Abstract

To better understand the cellular events that precede onset of clinically significant acute GVHD, a complication of allogeneic HSCT, we compared global gene expression profiles in patients 3 (days 18–22) and 4 (days 28–32) weeks after transplant. Patients in this study underwent myeloablative-conditioning regimen prior to receiving a T cell replete PBSCT from a related (n=9) or unrelated donor (5 HLA matched and 4 mismatched). Blood was obtained prospectively at scheduled times (prior to administration of glucocorticosteroids). RNA was isolated from nucleated blood cells and biotin-labeled cRNA hybridized on Affymetrix HG-U133A chips. MAS 5.0 software was used to extract gene expression values.

We initially compared gene expression profiles between 15 patients 3 weeks post-HSCT and 10 normal controls. A total of 1176 genes were differentially expressed with statistical criterion of NFD (number of false discovery) equal to 10. Gene profiles for these 1176 genes were compared between 8 patients who subsequently developed GVHD within 1–5 days and 7 patients who remained GVHD free for 90 days. A limited number of genes were differentially expressed with NFD=1: 3 genes in GVHD patients showed increased expression and 6 showed decreased expression.

A second set of experiments was performed to compare changes occurring within individual patients over an interval of 7 days (between weeks 3 and 4) prior to diagnosis of clinically significant GVHD (onset between days 27–32). We used a pair-wise comparison with selection criterion NFD=1. Increased expression prior to GVHD was observed in 55 genes and decreased expression in 88 genes. Approximately 50 of these genes were associated with inflammation and cellular stress response. Using the same statistical criterion we compared gene profiles between weeks 3 and 4 for 3 patients who remained GVHD-free for at least 90 days. Fewer changes were observed with increased expression occurring in 6 genes and decreased expression in 14 genes. These differentially expressed genes did not overlap with the candidate genes associated with the development of GVHD. Genes showing expression changes in GVHD included:

IncreasedDecreased
Inflamamtory Response IFN-α10, IL8, IL17  
Transcription Factors NFATC1 GATA3 
Cell Surface/Signal Transduction  CD6, CD7, CD8, TCR-interacting molecule, MAP4K1, TNFRSF25, 
Effectors Molecules  GRMM 
AICD/Apoptosis  TOSO, BAX 
Cellular Stress Response DDAH1 DLAT, PKC1, COX5B 
IncreasedDecreased
Inflamamtory Response IFN-α10, IL8, IL17  
Transcription Factors NFATC1 GATA3 
Cell Surface/Signal Transduction  CD6, CD7, CD8, TCR-interacting molecule, MAP4K1, TNFRSF25, 
Effectors Molecules  GRMM 
AICD/Apoptosis  TOSO, BAX 
Cellular Stress Response DDAH1 DLAT, PKC1, COX5B 

These results suggest that extensive complex gene expression changes occur among nucleated blood cells during the early post-transplant period presumably due to extensive alterations in cellular activation occurring during reconstitution. The preliminary results of the longitudinal analysis of changes occurring within individual patients indicate that early post-transplant studies are feasible and that they may be informative for yielding insight into the molecular events associated with development of clinically significant GVHD. These data also indicate a paradoxical decrease in certain T cell associated genes in GVHD. However alloimmune induced T cell activation may lead to AICD and previous studies have demonstrated increased apoptosis among peripheral blood T cells in GVHD patients. Further studies including gene expression profiling of isolated T cells will be necessary to determine if this approach can be useful in identifying a molecular “signature” for GVHD that may be useful for diagnosis and monitoring.

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