Molecular analysis of alloreactive CTL post-hemopoietic stem cell transplantation.

The development of laboratory tests for the diagnosis and monitoring of graft-vs-host disease (GVHD) is hampered by a lack of knowledge of minor histocompatibility Ags triggering alloresponses. We hypothesized that the unique molecular structure of the TCR could be used as a marker for the unidentified Ags and exploited for molecular monitoring of GVHD posttransplant. To identify alloreactive T cell clones, we performed in vitro allostimulation cultures for a cohort of patients undergoing hemopoietic stem cell transplantation and determined the sequence of the CDR3 of immunodominant alloreactive clones; 10 corresponding clonotypes restricted to activated T cells were identified. As an alternative method for the identification of alloreactive clones, molecular TCR analysis was applied to biopsies of GVHD-affected tissues. Culture- and biopsy-derived clonotypes were used to design sequence-specific quantitative PCR assays to monitor the levels of putative allospecific clonotypes in posttransplant blood samples and subsequent biopsies. Because of the rational design of the methods used to identify immunodominant clonotypes, we were able to follow the behavior of potentially GVHD-specific T cells during the transplant course. Based on our results, we conclude that molecular T cell diagnostics can be a powerful tool for monitoring immune responses posttransplantation.