Screening For Anti-Leukemic CTLs After Allogeneic Hematopoietic Stem Cell Transplantation Applying Streptamer Technology
Authors: Heidenreich Falk , Rücker-Braun Elke , Tuve Sebastian , Schmitz Marc , Wehner Rebekka , Tunger Antje , Odendahl Marcus , Torsten Tonn , Bornhäuser Martin , Schetelig Johannes , Link Cornelia S. 
Published: October 2019

Abstract

Introduction
Greiner et al. demonstrated data suggesting a promising link between CD8+ T cell responses with a specificity of NPM1mut and graft versus leukemia effect (GvL). In patients with acute myeloid leukemia (AML) who underwent hematopoietic stem cell (HSC) transplantation, effector cells of the newly established immune system are thought to elicit a potent graft versus leukemia (GvL) effect eradicating residual leukemic cells. Cytotoxic T-lymphocytes (CTLs) specific for leukemia associated MHC-presented peptides may play a crucial role in the anti-tumor reaction and in achieving complete molecular remission. Leukemic cells are detectable after allogeneic HSCT in AML at least at the level of minimal residual disease (MRD). Malignant cells could therefore be identified and targeted by effector cells of the new immune system and stimulation of CTLs by antigen is assured. Detectable leukemia-specific CTLs will allow studies which address the correlation of the course of CTLs and clinical events. Further, it could allow for an assessment of the functional properties of these cells with respect to GvL effects.

Methods
We screened HLA-A0201 positive patients with AML and chronic lymphocytic leukemia (CLL) biweekly from day 28 after transplantation until day 112 for antileukemic CTLs applying streptamer technology. For AML a panel of 7 MHC peptide complexes was used to label CTLs from peripheral blood: phycoerythrin-(PE)-labeled streptamers refolded with peptides (number of different peptides) derived from the amino acid sequence of the proteins WT1 (1), PR3 (1), RHAMM (1), mutated NPM1 (2), and survivin (2). For CLL peptides derived from RHAMM (1), survivin (2) and fibromodulin (4) were chosen for screening. Peptides from cytomegalovirus (CMV) protein pp65 and from Influenza matrix protein M1 were used as positive controls. A peptide of the HIV reverse transcriptase served as negative control.

Results
Up to now neither for AML (n = 9) nor for CLL (n = 1), leukemia specific CTLs were detected according to the respective panel at any time point. However, it was possible to detect CMV-specific CTLs in most of the CMV seropositive patient-recipient pairs and frequencies increased when CMV reactivation occurred. Increasing frequencies of CMV-specific CTLs were even measured in CMV seropositive patients who received allogeneic HSC grafts from CMV seronegative donors, proving the adequacy of the method and reflecting the stimulation and proliferation of CMV-specific CTLs.

Discussion
Different reasons may account for our negative results so far: the limited numbers of selected peptides, which may not be immunodominant or the small number of screened patients are two of them. It must be considered that the CTL immune response to virus infection and concomitant CMV clearance may not represent a proper model for the GvL effect, which possibly is characterized only by a weak proliferation of CTLs resulting in cell counts below the detection limit of the applied flowcytometric measurements. We will further extend our screening approach to a minimum of 18 individuals per specificity. If frequencies above the limit of detection will not be detected in at least one out of 18 patients the 95 % confidence interval of the true percentage of patients with measurable CTLs of this specificity should be < 15 %.

Conclusions
This study highlights the difficulties to study leukemia-specific CTLs. So far, leukemia-specific CTLs have only been measured in single patients after allogeneic HSCT. Information on cohorts of patients who have been monitored longitudinally for specific CTLs is very rare. The kinetics of leukemia-specific CTLs which mediate GvL effects are almost unknown. Further research on delineating T-cell mediated GvL-effects and realistic estimates for frequencies of specific CTLs at well defined time-points are urgently needed.

Disclosures:
No relevant conflicts of interest to declare.