Redirected EBV-Specific Stem Cell-Memory and Central-Memory CD8+ T Lymphocytes Exhibit Effective and Sustained Antileukemic Immunity to Acute Myeloid Leukemia in a Patient-Tailored Humanized NSG Mouse Model
Authors: Hartwig Udo F. , Weber Iuliia , Petry Janina , Khan Shamsul A. , Mades Andreas , Theobald Matthias 
Published: October 2019

Abstract
Introduction: Adoptive cellular therapy (ACT) of donor-derived cytolytic T lymphocytes (CTL) directed to leukemia or herpesvirus has proven promising to improve antiviral and antileukemic immunity in patients following allogeneic hematopoietic stem cell transplantation (AHSCT). However, durable clinical responses are often hampered by detrimental graft-versus host (GvH) reactivity and limited persistence of transferred, fully differentiated antileukemic effector T cells (TEFF). We thus explored memory and tumoricidal features of in vitro generated EBV-specific stem cell-memory T cells (TSCM) and central-memory T (TCM) cells, T cell-receptor (TCR) redirected to primary acute myeloid leukemia (AML) blasts, for improved immunotherapy using a patient-tailored minimal residual leukemia NOD/scidIL2Rcg-null (NSG) mouse model.
Methods: Purifiednaive CD8+CD45RA+ T cells from 2 EBV-negative healthy donors were stimulated with EBV-peptide loaded autologous DCs or EBV-transformed B cells (B-LCL) derived from a HLA-class I-matched AML patient in the presence of IL-12, -15, -21 and inhibitors targeting the glycogen synthase kinase-3β thereby blocking the canonical Wnt-signaling pathway for 3-4 weeks. EBV-specific CTL generated from unselected CD8+ T cells served as controls. Phenotype and function as well as metabolic and migratory properties of EBV-specific TSCM/TCM was analyzed by standard assays, glucose uptake and transmigration analyses. αβ-TCR genes isolated from AML-reactive CTL-clones (Albrecht et al. Cancer Immunol. Immunther. 2011), retroviral (RV) transduction and AML-reactivity of eGFP+ TCRVβ7.1+ and TCRVβß21.3+ RV-transduced T(SCM) and T(CM) was tested in vitro according to standard procedures. For in vivo studies redirected T cells were adoptively transferred into AML- and B-LCL-engrafted NSG mice, respectively, and monitored for engraftment, persistence and antileukemic reactivity. All experimental procedures were performed with human specimens obtained with informed consent and were approved by the local ethics committee in compliance of the Helsinki Declaration. Animal studies were approved by institutional supervisory boards and federal law.
Results: Upon 3 to 4 weekly stimulations TSCM and TCM expressing a CD8+CD45RA+CD45RO-CD62L+CCR7+ and CD8+CD45RA-CD45RO+CD62L+CCR7+ phenotype, respectively, elicited strong and to EBV-specific CD8+ TEFF comparable reactivity to HLA class I-matched B-LCL. Additionally, reduced glucose uptake but enhanced CCL-21 driven migration properties confirmed their TSCM and TCM status as described previously. Moreover, TCR-redirected EBV-specific CTL elicited comparable reactivity to AML blasts derived from the same patient as the B-LCL when compared with the original AML-reactive CTL. Finally, adoptive transfer of redirected TSCM and TCM into NSG mice engrafted with ≤ 5% of HLA-class I matched patient-derived AML blasts resembling a minimal residual disease situation upon odoptive transfer resulted in strong reduction of AML-burden and long term persistence (>90d) in NSG mice. Additional studies on boosting antileukemic immunity by additional transfer of irradiated autologous B-LCL into treated mice or supporting T cell homeostasis by weekly i.p. injection of a novel IL-15 superagonist are currently in progress.
Conclusion: These results show that EBV-specific TSCM and TCM redirected to AML can induce sustained antileukemic immunity and thus might represent a promising tool to improve ACT in AHSCT.

Disclosures
No relevant conflicts of interest to declare.