Graft-Versus-Leukemia Antigen CML66 Elicits Coordinated B and T Cell Immunity After Donor Lymphocyte Infusion.

Abstract 2449

Poster Board II-426

Donor lymphocyte infusion (DLI) is a highly effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic hematopoietic stem cell transplantation (HSCT), in which 70-80% patients achieve curative responses. Despite its efficacy and established clinical use, the mechanism by which DLI achieves anti-tumor immunity remains incompletely understood. CML66 was discovered as an overexpressed nonpolymorphic CML-associated antigen that elicited a high-titer (>1:50,000) antibody response in a patient who received CD4+ DLI for relapsed CML. CML66 is preferentially expressed on myeloid leukemia progenitor cells and the development of CML66 antibodies temporally correlated with attainment of molecular remission. Since CD4+ T cell responses are involved in generating both antigen-specific B cell and CD8+T cell responses, we queried whether development of potent B cell immunity was also associated with the development of cytolytic T cell immunity against CML66. To detect specific T cell responses, post-DLI PBMC from this patient were screened for reactivity against pools of overlapping 15- to 18-mer peptides encoding the entire protein sequence of CML66. Patient CD8+ T cells were found to be reactive against a single CML66-derived peptide pool, and specifically an 8 amino acid peptide (HDVDALLW), beginning at residue 459. This T cell epitope is positioned in a different region of the protein compared to the B cell epitope (at residues 198-217: GFYVSLEWVTISKKNQDNK). A peptide-specific T cell clone was isolated, whose MHC class I molecule binding was restricted to HLA B*4403. The CML66-reactive T cell clone recognized the HLA-B4403-expressing CD34+ AML cell line MUTZ-3, providing evidence that this epitope is naturally processed and presented by myeloid leukemia cells. Functional cytotoxic T cell responses to CML66 appeared to precede the appearance of antigen-specific antibody responses. While CML66-specific plasma antibody was only detectable starting 2-3 months after DLI, reactivity to the HLA-B4403-restricted epitope by peripheral blood-derived CD8+ T cells was detected by IFNγ ELISpot as early as one month following DLI. To sensitively detect CD8+ CML66-specific T cells in vivo, we sequenced the CDR3 region specific to the CML66-specific clone, and designed clone-specific quantitative PCR primers. Nested PCR of RNA directly extracted from patient PBMC revealed only minimal detection of the CML66-specific T cell clone prior to DLI, but then expansion between 1 to 6 months after DLI. Similar analysis of marrow-derived RNA also revealed the presence of CML66-specific T cell clone in patient marrow following DLI. Moreover, this clone was already detected in patient marrow prior to DLI, suggesting that marrow is a reservoir of leukemia-specific T cells following hematopoietic stem cell transplantation. Our studies provide the first description of coordinated adaptive immunity developing against a nonpolymorphic leukemia-associated antigen, in close temporal correlation with the attainment of long-lasting immune-mediated clinical remission after allogeneic HSCT. The detected cytolytic T cell response was associated with the secretion of high-titer IgG antibody specific for a distinct epitope in the same target protein. In this setting, our results indicate that infusion of CD4+ donor cells resulted in the rapid activation and expansion of pre-existing marrow-resident leukemia-specific CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in the periphery.