High Avidity Leukemia-Associated Antigen-Specific CD8+ T Cells Preferentially Localize to the Bone Marrow in Patients with Myeloid Malignancies.

Abstract

Both acute and chronic myeloid leukemias (AML and CML) and myelodysplastic syndromes (MDS) over-express and present self-antigens such as the HLA-A*0201-restricted proteinase 3 (PR1) and Wilm's tumor-1 (WT1) epitopes, making these leukemia-associated antigens selectively amenable to immunotherapeutic intervention. Here, we examined the antigen avidity properties of circulating and bone marrow-resident CD8+ T cells specific for PR1 and WT1 in patients with AML (n=11), CML (n=10) and MDS (n=3). A total of 19 bone marrow (BM) samples and 27 peripheral blood (PB) samples were studied both prior to and following stem cell transplantation (SCT). Cognate HLA-A*0201 tetramers with identical TCR docking platforms were produced using three distinct monomeric HLA-A*0201 complexes with differential coreceptor binding properties to dissect the avidity of antigen binding directly ex vivo:1.“CD8-null” tetramers, which contain a compound D227K/T228A mutation in the a3 domain of the heavy chain that abrogates CD8 binding;2.wildtype tetramers; and,3.“CD8-enhanced” tetramers, which contain a Q115E mutation in the a2 domain of the heavy chain that moderately increases CD8 binding.We have shown previously that CD8-null tetramers engage only high avidity antigen-specific CD8+ T cells; in contrast, CD8-enhanced tetramers can engage populations of antigen-specific CD8+ T cells with low avidities that fall below the threshold for detection with wildtype tetramers. Using these reagents, we developed a polychromatic flow cytometric panel that enabled the simultaneous assessment of phenotype, function and avidity within antigen-specific CD8+ T cell populations. Either PR1- and/or WT1-specific CD8+ T cells were identified in 12/19 BM samples and 6/27 PB samples. Notably, one of the pre-SCT samples contained only low avidity leukemia-associated antigen-specific CD8+ T cells; in contrast, all of the specific populations identified in the post-SCT samples engaged their cognate antigen with high avidity. In 5/7 patients, analysis of paired BM/PB samples revealed the presence of high avidity PR1- and/or WT1-specific CD8+ T cells confined almost exclusively to the BM. Phenotypic analysis demonstrated a mixture of central and effector memory cells in all cases, thereby confirming that these PR1- and WT1-specific CD8+ T cell populations were antigen-experienced. Thus, high avidity CD8+ T cells specific for leukemia-associated antigens are present in vivo and preferentially localize to BM in myeloid malignancies.