Graft-versus-leukemia antigen CML66 elicits coordinated B-cell and T-cell immunity after donor lymphocyte infusion.

PURPOSE: The target antigens of graft-versus-leukemia that are tumor associated are incompletely characterized. EXPERIMENTAL DESIGN: We examined responses developing against CML66, an immunogenic antigen preferentially expressed in myeloid progenitor cells identified from a patient with chronic myelogenous leukemia who attained long-lived remission following CD4+ donor lymphocyte infusion (DLI). RESULTS: From this patient, CML66-reactive CD8+ T-cell clones were detected against an endogenously presented HLA-B*4403-restricted epitope (HDVDALLW). Neither CML66-specific antibody nor T-cell responses were detectable in peripheral blood before DLI. However, by 1 month after DLI, CD8+ T cells were present in peripheral blood and at 10-fold higher frequency in marrow. Subsequently, plasma antibody to CML66 developed in association with disease remission. Donor-derived CML66-reactive T cells were detected at low levels in vivo in marrow before DLI by ELISpot and by a nested PCR-based assay to detect clonotypic T-cell receptor sequences but not in blood of the patient pre-DLI nor of the graft donor. CONCLUSIONS: CD4+ DLI results in rapid expansion of preexisting marrow-resident leukemia-specific donor CD8+ T cells, followed by a cascade of antigen-specific immune responses detectable in blood. Our single-antigen analysis thus shows that durable posttransplant tumor immunity is directed in part against nonpolymorphic overexpressed leukemia antigens that elicit coordinated cellular and humoral immunity.

Molecular assessment of erythroid lineage chimerism following nonmyeloablative allogeneic stem cell transplantation.

OBJECTIVE: Nonmyeloablative conditioning regimens for allogeneic stem cell transplantation are now commonly used in the treatment of patients with hematologic malignancies. Since this treatment often results in the establishment of mixed hematopoietic chimerism, this approach may also prove to be useful in the treatment of nonmalignant disorders, such as sickle cell disease and thalassemia major. To apply this approach to these diseases, it will be necessary to determine the levels of donor erythropoiesis required to correct hemolysis and ameliorate disease symptoms. Current methods for measuring hematopoietic chimerism are based on DNA polymorphisms that distinguish recipient from donor. These methods accurately measure donor leukocyte engraftment but do not quantify the relative contributions of recipient and donor erythropoiesis following transplant. METHODS: To specifically measure erythroid-lineage chimerism, we used pyrosequencing of the sickle cell mutation to quantify the relative levels of normal and sickle beta-globin mRNA in patient samples. Results of beta-globin RNA chimerism were compared to assessment of beta-globin DNA chimerism as well as analysis of short tandem repeat (STR) polymorphisms, cytogenetics, and hemoglobin electrophoresis. RESULTS: Donor engraftment was measured in two adult patients following nonmyeloablative stem cell transplant for sickle cell disease. In Patient 1, 25 to 30% of peripheral leukocytes were donor derived after day 41. In contrast, more than 55% of peripheral blood beta-globin mRNA was of donor origin, and these results correlated with posttransplant clinical improvement. Patient 2 achieved 40 to 50% donor leukocyte engraftment from day 33 onward. This was associated with 70 to 100% peripheral blood donor beta-globin mRNA. CONCLUSIONS: These studies demonstrate that relatively low levels of donor leukocyte engraftment can be associated with higher levels of donor erythropoiesis and with significant clinical improvement. Pyrosequencing of lineage-specific mRNA directly measures functional reconstitution of donor cells and provides valuable information that can affect clinical decisions in patients with nonmalignant diseases following allogeneic transplant.