ABSTRACT: In lupus autoimmunity, pathogenic IgG autoantibodies that fix complement and bind FcGammaR on inflammatory cells, are produced with help from T helper (Th1 and Th17) cells specific for peptides from nucleosomes of apoptotic cells; and these Th cells also infiltrate vital organs (1-9). Macrophages (e.g. tingible body MΦ), and DCs are normally tolerant to apoptotic cell antigens (10), but they are activated to present such autoantigens after binding to IgG immune complexes (IC) containing apoptotic cell derived DNA/RNA, which then dually stimulate their TLR and FcGammaR (11-16). Hence, to generate the activating IC, IgG-switched autoantibodies have to be made first by T cell help. Moreover, B cells become efficient antigen presenting cells (APC) to Th cells pre-primed by other APC (17), or if the B cells have developed high affinity somatically mutated receptors by T cell help (18). Thus, conventional APCs participate as disease progresses, but it is unknown who initially primes autoimmune Th cells. We fractionated spleen cells of lupus prone SNF1 mice in search of such APC. To produce lupus-prone SNF1 mice, NZB and SWR mice were purchased from The Jackson Laboratory, ME to breed the lupus-prone SNF1 hybrids (19). Female SNF1 mice, like BWF1, have high serum levels of IgG class anti-DNA and other anti-nuclear autoantibodies by 2 mo, and spontaneously begin to develop severe lupus nephritis by 5 mo age (20). We depleted spleen cells of lupus-prone SNF1 mice of cells with mature lineage markers, including conventional APC, and then isolated pure CD117+ (c-Kit+ or K+) cells from the lineage– (Lin– or L–) cells. These Lin–c-Kit+pure cells, which had morphology of hematopoietic progenitor cells, were the main type of APC inducing nuclear autoantigen-specific T helper cell (Th17) response upon feeding them with nucleosomes. These Lin–c-Kit+pure cell isolate is called "LinminuscKitplus pure" in the Sample titles shown below. CD117 (c-Kit) is also a marker for macrophage/dendritic cell precursors (MDP), which also express CX3CR1 (21-23). Therefore, from T and B-cell depleted spleen cells, we sorted out CD117+CX3CR1–, CD117+CX3CR1+, CD117–CX3CR1+, and CD117–CX3CR1– cell subsets, and then tested their abilities to induce Th responses to nucleosomes. The CD117+CX3CR1– cells were Lin– (Lin–c-Kit+CX3CR1– or L–K+Cx–; named as "LinminuscKitplusCX3CR1minus" in the sample titles below) were very similar to L–K+pure cells (LinminuscKitplus pure), which are also CX3CR1–, although isolated in a different way. Therefore, we compared gene expression profiles of nucleosome-pulsed APC that were isolated in those two ways, namely purified Lin–c-Kit+ pure (LinminuscKitplus pure) cells or the Lin–c-Kit+CX3CR1– (LinminuscKitplusCX3CR1minus) cell subset, with other types of APCs, namely the Lin–c-Kit+CX3CR1+ subset (named as "LinminuscKitminusCX3CR1plus" in sample titles below), or CD11c+CD11blowc-Kit– cells (Dendritic cells in the sample titles below). REFERENCES 1. Adams, S., P. Leblanc, and S. K. Datta. 1991. Junctional region sequences of T-cell receptor b chain genes expressed by pathogenic anti-DNA autoantibody-inducing T helper cells from lupus mice: Possible selection by cationic autoantigens. Proc. Natl. Acad. Sci. USA. 88:11271-11275. 2. Mohan, C., S. Adams, V. Stanik, and S. K. Datta. 1993. Nucleosome: A major immunogen for the pathogenic autoantibody-inducing T cells of lupus. J. Exp. Med. 177:1367-1381. 3. Kaliyaperumal, A., C. Mohan, W. Wu, and S. K. Datta. 1996. 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Lin. 2008. lumi: a pipeline for processing Illumina microarray. Bioinformatics 24:1547-1548. 25. Lin, S. M., P. Du, W. Huber, and W. A. Kibbe. 2008. Model-based variance-stabilizing transformation for Illumina microarray data. Nucleic Acids Res 36:e11. 26. Du, P., W. A. Kibbe, and S. M. Lin. 2007. nuID: a universal naming scheme of oligonucleotides for illumina, affymetrix, and other microarrays. Biol Direct 2:16. 27. Wettenhall, J. M., and G. K. Smyth. 2004. limmaGUI: a graphical user interface for linear modeling of microarray data. Bioinformatics 20:3705-3706. Gene profiling microarray and data analysis. Spleen cells from 3 batches of three 5 mo SNF1 female mice each time were used to obtain three independent isolations of each type of APC population. Total RNA was purified from each APC after incubation with nucleosomes (20µg/ml) for 6h. RNA expression analysis of each type of APC isolate in triplicate was performed at our Genomics Core Facility using Illumina Mouse WG-6 v2.0 Expression Beadchips, which provides coverage of around 45,281 genes and expressed sequence tags. Raw signal intensities of each probe were obtained using data analysis software (Beadstudio; Illumina) and imported to the Lumi package of Bioconductor for data analysis. Before transformation and normalization (24-26), A/P call detection was performed based on detection p value. 20,890 out of 45,281 probes with less than 0.01 were considered as valid signals. For each pairs of four comparisons: Lin–c-Kit+CX3CR1– versus Lin–c-Kit+ CX3CR1+; Lin–c-Kit+pure vs. Lin–c-Kit+CX3CR1+; Lin–c-Kit+CX3CR1– vs. DC; and Lin–c-Kit+pure vs. DC; differentially expressed genes were identified using an Analysis of Variance (ANOVA) model with empirical Bayesian variance estimation (27). Thus, 1,619 genes were identified as being differentially expressed (up or down) on the basis of a statistically significant (raw P-value < 0.01 and false discovery rate adjusted P-value < 0.05), and 1.5-fold change (up or down) in expression level in at least one of the comparisons, and out of these 230 genes were significantly UP-regulated by the above criteria in all 4 pair comparisons.
