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1.  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.
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.
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.
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.
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.
PMCID: PMC3575180  PMID: 21590683
2.  Function and regulation of self-reactive marginal zone B cells in autoimmune arthritis 
Cellular and Molecular Immunology  2015;12(4):493-504.
Polyreactive innate-type B cells account for many B cells expressing self-reactivity in the periphery. Improper regulation of these B cells may be an important factor that underlies autoimmune disease. Here we have explored the influence of self-reactive innate B cells in the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis. We show that splenic marginal zone (MZ), but not B-1 B cells exhibit spontaneous IgM reactivity to autologous collagen II in naïve mice. Upon immunization with heterologous collagen II in complete Freund's adjuvant the collagen-reactive MZ B cells expanded rapidly, while the B-1 B cells showed a modest anti-collagen response. The MZ B cells were easily activated by toll-like receptor (TLR) 4 and 9-ligands in vitro, inducing proliferation and cytokine secretion, implying that dual engagement of the B-cell receptor and TLRs may promote the immune response to self-antigen. Furthermore, collagen-primed MZ B cells showed significant antigen-presenting capacity as reflected by cognate T-cell proliferation in vitro and induction of IgG anti-collagen antibodies in vivo. MZ B cells that were deficient in complement receptors 1 and 2 demonstrated increased proliferation and cytokine production, while Fcγ receptor IIb deficiency of the cells lead to increased cytokine production and antigen presentation. In conclusion, our data highlight self-reactive MZ B cells as initiators of the autoimmune response in CIA, where complement and Fc receptors are relevant in controlling the self-reactivity in the cells.
PMCID: PMC4496548  PMID: 25958842
arthritis; complement receptors; Fc receptors; marginal zone B cells, mice
3.  T Cell Receptor-Signaling Induced by an Analog Peptide of type II Collagen Requires Activation of Syk 
Clinical immunology (Orlando, Fla.)  2009;133(1):145-153.
We have previously described an analog peptide of type II collagen (CII) that can suppress collagen-induced arthritis (CIA). This analog peptide represents CII245-270, the immunodominant epitope of CII, but with substitutions at 260, 261, and 263 - CII245-270 (A260, B261, and N263) (A9). To elucidate the mechanisms responsible for suppression, we used mice transgenic for a collagen-specific T cell receptor (TCR). When we found that APCs pulsed with A9 failed to induce T cell phosphorylation of TCR-? and ZAP-70, we explored alternative signaling pathways. We determined that A9 instead induced phosphorylation of spleen tyrosine kinase (Syk). The importance of Syk was confirmed by the use of chemical Syk inhibitors, which blocked both cytokine secretion and activation of GATA-3 mediated by peptide A9. In summary, T cells use an alternative pathway in response to A9 that involves Syk. This novel T cell pathway may represent an important means for altering T cell phenotypes.
PMCID: PMC2756788  PMID: 19596610
Collagen II; T cells; altered peptide ligands; T cell signaling; Syk (spleen tyrosine kinase); autoimmunity
4.  Characterization of T cell phenotype and function in a double transgenic (collagen-specific TCR/HLA-DR1) humanized model of arthritis 
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.
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.
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.
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.
PMCID: PMC3978884  PMID: 24405551
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.
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).
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.
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.
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.
PMCID: PMC3446484  PMID: 22569209
6.  Supernatants from culture of type I collagen-stimulated PBMC from patients with cutaneous systemic sclerosis versus localized scleroderma demonstrate suppression of MMP-1 by fibroblasts 
Clinical Rheumatology  2012;31(6):973-981.
Systemic sclerosis (SSc) is a chronic fibrosing disease characterized by vasculopathy, autoimmunity, and an accumulation of collagen in tissues. Numerous studies have shown that compared to healthy or diseased controls, the peripheral blood mononuclear cells (PBMC) from patients with SSc produce a variety of cytokines or proliferate when cultured with solubilized type I collagen (CI) or constituent α1(II) and α2(I) polypeptide chains. The purpose of this study was to determine whether PBMC isolated from patients with SSc and cultured in vitro with soluble CI elaborated soluble mediators that inhibit the production of collagenase (i.e., matrix metalloproteinase, MMP-1) by fibroblasts. Supernatants of CI-stimulated PBMC from juvenile and adult diffuse cutaneous (dc)SSc patients significantly reduced MMP-1 production by SSc dermal fibroblasts, while supernatants of CI-stimulated PBMC from patients with localized scleroderma (LS) did not. CI-stimulated PBMC culture supernatants from patients with dcSSc in contrast to patients with LS exhibited increased levels of platelet-derived growth factor (PDGF)-AA, PDGF-BB, TNF-α, IL-13, and EGF. Prolonged culture of SSc dermal fibroblasts with recombinant PDGF-BB or IL-13 inhibited the induction of MMP-1 in response to subsequent TNF-α stimulation. These data suggest that therapies aimed at reducing these cytokines may decrease collagen accumulation in SSc, preventing the development of chronic fibrosis.
PMCID: PMC3362697  PMID: 22367096
IL-13; Diffuse; Localized scleroderma; MMP-1; PBMC; PDGF-BB; Scleroderma
7.  Modulation of collagen-induced arthritis by adenovirus-mediated intra-articular expression of modified collagen type II 
Arthritis Research & Therapy  2010;12(4):R136.
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).
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.
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.
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.
PMCID: PMC2945026  PMID: 20615221
8.  46,X,del(X)(q13) Turner's Syndrome Female with Systemic Lupus Erythematosus in a Pedigree Multiplex for SLE 
Genes and immunity  2009;10(5):478-481.
Systemic lupus erythematosus (SLE) disproportionately affects females. Recent work demonstrates that men with Klinefelter's syndrome (47,XXY males) have a similar risk of developing SLE as do genotypic females. We present an unusual case of an African American family with two SLE affected individuals in which one of the SLE patients also has Turner's syndrome [46,X,del(X)(q13)]. While not definitive, this family raises interesting questions regarding the role of genes located on the X chromosome in the development of SLE. The paucity of case reports documenting the overlap of SLE with Turner's syndrome while there is and association of male SLE with Klinefelter's syndrome suggests a lower risk of SLE in Turner's females. These observations are consistent with a gene dose effect at X with two X chromosomes (46,XX or 47,XXY) conferring higher risk and one X chromosome (46,XY or 45,XO) conferring lower risk of SLE.
PMCID: PMC2722751  PMID: 19458623
9.  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.
PMCID: PMC1779432  PMID: 16982003
10.  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 1-10 (10)