Molecular Identification of Alloreactive CTL Precursors in Hematopoietic Stem Cell Transplantation.
Authors: O’Keefe Christine , Gondek Lukasz , Sobecks Ronald , Rodriguez Alexander , Narvaez Yadira , Tuthill Ralph , Bolwell Brian , Maciejewski Jaroslaw 
Published: April 2020

Abstract
One of the leading complications in HSCT is GvHD mediated by allospecific cytotoxic T cell (CTL) clones that develop from pre-primed or naïve alloreactive precursors present in the graft. The ability to identify GvHD-specific CTL may facilitate development of monitoring tools for management of GvHD. The sequence (or clonotype) of the complementarity determining region 3 of the variable beta (VB) chain of T cell receptor (TCR) can be used as a molecular marker of individual T cell clones that can be detected in blood or tissues affected by GvHD. We have previously shown that although GvHD is a polyclonal process, immunodominant clonotypes and consequently CTL clones can be identified and quantitated (Exp. Hem.32: 1010–1022; Br. J. Haematol.129: 411–419). Although these clonotypes likely correspond to CTL clones participating in the GvHD process their specificity is unknown and may be directed against infectious agents or autoantigens.
To more rationally utilize clonotypic diagnostics using truly allospecific CTL clones we developed new methods of selection of immunodominant alloreactive clonotypes. These clonotypes can be isolated from in vitro activated CTL populations following an allostimulation assay or from tissues affected by GvHD, indirectly indicating their target specificity. In 8 HSCT donor/patient pairs we performed allostimulation cocultures using recipient cells as targets and graft lymphocytes as effectors; activated donor CD8+ T cells were sorted based on CD69 expression and immunodominant CTL clonotypes were analyzed in the activated fraction. were studied. Several immunodominant clonotypes were identified and 11 were found to be restricted to activated CTL. The frequency of 5 immunodominant clonotypes from 2 patients was followed in the post-transplant course by direct sequencing of the appropriate VB family-specific PCR. For 4 of the 5 putatively allospecific clones a clonotype-specific quantitative PCR assay was designed and performed, illustrating that levels of the clonotypes in blood can be monitored using this technology. By direct sequencing, allospecific clonotypes were not detected in biopsies of GvHD-affected tissues. By more sensitive clonotype-specific quantitative PCR assay allostimulation-derived clonotype was also detected in skin biopsy obtained for GvHD diagnosis following transplantation. We have also used a reverse strategy for detection of allsopecific clonotypes. GvHD affected tissues were used as a source of clonotype isolation; VB-specific multiplex PCR assay performed on biopsy-derived DNA from 6/8 of the HSCT recipients was performed and a total of 12 immunodominant clonotypes were identified in 5 patients. We showed that identical CTL clones can be detected in various tissues affected by GvHD (e.g. gut and skin). Moreover, we demonstrate that tissue-derived clonotypes can also be used for clonotype-specific quantitative PCR. For example, 1/3 patients studied tissue-derived clonotype was detected also in blood.
Our studies demonstrate the feasibility of identifying alloreactive precursors in HSC grafts pre-transplant or during the first bout of GvHD. Once such CTL marker clonotypes are identified, they may be selectively depleted from the graft or simply be used as an individualized marker of GvHD activity.