Treg cells.
doi:10.4049/jimmunol.1300024
PMCID: PMC3673549  PMID: 23630354
2.  Characterization of T cell phenotype and function in a double transgenic (collagen-specific TCR/HLA-DR1) humanized model of arthritis 
Introduction
T cells orchestrate joint inflammation in rheumatoid arthritis (RA), yet they are difficult to study due to the small numbers of antigen-specific cells. The goal of this study was to characterize a new humanized model of autoimmune arthritis and to describe the phenotypic and functional changes that occur in autoimmune T cells following the induction of pathological events.
Methods
We developed a double transgenic mouse containing both the HLA-DR1 transgene and an HLA-DR1-restricted collagen-specific TCR in order to obtain large numbers of antigen-specific T cells that can be used for immunologic studies.
Results
In vitro, CII-specific T cells from this mouse proliferated vigorously in response to the CII immunodominant peptide A2 and the cells altered their phenotype to become predominately CD62Llow and CD44high “activated” T cells. The response was accompanied by the production of Th1, Th2, and Th17-type cytokines. Following immunization with bovine CII/CFA, these mice develop an accelerated arthritis compared to single transgenic HLA-DR1 mice. On the other hand, when the mice were treated orally with the analog peptide A12, (a suppressive analog of collagen we have previously described), arthritis was significantly suppressed, despite the fact that >90% of the CD4+ T cells express the TCR Tg. In GALT tissues taken from the A12-treated mice, IL-2, IFN-γ, and IL-17 production to the autoimmune collagen determinant dropped while high levels of IL-10 and IL-4 were produced.
Conclusions
We have developed a humanized model of autoimmune arthritis that will be useful for the study of T cell directed therapies as well as T cell mediated mechanisms of autoimmune diseases.
doi:10.1186/ar4433
PMCID: PMC3978884  PMID: 24405551
3.  T Cells Stimulated by an Analog Peptide of Type II Collagen Require FcRγ to Secrete IL-4 and Suppress Autoimmune Arthritis 
Arthritis and rheumatism  2011;63(9):2661-2670.
Objective
Using the collagen-induced arthritis (CIA) model, we explored the characteristics of the T cell population which responds to an analog peptide (A9) of type II collagen (CII) and regulates autoimmunity.
Methods
A9 is a 26 amino acid peptide analogous to the sequence of a segment of CII (CII 245-270) but with substitutions made at amino acid positions 260 (alanine for isoleucine), 261 (hydroxyproline for alanine), and 263 (asparagine for phenylalanine). We have previously shown that A9 profoundly suppresses immunity to CII and CIA. In order to determine the mechanism of suppression, we used a transgenic mouse whose T cells express a CII specific receptor (TCR) and performed passive cell transfer experiments.
Results
The results demonstrate that suppression of CIA by the A9 is dependent upon T cells. Using multiparameter flow cytometry, we determined that the cells responsible for suppression were CD4+ and expressed high levels of FcεRIγ(FcRγ). To establish the significance of this finding, we obtained mice genetically deficient in FcRγ to perform passive transfer experiments. The resulting FcRγ-/- CD4+ T cells when primed by culture with A9 could not transfer the suppression of arthritis nor secrete cytokines in response to A9.
Conclusion
Taken together, these data suggest that the suppression of arthritis and the Th2 cytokine profile elicited by A9 is dependent upon the presence of FcRγ in the T cells. These findings are novel and may have therapeutic potential for patients with autoimmune arthritis.
doi:10.1002/art.30454
PMCID: PMC3575180  PMID: 21590683
4.  Nucleosides from Phlebotomus papatasi salivary gland ameliorate murine collagen-induced arthritis by impairing dendritic cell functions 
Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. Herein, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and pro-inflammatory cytokines release. In vitro incubation of dendritic cells (DC) with SGE limited specific CD4+Th17 cell response. We identified adenosine (ADO) and adenosine monophosphate (5′AMP) as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2A receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5′nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5′AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5′AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.
doi:10.4049/jimmunol.1003404
PMCID: PMC3195336  PMID: 21930966
5.  Characterization of inhibitory T cells induced by an analog of type II collagen in an HLA-DR1 humanized mouse model of autoimmune arthritis 
Arthritis Research & Therapy  2012;14(3):R107.
Introduction
We used DR1 transgenic mice and covalently linked DR1 multimers to characterize analog-specific inhibitory T cells in collagen-induced arthritis (CIA). Because of the low numbers of antigen-specific T cells in wild-type mice, functional T-cell studies in autoimmune arthritis have been challenging. The use of T-cell receptor (TCR) transgenic mice has provided useful information, but such T cells may not represent the heterogeneous T-cell response that occurs in natural settings. Our focus was to develop tools to identify and characterize the population of immunoregulatory T cells induced in wild-type mice by an analog peptide of CII259-273, which contains amino acid substitutions at positions 263 (N) and 266 (D) (analog peptide A12).
