Liu et al. A Genomic View of Systemic Autoimmunity in MRL lpr Mice
Supplementary Materials
1) Identifying splenic genes that change with normal aging in mice
In order to identify the gene expression patterns in spleen associated with autoimmunity, it was first important to document the changes in splenic gene expression in non-autoimmune mice over time. We sampled 3 C57BL/6J female mice at each of the following timepoints: 6, 12, 16,
24, 36 and 48 weeks. As shown in Figure 1 (see primary manuscript), there were only minor changes in the overall numbers of splenocytes and in the cellular composition of the control
C57BL/6 spleens, and these mice showed no evidence for autoantibody production, renal damage, or renal inflammation at any timepoint examined. Complementary RNA (cRNA) probes were generated from splenic RNA, and expression levels of ~ 11,000 transcripts were measured using Affymetrix U74Av2 chips. The data were analyzed to identify genes undergoing significant changes in expression over time. This analysis identified 91 genes that change in response to normal aging and/or those that may be particularly sensitive to environmental factors
(fold-changes ranging from –11.8 to +15.7 - see Table S1 for details). These genes were eliminated from further consideration in subsequent analyses.
2) Genes differentially expressed among MRL +/+ and MRL lpr mice
There were 236 genes that were differentially expressed among MRL +/+ and MRL lpr mice
(Figure S1 and Table S2). Selective differentially expressed genes, that have also been previously reported in MRL lpr mice, were summarized in Table S3. This further validated our approaches in revealing interesting candidate genes and pathways.

3) Within-group verification of gene profiles
We tested ‘within-group variation’ in the splenic gene expression profiles of each cohort of 3-4 mice at each of the sampled timepoints by measuring correlation coefficients for the differentially expressed genes (n=236) between the replicate samples. Both C57BL/6 and MRL
+/+ samples showed highly concordant expression patterns at each time point, whereas MRL lpr showed increasing variance as disease developed and progressed, as demonstrated by diminished correlation coefficient constants (Figure S2A). Similarly, MRL lpr samples showed more variability in gene expression between adjacent time points than either C57BL/6 or MRL +/+ mice (Figure S2B). When comparing splenic gene profiles to the 6-week timepoint, MRL +/+ mice showed greater differences compared to C57BL/6, and MRL lpr mice showed even higher levels of variance (Figure S2C). Overall, the correlations were judged to be sufficient to be able to examine the individual groups of mice by age. However, the variability in gene expression at later time points in MRL lpr mice is significant, likely reflecting secondary events during endstage disease. We believe that some caution is warranted in interpreting late expression (e.g. 24 weeks) MRL lpr results.
4) Representative real-time PCR and flow cytometric verification of gene expression differences
Shown in Figure S3 are representative paired data from microarray analysis and real-time PCR of 16 week old C57BL/6, MRL, and MRL lpr mice for (A) CXCL9, (B) IL-10, (C) Fig1 , and (D)
Ddc expression levels. Real-time PCR data were normalized by  -Actin and calculated as  Ct.
Microarray data are compared to flow cytometry findings for (E) Lag-3 and (F) 4-1BB

expression levels. Shown are T cells gated by CD3 and CD4. Solid lines (  ) indicate
C57BL/6 samples, dotted lines (  ) are MRL samples, and bold dash lines ( -----) are MRL lpr samples. These data indicate that the identified changes in gene expression can be validated by real-time PCR and at the protein level.
5) Genes dysregulated in MRL +/+ spleen
We next identified the genes that showed the largest changes in expression in MRL +/+ spleens over time. We compared MRL +/+ splenic expression profiles at 12, 16, 24, 36, and 60 weeks to levels at 6 weeks. Figure S4 contains 57 genes (not including immunoglobulin genes) that showed the largest changes in gene expression over time in MRL +/+ mice, using the following filtering criteria: ≥ 3-fold difference in gene expression level between any two timepoints, p<0.01, and average signal difference of > 200 expression units. Among these genes, only one was IFN-regulated, and overall 23/57 (40.4%) were shared with the MRL lpr gene list.

Titles and legends to supplemental figures
Figure S1.
Dynamic changes in splenic gene expression during disease progression in MRL lpr mice. Shown are 236 genes. Each column represents an individual spleen sample. Clustering of the genes (rows) was unsupervised.
Figure S2.
Within-group verification of gene expression profiles in C57BL/6, MRL +/+, and
MRL lpr mice. (A) Correlation coefficient of each age group, (B) Correlation coefficient between adjacent age groups, (C) Number of differentially expressed genes comparing to strain specific 6 weeks old mice.
Figure S3.
Representative real-time PCR and flow cytometric verification of gene expression differences. Representative paired data from microarray analysis and real-time PCR of 16 week old C57BL/6, MRL, and MRL lpr mice for (A) CXCL9, (B) IL-10, (C) Fig1 , and (D) Ddc expression levels. Representative paired data from microarray analysis and flow cytometry findings for (E) Lag-3 and (F) 4-1BB expression levels. Flow cytometry results were gated on
CD3+ and CD4+ cells. Solid lines (  ) indicate C57BL/6 samples, dotted lines (  ) are
MRL samples, and bold dash lines ( -----) are MRL lpr samples.
Figure S4.
Dynamic changes in splenic gene expression in MRL +/+ mice along with aging.
Shown are 57 genes. Each column represents an individual spleen sample. Clustering of the genes (rows) was unsupervised.