Cathleen Rich

Three Novel Collagen VI Chains, α4(VI), α5(VI), and α6(VI)

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the α4(VI), α5(VI), and α6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (α4 and α6 chains) or three (α5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the α6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse α4(VI)N6-C2 chain co-assembled with endogenous α1(VI) and α2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, α5(VI)N5-C1 and α6(VI)N6-C2 chains did not assemble with α1(VI) and α2(VI) chains and accumulated intracellularly. We conclude that the α4(VI)N6-C2 chain contains all the elements necessary for trimerization with α1(VI) and α2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.

Recombinant TCR Ligand Induces Tolerance to Myelin Oligodendrocyte Glycoprotein 35-55 Peptide and Reverses Clinical and Histological Signs of Chronic Experimental Autoimmune Encephalomyelitis in HLA-DR2 Transgenic Mice

In a previous study, we demonstrated that myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide could induce severe chronic experimental autoimmune encephalomyelitis (EAE) in HLA-DR2+ transgenic mice lacking all mouse MHC class II genes. We used this model to evaluate clinical efficacy and mechanism of action of a novel recombinant TCR ligand (RTL) comprised of the α1 and β1 domains of DR2 (DRB1*1501) covalently linked to the encephalitogenic MOG-35-55 peptide (VG312). We found that the MOG/DR2 VG312 RTL could induce long-term tolerance to MOG-35-55 peptide and reverse clinical and histological signs of EAE in a dose- and peptide-dependent manner. Some mice treated with lower doses of VG312 relapsed after cessation of daily treatment, but the mice could be successfully re-treated with a higher dose of VG312. Treatment with VG312 strongly reduced secretion of Th1 cytokines (TNF-α and IFN-γ) produced in response to MOG-35-55 peptide, and to a lesser degree purified protein derivative and Con A, but had no inhibitory effect on serum Ab levels to MOG-35-55 peptide. Abs specific for both the peptide and MHC moieties of the RTLs were also present after treatment with EAE, but these Abs had only a minor enhancing effect on T cell activation in vitro. These data demonstrate the powerful tolerance-inducing therapeutic effects of VG312 on MOG peptide-induced EAE in transgenic DR2 mice and support the potential of this approach to inhibit myelin Ag-specific responses in multiple sclerosis patients.

T lymphocytes do not directly mediate the protective effect of estrogen on experimental autoimmune encephalomyelitis.

Gender influences mediated by 17 beta-estradiol (E2) have been associated with susceptibility to and severity of autoimmune diseases such as diabetes, arthritis, and multiple sclerosis. In this regard, we have shown that estrogen receptor-alpha (Esr1) is crucial for the protective effect of 17 beta-estradiol (E2) in murine experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis. The expression of estrogen receptors among various immune cells (eg, T and B lymphocytes, antigen-presenting cells) suggests that the therapeutic effect of E2 is likely mediated directly through specific receptor binding. However, the target immune cell populations responsive to E2 treatment have not been identified. In the current study, we induced EAE in T-cell-deficient, severe combined immunodeficient mice or in immunocompetent mice with encephalitogenic T cells from wild-type Esr1+/+ or Esr1 knockout (Esr1-/-) donors and compared the protective E2 responses. The results showed that E2-responsive, Esr1+/+ disease-inducing encephalitogenic T cells were neither necessary nor sufficient for E2-mediated protection from EAE. Instead, the therapeutic response appeared to be mediated through direct effects on nonlymphocytic, E2-responsive cells and down-regulation of the inflammatory response in the central nervous system. These results provide the first demonstration that the protective effect of E2 on EAE is not mediated directly through E2-responsive T cells and raise the alternative possibility that nonlymphocytic cells such as macrophages, dendritic cells, or other nonlymphocytic cells are primarily responsive to E2 treatment in EAE.

Myelin oligodendrocyte glycoprotein-35–55 peptide induces severe chronic experimental autoimmune encephalomyelitis in HLA-DR2-transgenic mice

The use of HLA class II‐transgenic (Tg) mice has facilitated identification of antigenic T cell epitopes that may contribute to inflammation in T cell‐mediated diseases such as rheumatoid arthritis and multiple sclerosis (MS). In this study, we compared the encephalitogenic activity of three DR2‐restricted myelin determinants [mouse (m) myelin oligodendrocyte glycoprotein (MOG)‐35–55, human (h)MOG‐35–55 and myelin basic protein (MBP)‐87–99] in Tg mice expressing the MS‐associated DR2 allele, DRB1*1501. We found that mMOG‐35–55 peptide was strongly immunogenic and induced moderatelysevere chronic experimental autoimmune encephalomyelitis (EAE) with white matter lesions after a single injection in Freunds complete adjuvant followed by pertussis toxin. hMOG‐35–55 peptide,which differs from mMOG‐35–55 peptide by a proline for serine substitution at position 42, was also immunogenic, but not encephalitogenic, and was only partially cross‐reactive with mMOG‐35–55. In contrast, MBP‐87–99, which can induce EAE in double‐Tg mice expressing both HLA‐DR2 and a human MBP‐specific TCR, was completely non‐encephalitogenic in HLA‐DR2‐Tg mice lacking the human TCR transgene. These findings demonstrate potent encephalitogenic activity of the mMOG‐35–55 peptide in association with HLA‐DR2, thus providing a strong rationale for further study of hMOG‐35–55 peptide as a potential pathogenic determinant in humans.

