us, the data indicate gene expression and pathways can change as a function of time following transplant and this is visualized in Fig 1. At month 6, we also observed lower SNDX 275 levels of genes involved in T-cell signaling pathways which were possibly due to the standard reduction in immune suppressant drugs that occurs after month 3 and a gradual T-cell recovery which occurs as induction therapy effects wane. Discussion This is the first study to conduct whole transcriptome sequencing in PBMCs and characterize changes in expression at multiple times post-transplant. This research design is distinctly unique compared to other studies using microarray of kidney biopsies or blood at one time point following transplant usually at the time of a rejection event. As we hypothesized, PBMC transcripts vary after the initiation of immunosuppression and at different times following kidney allograft transplantation. We showed that many genes had altered expression levels at week 1 and then slowly move towards baseline expression levels as time passes post-transplant. Of major importance, our data show there are substantial transcript expression changes in the blood of patients not experiencing rejection events. For instance, the T-cell signaling components CD3D, CD3E, and CD3G had decreased levels at week 1 post-transplant, but levels increase towards pre-transplant levels at months 3 and 6 in the blood. Speculatively, thymoglobulin induction therapy may be depleting T-cells in the blood that typically express CD3E, CD3G and CD3D. Most pathways among PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19775307 genes with lower levels compared to baseline were T-cell related possibly due to T-cell depleting induction agents or calcineurin inhibitor therapy early after transplant, but the up regulated pathways involve genes in axonal guidance or complement activation. It is possible that axonal guidance genes are involved in signaling the leukocytes or as a cross talk mechanism with the nervous system. However, since our patients did not have rejection, this pathway may represent an allograft tolerance mechanism. Many genes with lower levels compared to baseline, such as killer PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19777456 cell lectin receptors or CD3 signaling components at week 1, or genes with higher levels, such as MMP8 at week 1 or chemokines at month 6 could be expressed by specific cell types. Therefore, it is possible that the expression we observed are due to changes in the abundance of certain lymphocyte subtypes in the blood which express these transcripts. 
It is important that in the future we understand which lymphocytes are responsible for the altered transcripts and their relative frequency in the blood. It is difficult to directly compare our results to published gene expression data since ours is the first study using RNAseq longitudinally in non-rejecting kidney patients’ blood. Many patients at baseline were not on immunosuppression. Comparison of gene expression among the baseline patients on immunosuppression versus those not on immunosuppression did not show any significantly different genes at FDR < 0.01. One previous study did investigate, longitudinally RNA expression by microarray in biopsy and blood, but it is not directly comparable as the study used experimental donor hematopoietic stem cells to induce tolerance in HLA-identical transplants. Another study by Sarwaal and colleagues used microarray and qRT-PCR to detect a set of genes for AR called a kSORT assay. Not surprising, significant genes in our study were not the same as th
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