Expression Of TCR Vβ Gene And T Cell Clonality Analysis In Murine Models Of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of pathogenic autoantibodies and tissue deposition of immune complexes, which result in multiple organ damage. The MRL/lpr and (NZBNZW)F1 mouse that spontaneously develop a lupus syndrome closely resembling human SLE are considered to be excellent models for investigating the pathogenesis of the human disease.It has been suggested that T-cell-dependent antigen-specific immune responses play a key role in the pathogenesis of these two strains. Published data have documented the involvement of T cells, especially CD4+ T cells, in lupus in the mouse. This involvement includes: 1) The infiltration of affected tissues with predominantly CD4+ T cells; 2) The development of lymphadenopathy but not autoimmune renal disease or autoantibodies in CD4+ T cell-deficient or MHC class II-deficient lupus mice; 3) Improvement in the disease manifestations following treatment with anti-CD4 mAb. These results suggest that CD4+ T cells play an important role in the development of lupus. Although the participation and importance of CD4+ T cells in lupus mice has been emphasized, the pathogenic CD4+ T cell clonotypes that expanded in different sites of these murine models have not yet been identified and characterized. Also, the nature of antigen-specific T cells in SLE still remains poorly defined. Detection and characterization of the pathogenic T cell clonotypes that expanded in lupus mice may help to illustrate the contribution of CD4+ T cell clones to the antigen-specific immune responses in SLE, give insight into the nature of the T cell Ags involved and provide possible immunoregulatory therapeutic way to SLE as well.-1-In order to address these questions and to further investigate the pathogenic role of CD4+ T cells clonally expanded in SLE, we compared the T cell clonality in different organs derived from young and nephritic (NZBNZW)F1 as well as MRL/lpr mice using the established RT-PCR-SSCP study in combination with CDR3 sequence analysis of the TCR VP family. Furthermore we use MACS and FACS cell sorting method combined with RT-PCR-SSCP analysis to identify the phenotype of the T cell commonly infiltrating in different organs. To test the accuracy of CDR3 sequence, we also perform the density analysis of T cell clonality based on SSCP image using Scion Image software.Our RT-PCR-SSCP study of TCR Vp chain demonstrated that there were some identical T cell clonotypes expanded and accumulated in different organs in aged diseased mice. Most of these identical clonotypes were CD4+ T cells in both of the two strains. In contrast, young lupus mice exhibited little accumulation of common clone in different organs. The TCR VP usage of these identical clonotypes was limited in Vp2, VP6, VfJS.l, vpio, VP16, Vpl8 in MRL/lpr mouse and Vp6, VP? in (NZBNZW)F1 mouse respectively. Furthermore, some conserved amino acid motifs such as I and D were observed in CDR3 loops of TCR Vp from these identical CD4+ clonotypes in (NZBNZW)F1 mice. Likewise, I, G and D or E were also found in MRL/lpr mice. Density analysis of T cell clonality based on SSCP image using Scion Image software revealed the accurate result of CDR3 sequence analysis. The T cell phenotypic analysis showed the existence of the activated CD4+ T cell populations in different organs in aged (NZBNZW)F1 mouse and the FACS sorted CD4+CD69+ cells from right kidney of this strain displayed the identical expanded clonotypes with the left kidney and other organs from the same individual.These findings suggest that activated and clonally expanded CD4+ T cells commonly accumulated in different tissues in aged murine lupus models, and these CD4+ T cell clonotypes might recognize restricted T cell epitopes on autoantigens and are involved in specific immune responses of SLE, thus playing a pathogenic role in these lupus mice.