Identification of systemically expanded activated T cell clones in MRL/lpr and NZB/W F1 lupus model mice

Abstract

CD4(+) T lymphocytes play an important role in the pathogenesis of systemic lupus erythematosus (SLE). To characterize the clonal expansion of CD4(+) T cells in murine lupus models, we analysed the T cell clonality in various organs of young and nephritic MRL/lpr and NZB/W F1 mice using reverse transcription-polymerase chain reaction (RT-PCR) and subsequent single-strand conformation polymorphism (SSCP) analysis. We demonstrated that some identical T cell clonotypes expanded and accumulated in different organs (the bilateral kidneys, brain, lung and intestine) in nephritic diseased mice, and that a number of these identical clonotypes were CD4(+) T cells. In contrast, young mice exhibited little accumulation of common clones in different organs. The T cell receptor (TCR) V beta usage of these identical clonotypes was limited to V beta 2, 6, 8.1, 10, 16 and 18 in MRL/lpr mice and to V beta 6 and 7 in NZB/W F1 mice. Furthermore, some conserved amino acid motifs such as I, D or E and G were observed in CDR3 loops of TCR beta chains from these identical CD4(+) clonotypes. The existence of systemically expanding CD4(+) T cell clones in the central nervous system (CNS) suggests the involvement of the systemic autoimmunity in CNS lesions of lupus. FACS-sorted CD4(+)CD69(+) cells from the kidney displayed expanded clonotypes identical to those obtained from the whole kidney and other organs from the same individual. These findings suggest that activated and clonally expanded CD4(+) T cells accumulate in different tissues of nephritic lupus mice, and these clonotypes might recognize restricted T cell epitopes on autoantigens involved in specific immune responses of SLE, thus playing a pathogenic role in these lupus mice.

Fig. 1

Analysis of T cell clonality in various organs from young and nephritic lupus mice by RT-PCR–SSCP. Different organs such as the spleen, LNs, bilateral kidneys, brain, lung, small intestine and heart isolated using the magnetic cell sorting method from nephritic and young MRL/lpr as well as NZB/W F1 mice and CD4-depleted, CD8-depleted or CD4- and CD8-depleted populations from lymph nodes were subjected to RT-PCR–SSCP. (a) Comparison of T cell clonality in different tissues between nephritic and young MRL/lpr mice. (b) Comparison of T cell clonality in different tissues between nephritic and young NZB/W F1 mice. Black triangles indicate the identical TCR Vβ clonotypes expanded in different organs. Lanes representative of different organs are indicated as arrows. Three individual mice were analysed in each group, and all mice yielded essentially the same results within each group.

Fig. 2

The CDR3 sequence analysis of the TCR Vβ6 chain expressed by the identical clonotype accumulated in different organs from nephritic mice. Nucleotides and corresponding deduced amino acid sequences of the V(D)J junctional region of the identical TCR Vβ6 clones accumulated in the kidney and brain of nephritic NZB/W F1 and MRL/lpr mice are shown. Amino acid sequences are displayed in single letter code above the nucleotide sequences. Arrows indicate the identical bands at different sites that were sequenced. The indicated bands with the same migration from the left kidney and brain exhibited entirely identical CDR3 sequences. The common amino acid motif of ‘I’, ‘D’ or ‘E’ and ‘G’ found in these identical clonotypes from different individuals of the two strains were boxed, enlarged and underlined or simply underlined, respectively.

Fig. 3

Analysis of T cell clonality of FACS-sorted CD4⁺CD69⁺ from the right kidney of a NZB/W F1 nephritic mice at 47 weeks by RT-PCR–SSCP. FACS-sorted CD4⁺CD69⁺ cells from the right kidney and the whole T cell populations of other organs were subjected to RT-PCR–SSCP analysis to compare the expanded clonotypes of the sorted CD4⁺CD69⁺ population with those of the whole T cell populations in other organs, including the left kidney. Black triangles indicate identical expanded bands shared by the CD4⁺CD69⁺ population and the whole T cell populations in other organs. Lanes representative of different organs or sorted cells are indicated by arrows.