Dept. of Microbiology & Immunology

University of Tennessee, Memphis

Tony Marion, Ph.D.

Office: (901) 448-6527

Lab:  (901) 448-6188

tmarion@utmem.edu

Office:  201F M.S.B.

Lab:  221 M.S.B.

Laboratory web page

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Research Program

The Molecular and Cellular Basis for Autoimmunity to DNA in Mouse Models for Systemic Autoimmune Disease. During their development, lymphocytes are able to generate a large repertoire of antigen receptors by of somatic recombination. The somatic processes by which T and B cell receptors are generated is stochastic. Subsequent to the development and cell-surface expression of these clonotypic receptors for antigen, both T lymphocytes and B lymphocytes are subjected to clonotypic selection. Those lymphocytes whose receptors are by chance specific for self antigens are either eliminated or functionally silenced. These mechanisms are important to avoid autoimmunity in the respective host. Immature B and T cells with receptors specific for non-self antigens continue to develop into mature, functional lymphocytes and enter the circulating pool of immunocompetent cells. When specific foreign antigen binds to the antibody receptor of an immunocompetent B cell, the B cell, if it receives the necessary T cell help, will be stimulated to differentiate into an antibody secreting cell. When T and/or B cells specific for self antigens escape tolerance induction for whatever reason, they may be stimulated to initiate an autoimmune response and autoimmune disease. Systemic lupus erythematosus (SLE) is such an autoimmune disease. The autoimmune disease systemic lupus erythematosus is characterized at least in part by autoimmunity to nuclear antigens including DNA. The anti-DNA antibodies produced in this autoimmune response are a major contributor to the immunopathological consequences of lupus which is predominantly chronic inflammation, most notably glomerulonephritis.

The general goal of the research in my laboratory has been to elucidate the cellular and molecular basis for autoimmunity to DNA in an experimental mouse model for lupus. Results emanating from our laboratory as well as those from other laboratories over the past 10 years have clearly established that autoimmunity to DNA is both initiated and sustained as an antigen-specific immune response to DNA or DNA-containing complexes. The conclusion derived from those results has been further confirmed with an experimental immunization system for inducing antibodies to DNA in mice that are not genetically predisposed to autoimmunity. Those efforts have been based upon our ability to induce anti-DNA antibody in normal, non-autoimmune mice by immunization with DNA-peptide (Fus1) complexes. The variable-region structures of the induced anti-DNA antibodies are similar if not identical to those of autoimmune anti-DNA antibodies. Likewise the serological characteristics, such as heavy-chain isotype and antigen specificity, of the induced anti-DNA antibodies are identical to those of autoimmune anti-DNA. Moreover, the immunopathological function of the induced antibody is similar to that of autoimmune anti-DNA antibody. Results derived from this experimental immunization system have provided new insight about the necessary requirements for autoimmune DNA-specific B cells to initiate an autoimmune anti-DNA antibody response.

More recently we have initiated experiments to understand DNA-specific memory B cell and germinal center development and function in autoimmune-prone (NZB x NZW)F₁ mice. In particular, we are determining whether anti-DNA antibody production is dependent upon germinal center formation and if so, whether the autoimmune, DNA-specific germinal centers are similar in structure and function to germinal centers that develop in response to conventional haptenated antigens such as DNP (dinitrophenyl)-ovalbumin. We already know that there is a spontaneous, precocious development of germinal centers in autoimmune (NZB x NZW)F₁ mice as early as two months of age. Rarely have we seen germinal center development in unimmunized normal mice, and never at only two months of age. Using biotinylated DNA as a probe for DNA-binding B cells, germinal centers containing DNA-binding B cells have been identified. Such germinal centers are readily identified in (NZB x NZW)F₁ mice that are producing IgG anti-DNA antibody; however, DNA-binding germinal center B cells have not been identified in normal, non autoimmune mice. Although normal, non autoimmune-prone mice produce IgG anti-DNA antibody in response to DNA-Fus1 immunization, they do not appear to develop B cell memory to DNA even after repeated immunizations with DNA-Fus1. We have hypothesized that the serum IgG anti-DNA produced in response to DNA-Fus1 may be produced by B cells stimulated to differentiate directly into IgG plasma cells. Such differentiation could be directed by helper T cells in splenic PALS or interfollicular regions of lymph nodes. DNA-specific memory B cells that would normally be generated in germinal centers after specific immunization are probably made tolerant when their surface immunoglobulin receptors bind to soluble nucleosomal DNA. Since such B cells would be unlikely to receive T-cell help from Fus1-specific T cells, they would be eliminated by apoptosis. Why such B cells would not also be eliminated in autoimmune (NZB x NZW)F₁ mice is one of the obvious questions we are continuing to pursue.