Methods
DR1 multimers, developed by loading empty class II molecules with exogenous peptide, provide a method for visualizing antigen-specific T cells with flow cytometry. However, the low binding avidity of A12 for the major histocompatibility complex (MHC) made this strategy untenable. To overcome this problem, we generated DR1 multimers in which the analog peptide A12 was covalently linked, hoping that the low-avidity analog would occupy enough binding clefts to allow detection of the responsive T cells.
Results
Staining with the tetramer revealed that A12-specific T cells were readily detectable at 10 days after immunization. These CD4(+) T cells are a highly selective subset of the TCR repertoire and have a limited clonality. Analysis of cytokine expression showed that cells detected by tetramer (A12) expressed primarily suppressive cytokines (interleukin-4 (IL-4) and IL-10) in response to collagen, compared with control cells. Although they did not express Fox-p3, they were extremely effective in preventing and suppressing inflammatory arthritis.
Conclusions
In summary, our studies showed that the use of covalently linked multimers allows characterization of analog-specific T cells that are otherwise difficult to detect. The suppressive character of the analog-specific T-cell response suggests that these cells attenuate autoimmunity and differ significantly in phenotype from the inflammatory T cells predominantly found in arthritic joints. Such reagents will become powerful tools to study T-cell responses in RA patients in upcoming clinical trials.
doi:10.1186/ar3832
PMCID: PMC3446484  PMID: 22569209
6.  Protein kinase D1 is essential for the pro-inflammatory response induced by hypersensitivity pneumonitis-causing thermophilic actinomycetes Saccharopolyspora rectivirgula 
Hypersensitivity pneumonitis is an interstitial lung disease that results from repeated pulmonary exposure to various organic antigens, including Saccharopolyspora rectivirgula (SR, the causative agent of farmer's lung disease). Although the contributions of pro-inflammatory mediators to the disease pathogenesis are relatively well documented, the mechanism(s) involved in initiation of pro-inflammatory responses against the causative microorganisms, and the contribution of signaling molecules involved in host immune defense have not been fully elucidated. In the present study, we found that SR induces activation of protein kinase D1 (PKD1) in lung cells in vitro and in vivo. Activation of PKD1 by SR was dependent on MyD88. Inhibition of PKD by pharmacological PKD inhibitor Gö6976, and silencing of PKD1 expression by siRNA, revealed that PKD1 is indispensable for SR-mediated activation of MAPKs and NF-κB and expression of various pro-inflammatory cytokines and chemokines. In addition, compared to controls, mice pretreated with Gö6976 showed significantly suppressed alveolitis and neutrophil influx in bronchial alveolar lavage fluid and interstitial lung tissue, and substantially decreased myeloperoxidase activity in the lung after pulmonary exposure to SR. These results demonstrate that PKD1 is essential for SR-mediated pro-inflammatory immune responses and neutrophil influx in the lung. Our findings also imply the possibility that PKD1 might be one of the critical factors that play a regulatory role in development of hypersensitivity pneumonitis caused by microbial antigens, and that inhibition of PKD1 activation could be an effective way to control microbial antigen-induced hypersensitivity pneumonitis.
doi:10.4049/jimmunol.0903718
PMCID: PMC2987577  PMID: 20142359
Protein kinase D1; MyD88; Cytokines/Chemokines; Inflammation; Saccharopolyspora rectivirgula; hypersensitivity pneumonitis
7.  Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ regulatory T cells1 
European journal of immunology  2010;40(1):142-152.
Summary
TGF-β plays an important role in the induction of regulatory T cells (Treg) and maintenance of immunologic tolerance, but whether other members of TGF-β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF-β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP-2 and BMP-4, are unable to induce non-regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-β to induce naive CD4+CD25- cells to express Foxp3; however, BMP-2/4 and TGF-β have a synergistic effect on the induction of Foxp3+ Treg. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF-β. Data further advocate that TGF-β is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP-2/4 and TGF-β indicates that the simultaneous manipulation of TGF-β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.
doi:10.1002/eji.200939618
PMCID: PMC2837277  PMID: 19943263
BMP; TGF-β; Foxp3; regulatory T cells
8.  Transfer of tolerance to collagen type V suppresses Th-17 lymphocyte mediated acute lung transplant rejection 
Transplantation  2009;88(12):1341-1348.
Background
Rat lung allograft rejection is mediated by collagen type V (col(V)) specific Th17 cells. Adoptive transfer of these cells is sufficient to induce rejection pathology in isografts, whereas tolerance to col(V) suppresses allograft rejection. We therefore tested if regulatory T cells from tolerant rats could suppress the Th17 mediated rejection in the syngeneic model of lung transplantation.