Monomeric Recombinant TCR Ligand Reduces Relapse Rate and Severity of Experimental Autoimmune Encephalomyelitis in SJL/J Mice through Cytokine Switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-As/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 × SJL)F1 mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 × SJL/J)F1 mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-β3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.

Identification of HLA-DRB1*1501^ Restricted T-cell Epitopes from Prostate-Specific Antigen

The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate tissue is very attractive because prostate is not a vital organ beyond the reproductive years. CD4 T cells play an important role in the development of antitumor immune responses, yet the identification of naturally processed MHC Class II–restricted epitopes derived from prostate differentiation antigens has not been described. To facilitate the search for prostate-specific antigen (PSA)–derived MHC class II–restricted peptides, we immunized mice transgenic for HLA-DRB1*1501 with human PSA and showed a robust dose-dependent immune response to the antigen. Screening a library of overlapping 20-mer peptides that span the entire PSA sequence identified two 20-mer peptides, PSA171-190 and PSA221-240, which were responsible for this reactivity. Immunization of DR2b transgenic mice with these peptides induced specific responses to the peptide and whole PSA. Identified peptides were used to stimulate CD4 T cells from HLA-DRB1*1501+ patients with a rare condition, granulomatous prostatitis, and who seem to have a preexisting immune response directed against the prostate gland. We previously showed a linkage of granulomatous prostatitis to HLA-DRB1*1501, suggesting that this disease may have an autoimmune etiology. Peptide-specific CD4 T-cell lines were generated from the peripheral blood of these patients as well as one patient with prostate cancer. These lines also recognized whole, processed PSA in the context of HLA-DRB1*1501. This study will be instrumental in understanding the interaction between circulating self-reactive T cells, organ-specific autoimmunity, and antitumor immune response. The use of these peptides for the immunotherapy of prostate cancer is under investigation.

T‐cell hybridoma specific for myelin oligodendrocyte glycoprotein‐35–55 peptide produced from HLA‐DRB1*1501‐transgenic mice

The goal of this study was to establish an unlimited and standardized source of humanized myelin peptide‐specific T cells for in vitro testing of biological function. Thus, we perpetuated myelin oligodendrocyte glycoprotein (MOG)‐35–55 peptide‐specific T cells obtained from immunized HLA‐DRB1*1501‐transgenic (Tg) mice by somatic fusions with BW5147 thymoma cells or BW5147 T‐cell receptor (TCR) α−β− variant (BW5147 variant) cells. The resulting T‐cell hybridomas responded strongly to both mouse MOG‐35–55 (42S) and human MOG‐35–55 peptide (42P), regardless of which peptide was used for initial immunization, and were DRB1*1501 restricted. The MOG‐35–55‐reactive T‐cell hybridomas were CD3+CD4+CD8− and expressed intracellular Th1 cytokines upon concanavalin A stimulation. Clones from either human MOG‐35–55‐ or mouse MOG‐35–55‐selected hybridomas uniquely expressed the TCR BV8 gene in combination with AV17 and AV11 genes. V gene analyses confirmed the expression of TCR AV1, AV11, AV16, BV1, and BV5 gene segments in the widely used fusion partner BW5147 and demonstrated deletion of TCR AV1, AV11, and BV1 in the BW5147 variant. T‐cell hybridomas were positively stained with anti‐TCR β‐chain antibody on the cell surface, whereas neither BW5147 nor its variant had positive TCR surface expression. For functional application, we found that a monomeric form of the human HLA‐DR2‐derived recombinant T‐cell receptor ligand (RTL) covalently linked to human MOG‐35–55 peptide specifically inhibited proliferation of a hybridoma clone selected with human MOG‐35–55 but not a different hybridoma clone selected with myelin basic protein. The RTL‐induced inhibition in vitro of the human MOG‐35–55 peptide‐specific hybridoma reflected the ability of the RTL to inhibit experimental autoimmune encephalomyelitis induced by human MOG‐35–55 peptide in HLA‐DR2 transgenic mice. Thus, the MOG‐35–55 peptide‐specific T‐cell hybridoma from DR2‐Tg mice represents a novel humanized T‐cell reagent useful for standardized biological screening of both DR2‐restricted stimulation and RTL‐dependent inhibition of response to human MOG‐35–55 peptide.