In order to more easily study germinal center development in autoimmune mice, we have generated (NZB x NZW)F₁ mice transgenic for expression of an IgM anti-DNA antibody. The 3H9 heavy chain variable region together with a V8 light chain encode an anti-DNA with specificity for ssDNA. The 3H9-µ heavy chain has been backcrossed into NZW mice, and the V8- light chain, into NZB. F₁ mice between these two transgenic strains will inherit both the light and heavy chain transgenes, only the light or the heavy chain transgene, or neither transgene. All of the B cells in double transgenic F₁ mice would presumably express an anti-ssDNA antibody. Preliminary results have indicated that all of the F₁ mice, whether they inherited either or both transgenes, had normal serum levels of both IgM and IgG. F₁ mice that inherited only the light chain transgene or neither transgene had autoimmune phenotypes similar to normal, non transgenic (NZB x NZW)F₁ mice. F₁ mice that inherited both the heavy and light chain transgenes produced IgG antinuclear antibodies but not IgG anti-DNA. Less than fifty percent of the double transgenic F₁ mice had serum titers of IgM anti-DNA higher than those detected in non autoimmune BALB/c mice. The double transgenic F₁ mice have generally lived to ages of over one year. (NZB x NZW)F₁ that inherit neither transgene have rarely survived past ten months of age. Even though F₁ mice transgenic for either the 3H9 heavy chain alone or both the 3H9 heavy chain and the V8 light chain did not spontaneously produce IgG anti-DNA, all of the transgenic F₁ mice produced IgG anti-DNA when immunized with DNA-peptide complexes. Why the 3H9 transgenic F₁ mice do not spontaneously produce IgG anti-DNA is presently not clear. Although B cells from the anti-DNA transgenic (NZB x NZW)F₁ mice appear initially to be allelically excluded for endogenous immunoglobulin heavy chain expression, serum IgG levels in the transgenic mice are identical to those of non transgenic littermates. Many if not most of the B cells eventually express both the transgene encoded µ heavy chain and a second heavy chain derived from endogenous variable region gene rearrangement. What has been surprising, at least to us, is the transgenic mice have serum anti-DNA titers no higher than those of non autoimmune, non anti-DNA transgenic BALB/c mice. The anti-DNA transgenic (NZB x NZW)F₁ mice are autoimmune since their serum titers of other antinuclear antibodies, such as anti-Scl70, are similar to those of non transgenic (NZB x NZW)F₁ mice. The anti-DNA transgenic (NZB x NZW)F₁ mice are also able to generate immune IgG antibody in response to antigens unrelated to DNA. We do not at present understand why such mice would not also generate IgG anti-DNA from endogenous immunoglobulin gene expression; however, an understanding of this experimental system will be one of our future goals.

Marion, T. N., D. M. Tillman, N.-T. Jou, and R. J. Hill. 1992. Selection of immunoglobulin variable regions in autoimmunity to DNA. Immunol. Rev. 128: 123-149.

Marion, T.N., M. R. Krishnan, D. D. Desai, N.-T. Jou, and D. M. Tillman. 1997. Monoclonal anti-DNA antibodies: Structure, specificity, and biology. In Anti-DNA Antibodies: Induction and Role in Autoimmunity, Ed. T. N. Marion. Methods: A Companion to Methods in Enzymology 11:3-11.

Krishnan, M. R., N.-T. Jou, and T. N. Marion. 1996. Correlation between the amino acid position of arginine in VH-CDR3 and specificity for native DNA among autoimmune antibodies to DNA. J. Immunol. 157:2430-2439.

Krishnan, M. R. and T. N. Marion. 1998. Comparison of the frequencies of arginines in heavy chain CDR3 of antibodies expressed in the primary B cell repertoires of autoimmune-prone and normal mice. Scand. J. Immunol. 48:223-232.