Methods
Rats were subjected to syngeneic left lung transplantation and acute rejection was induced by adoptive transfer of lymph node cells from col(V)-immunized rats. Tolerance was induced by intravenous (iv) injection of col(V) and spleen lymphocytes were used for adoptive transfer. CD4+ T cells were depleted using magnetic beads. Lung isografts were analyzed using micro-PET imaging and histochemistry. The transvivo delayed type hypersensitivity (TV-DTH) assay was used to analyze the Th17 response.
Results
Adoptive co-transfer of col(V)-specific effector cells with cells from col(V) tolerized rats suppressed severe vasculitis and bronchiolitis with parenchymal inflammation, and the expression of IL-17 transcripts in mediastinal lymph nodes induced by effector cells alone. Analysis by TV-DTH showed that the reactivity to col(V) was dependent on the presence of TNF-α and IL-17, but not IFN-γ. Depletion of CD4+ T cells from the suppressor cell population abrogated the col(V)-specific protection.
Conclusion
Th17 mediated acute rejection after lung transplantation is ameliorated by CD4+ col(V)-specific regulatory T cells. The mechanism for this Th17 suppression is consistent with tolerance induction to col(V). The goal of transplantation treatment therefore should target Th17 development and not suppression of T cell activation by suppressing IL-2.
doi:10.1097/TP.0b013e3181bcde7b
PMCID: PMC2804859  PMID: 20029330
lung transplantation; animal model; IL-17; autoimmunity; adoptive transfer
9.  Type V Collagen-Induced Oral Tolerance Plus Low-Dose Cyclosporine Prevents Rejection of MHC Class I and II Incompatible Lung Allografts1 
Autoimmunity to type V collagen (col(V)) is a major risk factor for lung allograft rejection. Although col(V)-induced oral tolerance abrogates rejection of minor histoincompatible lung transplants, its ability to prevent rejection of fully MHC incompatible lung allografts is unknown. Rat lung allografts fully incompatible at MHC class I and II loci (Brown Norway (RT1n)) were transplanted into untreated Wistar Kyoto rat recipients (WKY, RT1l), or WKY rats were fed col(V) pretransplantation. To determine whether col(V) enhanced cyclosporine (CsA)-mediated immune suppression, WKY rats were treated with low-dose CsA (5 mg/kg), post-transplant, or oral col(V) plus CsA. The data showed that in contrast to col(V) or CsA, col(V) plus low-dose CsA significantly prevented rejection pathology, down-regulated alloantigen-induced production of IFN-γ and IL-17A, and suppressed chemotaxis for lung macrophages in allograft bronchoalveolar lavage fluid that was associated with lower local levels of MCP-1 (CCL2). Col(V) plus CsA was associated with alloantigen-induced expression of IL-10 in mediastinal lymph node or splenic T cells, intragraft expression of IL-10 and Foxp3 in perivascular and peribronchiolar mononuclear cells, and constitutive production of IL-10 from allograft alveolar macrophages. These data demonstrate that col(V) enhances low-dose CsA-mediated immune suppression, and suggest a role for oral col(V) in immune modulation in lung transplantation.
doi:10.4049/jimmunol.0804028
PMCID: PMC2997997  PMID: 19542435
10.  Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II 
Arthritis Research & Therapy  2010;12(4):R136.
Introduction
Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).
Methods
We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII124-402, 260A, 261B, 263D), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence.
Results
We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint.
Conclusions
Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.
doi:10.1186/ar3074
PMCID: PMC2945026  PMID: 20615221
11.  Direct Visualization of Protease Action on Collagen Triple Helical Structure 
PLoS ONE  2010;5(6):e11043.
Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease.
doi:10.1371/journal.pone.0011043
PMCID: PMC2886829  PMID: 20585385
12.  Isolation of Purified and Live Foxp3+ Regulatory T Cells using FACS Sorting on Scatter Plot 
There are no ideal ways to identify and isolate viable and purified Foxp3+ regulatory T cells so far. Here we developed a novel procedure for the isolation of highly purified Foxp3+ cells using flow cytometry. This method relies on an identification and sorting of the lymphoblast cell population identified on a scatter plot using flow cytometry. We confirmed that greater than 98% of the cells sorted using this technique expressed Foxp3 and displayed a potent suppressive activity. This method provides a valuable tool for the study of the T regulatory cell biology and their therapeutic manipulation.
doi:10.1093/jmcb/mjq007
PMCID: PMC2905066  PMID: 20430855
Foxp3; TGF-β; regulatory T cells
13.  IL-17–dependent cellular immunity to collagen type V predisposes to obliterative bronchiolitis in human lung transplants 
The Journal of Clinical Investigation  2007;117(11):3498-3506.
