Efficient Identification of T-Cell Clones Associated with Graft-Versus-Host Disease (GvHD) in Target Tissue for Subsequent Detection in Peripheral Blood.

We hypothesized that after allogeneic hematopoietic stem cell transplant (HSCT), GvHD-affected tissues harbor expanded “immunodominant” T-cell clones, and characterization of these clones can be used to develop markers of disease. A multiplex PCR was used to detect T-cell receptor variable beta (VB) chain rearrangements in target tissue. Molecular analysis of the amplified VB CDR3 sequences allowed for identification and quantitation of putative disease-associated “clonotypes” and for the development of clone-specific PCR. We studied 5 HSCT patients for the presence of signature clonotypes in 10 skin biopsies taken during diagnostic GvHD work-up. Size distribution analysis of VB PCR products showed a skewed peak pattern in 9 biopsies; immunodominant clones (per definition frequency ≥30%) were detected in 6/7 biopsies with histologically confirmed GvHD, consistent with the presence of expanded clonotypes and the oligoclonal nature of the tissue-specific alloresponse. Immunodominant clones were also found in 2 of 3 biopsies not diagnostic for GvHD but obtained based on strong clinical suspicion, raising the possibility that they were associated with early evolving GvHD not distinguishable by histology. For example, when serial skin biopsies were analyzed, a GvHD-positive post-transplant d63 biopsy contained an immunodominant clone (frequency 60%), which was also detected in a subsequent biopsy positive for GvHD (frequency 33%). Similar results were seen in another patient, in whom serial biopsies taken on d214 (not diagnostic) and d217 (GvHD-positive) showed an identical immunodominant clone, not present in a d13 GVHD-negative biopsy. This finding suggests that the d214 biopsy might have contained early GvHD that was not detectable morphologically. In a patient who rejected an initial MUD graft (Tx 1) and then received a MUD SCT (Tx 2) from a different, unrelated donor, immunodominant clones were identified in GvHD-positive biopsies following each transplant, that were distinct for each graft. To examine whether immunodominant clonotypes derived from biopsies could be used as markers of disease, clonotypic PCR was developed for an immunodominant biopsy-derived clonotype for each transplant. The Tx 1 clonotype was detected in blood and skin following Tx 1, but not in tissue or blood taken after Tx 2. Specificity and correct size of the clonotypic PCR product were confirmed by both Genescan analysis and sequencing. Clonotypic PCR designed for an immunodominant clonotype from the Tx 2 donor detected the putative allospecific clonotype in serial samples after the second engraftment. Neither clonotype could be found in either donor, indicating that the disease-associated clones expanded to detectable levels following transplant. These results indicate that clonotypic PCR can distinguish distinct GvHD-associated clonotypes from different donors in both blood and tissue following transplant. Monitoring of the relative frequency of disease-associated clones in recipient blood indicated a significant peripheral expansion of disease-associated clones at the time of active GvHD. Our results demonstrate an efficient method for identification of disease-associated clonotypic markers, which can be used to aid diagnosis and monitoring of GvHD.