Molecular Analysis of Alloreactive CTL Post-Hemopoietic Stem Cell Transplantation

Abstract

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.

Raising the spectra of T-cell profiling

“…Spectratyping has been used in the past to assess T-cell repertoire recovery after HSCT by several groups. 6,7 The present study by Friedman et al is notable for 2 reasons: First, using spectratyping, they show significant skewing of multiple V␤ families after MLC, and second, they observed the same pattern of skewing in patients after bone marrow transplantation and this correlated with GVHD occurrence.…”

Alloreactive microenvironment after human hematopoietic cell transplantation induces genomic alterations in epithelium through an ROS-mediated mechanism: in vivo and in vitro study and implications to secondary neoplasia

“…Although the same allospecific T-cell clones have been identified both in GvHD lesions and peripheral blood, 35 the frequency of alloreactive clonotypes is much lower in the circulation as compared to target tissues. 36,37 In addition, the degree of human leukocyte antigen mismatches in random MLCs results in higher level of T cell activation as compared to the human leukocyte antigen matched setting of the donor/recipient pair. 38,39 Instead of using post-transplant heterogeneic PBMCs and HaCaT cells as targets, testing of GvHD-related immunodominant T-cell clones isolated from GvHD lesions in a human skin explant model, 40 will probably be more appropriate to predict which patients will develop MSI.…”

Oral graft‐versus‐host disease

“…The increasing success of allogeneic hematopoietic stem cell transplantation (thereafter referred to as HSCT) for treatment of neoplastic and non‐neoplastic hematologic diseases has contributed to its steady increase in use (Flowers et al , 2002; Laughlin et al , 2004; Thomas et al , 2004; Appelbaum, 2007; O’Keefe et al , 2007). Worldwide over 40 000 individuals receive HSCT annually, out of which 15 000 are performed with cells from an allogeneic donor (Flowers et al , 2002).…”