Object: Acute leukemia is the most common malignancy in children. Despite recent therapeutic advances patients with relapsed or refractory disease require new treatment options. While synthetic immunotherapies such as chimeric antigen receptor (CAR) T-cells have shown impressive efficacy in B-precursor acute lymphoblastic leukemia (BCP-ALL) patients, the interaction between leukemic blasts and bone marrow T-cells remains largely unknown. Therefore, the role for immune response amplifiers in leukemia patients is currently unclear. Leukemia outgrowth leads to low frequency of physiologic bone marrow populations such as T-cells. Those T-cells are consequently within the site of tumor development and can thus be defined as tumor-infiltrating lymphocytes (TILs). Dysfunction of TILs has been described in a variety of solid and in some hematologic malignancies. To determine the changes driven by leukemia blasts we analyzed T-cells in bone marrow samples from pediatric patients with BCP-ALL, T-precursor ALL (TCP-ALL) and acute myelogenous leukemia (AML) at the time of diagnosis and relapse in comparison to healthy bone marrow donors. Material and Methods: In pilot experiments, any artificial changes in marker expression due to cryopreservation and thawing were excluded (n=5). Then, cryopreserved bone marrow samples from both pediatric patients with acute leukemia (n= 77; BCP-ALL: 18, TCP-ALL: 23, AML: 36) and age-matched healthy bone marrow donors (n=23) were identified in our local biobank. Multicolor flow cytometry was performed to quantify co-inhibitory markers on CD4 and CD8 T-cells in primary (n=49) and relapse leukemia samples (n=28). Results: The frequency of bone marrow T-cells was reduced in patients with acute leukemia in comparison with healthy controls (5.9% vs. 24.4%, mean values, p<0.001). This reduction was more pronounced in BCP-ALL than in AML (0.9% vs. 8.4%, p<0.001). The CD4/CD8 ratio of bone marrow T-cells in leukemia patients was not altered compared with healthy controls (1.27 vs. 1.09, p=0.82). The frequency of regulatory T-cells (Tregs, defined as CD4+ CD25+ CD127low T-cells) was decreased in leukemic bone marrow (7.5% vs. 9.8%, p=0.022). However, while BCP-ALL samples did not show a difference in Treg frequency between initial diagnosis and relapse (8.0 vs. 7.2, p=0.86), there was an increase of Tregs at relapse in AML samples (9.5% vs. 6.2%, p=0.004). Surface markers of T-cell exhaustion such as PD1, TIM-3 and LAG3 were found to be consistently more highly expressed on T-cells of leukemia patients than in healthy controls, both on CD4 and CD8 T-cells. PD1 was more highly expressed in relapse samples than in primary diagnosis samples than in healthy controls: (CD4: 42.3% vs. 28.9% vs. 19.8%, p<0.001; CD8: 45.2% vs. 33.3% vs. 26.5%, p=0.002). This observation was consistent for relapse samples in all three different leukemia subtypes both on CD4 and CD8 T-cells. LAG3 expression on T-cells was increased in leukemia patients vs. healthy controls (CD4: 2.6% vs. 0.7%, p<0.001; CD8: 8.6% vs. 2.2%, p<0.001). The same was observed for TIM3 (CD4: 3.7% vs. 1.3%, p=0.002; CD8: 8.5% vs. 3.3%, p<0.001). However, no difference in LAG3 or TIM-3 expression could be observed between primary disease and relapse. Conclusion: By analyzing bone marrow samples from pediatric leukemia patients and healthy controls, we confirm that bone marrow T-cells of leukemia patients show significant changes compared to healthy individuals. Clinical parameters such as relapse status or leukemia subtype are associated with changes in the T-cell phenotype. Most importantly, PD1 surface expression on T-cells was identified as a marker that correlates with disease status (relapse > primary > healthy). These findings could reflect insufficient immune surveillance of pediatric leukemia by bone marrow T-cells and may provide a rationale for future therapeutic interventions.

Citation Format: Semjon Manuel Willier, Paula Rothaemel, Jonas Wilhelm, Dana Stenger, Theresa Käuferle, Irene Schmid, Michael H. Albert, Vera Binder, Franziska Blaeschke, Tobias Feuchtinger. Bone marrow T-cells are tumor-infiltrating T-cells in pediatric patients with acute leukemia and their phenotype reflects immune evasion of leukemic blasts [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A224.