αB-Crystallin-reactive T cells from knockout mice are not encephalitogenic

Alpha B-crystallin (alphaB) is a small heat shock protein that is strongly up-regulated in multiple sclerosis (MS) brain tissue, and can induce strong T cell responses. Assessing a potential encephalitogenic function for alphaB protein in MS and experimental autoimmune encephalomyelitis (EAE) has been challenging due to its ubiquitous expression that likely maintains central and peripheral tolerance to this protein in mice. To address this issue, we obtained alphaB-knockout (alphaB-KO) mice in H-2b background that lack immune tolerance to alphaB protein, and thus are capable of developing alphaB-specific T cells that could be tested for encephalitogenic activity after transfer into alphaB-expressing wild type (WT) mice. We found that T cell lines from spleens of alphaB protein-immunized alphaB-KO mice proliferated strongly to alphaB protein itself, and the majority of T cells were CD4+ and capable of secreting pro-inflammatory Th1 cytokines upon restimulation. However, transfer of such alphaB-reactive T cells back into WT recipients was not sufficient to induce EAE, compared to the transfer of mouse MOG-35-55 peptide-reactive T cells from the same donors that induced severe EAE in recipients. Moreover, alphaB-specific T cells failed to augment severity of actively induced EAE in WT mice that were expressing high levels of alphaB message in the CNS at the time of transfer. These results suggest that alphaB-specific T cells are immunocompetent but not encephalitogenic in 129SvEv mice, and that immune tolerance may not be the main factor that limits the encephalitogenic potential of alphaB.

HLA-DRB1*1501 risk association in multiple sclerosis may not be related to presentation of myelin epitopes

Susceptibility to multiple sclerosis (MS) is associated genetically with human leucocyte antigen (HLA) class II alleles, including DRB1*1501, DRB5*0101, and DQB1*0602, and it is possible that these alleles contribute to MS through an enhanced ability to present encephalitogenic myelin peptides to pathogenic T cells. HLA‐DRB1*1502, which contains glycine instead of valine at position 86 of the P1 peptide‐binding pocket, is apparently not genetically associated with MS. To identify possible differences between these alleles in their antigen‐presenting function, we determined if T‐cell responses to known DRB1*1501‐restricted myelin peptides might be diminished or absent in transgenic (Tg) DRB1*1502‐expressing mice. We found that Tg DRB1*1502 mice had moderate to strong T‐cell responses to several myelin peptides with favorable DRB1*1501 binding motifs, notably myelin oligodendrocyte glycoprotein (MOG)‐35‐55 (which was also encephalitogenic), proteolipid protein (PLP)‐95‐116, and MOG‐194‐208, as well as other PLP and MOG peptides. These peptides, with the exception of MOG‐194‐208, were also immunogenic in healthy human donors expressing either DRB1*1502 or DRB1*1501. In contrast, the DRB1*1502 mice had weak or absent responses to peptides with unfavorable DRB1*1501 binding motifs. Overall, none of the DRB1*1501‐restricted myelin peptides tested selectively lacked immunogenicity in association with DRB1*1502. These results indicate that the difference in risk association with MS of DRB1*1501 versus DRB1*1502 is not due to a lack of antigen presentation by DRB1*1502, at least for this set of myelin peptides, and suggest that other mechanisms involving DRB1*1501 may account for increased susceptibility to MS.

Identification of HLA-DRB1*1501-Restricted T Cell Epitopes from Prostate-Specific Antigen (PSA) Using DR2b Transgenic Mice

augment IFN-DC secretion of inhibitory cytokine IL-10, and down-regulate IL-12p70. Previously, we have shown that a low concentration of IFNa (100 IU/ml) induces IFN-DCs to secrete a large amount of IL-10 and very little, if any, IL-12p70 in response to CD40 ligand. With the increase of IFNa concentration (up to 100 times), however, the production of IL-10 declined, which was associated with the upregulation of IL-12p70. In this study, we explored the possibility that poor secretion of IL-12p70 was due to endogenously produced IL-10. We implemented serum-free tissue culture conditions to minimize the effect of serum-related factors. Human peripheral mononuclear blood cells from breast cancer patients were grown in macrophage serum-free medium with granulocyte-macrophage colonystimulating factor (GM-CSF, 1000 IU/ml) and increasing concentrations of IFNa (100 IU/ml to 10,000 IU/ml) for 5–7 days. The CD40 ligand–induced IL-10 and IL-12p70 production in the presence of IL-10 neutralizing antibody or control antibody was measured by ELISA. We found that CD40L-induced production of IL-12p70 by low dose IFN-DCs was enhanced after incubation with anti-IL-10 antibody but still remained well below the levels of high dose IFN-DCs incubated with isotype control. We conclude that the endogenously produced IL-10 is only partially responsible for poor secretion of IL-12p70 by low dose IFN-DCs and the additional DC maturation signals provided by high concentrations of IFNa are necessary for production of immunostimulatory molecules.