Bronchiolitis obliterans syndrome (BOS), a process of fibro-obliterative occlusion of the small airways in the transplanted lung, is the most common cause of lung transplant failure. We tested the role of cell-mediated immunity to collagen type V [col(V)] in this process. PBMC responses to col(II) and col(V) were monitored prospectively over a 7-year period. PBMCs from lung transplant recipients, but not from healthy controls or col(IV)-reactive Goodpasture’s syndrome patients after renal transplant, were frequently col(V) reactive. Col(V)-specific responses were dependent on both CD4+ T cells and monocytes and required both IL-17 and the monokines TNF-α and IL-1β. Strong col(V)-specific responses were associated with substantially increased incidence and severity of BOS. Incidences of acute rejection, HLA-DR mismatched transplants, and induction of HLA-specific antibodies in the transplant recipient were not as strongly associated with a risk of BOS. These data suggest that while alloimmunity initiates lung transplant rejection, de novo autoimmunity mediated by col(V)-specific Th17 cells and monocyte/macrophage accessory cells ultimately causes progressive airway obliteration.
doi:10.1172/JCI28031
PMCID: PMC2040314  PMID: 17965778
14.  Analog peptides of type II collagen can suppress arthritis in HLA-DR4 (DRB1*0401) transgenic mice 
Rheumatoid arthritis (RA) is an autoimmune disease associated with the recognition of self proteins secluded in diarthrodial joints. We have previously established that mice transgenic for the human DR genes associated with RA are susceptible to collagen-induced arthritis (CIA) and we have identified a determinant of type II collagen (CII263–270) that triggers T-cell immune responses in these mice. We have also determined that an analog of CII263–270 would suppress disease in DR1 transgenic mice. Because the immunodominant determinant is the same for both DR1 transgenic and DR4 transgenic mice, we attempted to determine whether the analog peptide that was suppressive in DR1 transgenic mice would also be effective in suppressing CIA in DR4 transgenic mice. We treated DR4 transgenic mice with two analog peptides of CII that contained substitutions in the core of the immunodominant determinant: CII256–276 (F263N, E266D) and CII256–270 (F263N, E266A). Mice were observed for CIA, and T-cell proliferative responses were determined. Either peptide administered at the time of immunization with CII significantly downregulated arthritis. Binding studies demonstrated that replacement of the phenylalanine residue in position 263 of the CII peptide with asparagine significantly decreased the affinity of the peptide for the DR4 molecule. In contrast, replacement of the glutamic acid residue in position 266 with aspartic acid or with alanine had differing results. Aspartic acid reduced the affinity (35-fold) whereas alanine did not. Both peptides were capable of suppressing CIA. With the use of either peptide, CII256–276 (F263N, E266D) or CII256–270 (F263N, E266A), the modulation of CIA was associated with an increase in T-cell secretion of IL-4 together with a decrease in IFN-γ. We have identified two analog peptides that are potent suppressors of CIA in DR4 transgenic mice. These experiments represent the first description of an analog peptide of CII recognized by T cells in the context of HLA-DR4 that can suppress autoimmune arthritis.
doi:10.1186/ar2043
PMCID: PMC1779432  PMID: 16982003
15.  An HLA-DR1 Transgene Confers Susceptibility to Collagen-induced Arthritis Elicited with Human Type II Collagen 
The Journal of Experimental Medicine  1997;185(6):1113-1122.
Rheumatoid arthritis (RA) is an autoimmune disease that is strongly associated with the expression of several HLA-DR haplotypes, including DR1 (DRB1*0101). Although the antigen that initiates RA remains elusive, it has been shown that many patients have autoimmunity directed to type II collagen (CII). To test the hypothesis that HLA-DR1 is capable of mediating an immune response to CII, we have generated transgenic mice expressing chimeric (human/ mouse) HLA-DR1. When the DR1 transgenic mice were immunized with human CII (hCII), they developed a severe autoimmune arthritis, evidenced by severe swelling and erythema of the limbs and marked inflammation and erosion of articular joints. The development of the autoimmune arthritis was accompanied by strong DR1-restricted T and B cell responses to hCII. The T cell response was focused on a dominant determinant contained within CII(259–273) from which an eight amino acid core was defined. The B cell response was characterized by high titers of antibody specific for hCII, and a high degree of cross-reactivity with murine type II collagen. These data demonstrate that HLA-DR1 is capable of presenting peptides derived from hCII, and suggest that this DR1 transgenic model will be useful in the development of DR1-specific therapies for RA.
PMCID: PMC2196244  PMID: 9091584

Results 1-15 (15)