Tumor-initiating cells have been identified for several hematologic malignancies, including CML.20
To date, approved pharmacologic therapies for CML do not effectively eliminate the malignant progenitor cell.21, 22
A persistent malignant progenitor population ensures a continued risk for developing disease resistance and relapse, as evidenced by a continuous relapse rate of 2–3% per year in patients on imatinib therapy.23
Given the susceptibility of CML to immune-mediated destruction and the exquisite specificity of the adaptive immune system, immunotherapy directed at CML progenitor cell targets has the potential to effectively eliminate these cells without concurrent toxicity. Critical to this effort is the identification of antigens specific to the malignant progenitor cell population.
We focused on natural immune responses developing within a clinically homogenous group of patients who experienced clear curative responses (without GvHD) following DLI to reveal targets expressed on progenitor cells. We observed that patients responsive to DLI consistently developed B cell lymphocytosis and high-titer plasma antibodies that were not present prior to therapy, or in patients unresponsive to DLI. The timing of these B cell responses closely correlates with clinical and cytogenetic responses against CML, and potentially represents developing immunity against lysed tumor cells or their extruded contents. We therefore used established and recently available immunoproteomic platforms to identify 62 novel targets of B cell immunity following DLI. Some patient samples were concurrently screened by both cDNA expression library and by protein microarray, and each platform yielded different collections of target antigens. In particular, bacteriophage expression library screening exclusively identified intracellular antigens, similar to previously published studies using this screening strategy.24, 25
In contrast, protein microarray screening yielded antigens with both intracellular and surface expression. The two platforms thus represent complementary tools for detecting serologic responses. Nonetheless, the union of the datasets revealed that, irrespective of platform, a feature common to the entire collection was detectable expression on hematopoietic cells generally, and myeloid progenitor cells specifically. By using the strategy of first discovering immunogenic targets by serologic screening, and then focusing on those with high transcript expression in CML CD34+ cells, we successfully identified a subset of antigens that commonly elicit immune responses among other CML patients with GvL responses.
This antigen subset included RAB38, TBCE and DUSP12. RAB38, a small G-protein involved in endoplasmic reticulum-related vesicle transport, generates specific antibody26
and T cell27
responses in melanoma patients, and is highly expressed in melanoma tissue and absent in normal tissue.28
TBCE is a chaperone for tubulin heterodimerization and microtubule polymerization; individuals with mutations in this gene show defective neutrophil migration and phagocytosis.29
DUSP12, the human orthologue of the yeast YHV1 protein tyrosine phosphatase, may be involved in cell growth regulation.30
Of the three candidate antigens, antibody responses against DUSP12 were the most clearly associated with CML; antibody responses against this antigen were seen in over half the CML patients with GvL studied. Antibody responses against RAB38 also appeared specific to CML. Interestingly, patients with effective immunity against another hematologic malignancy, chronic lymphocytic leukemia, had a similar frequency of antibody responses against TBCE compared to CML patients with effective immunity. TBCE may represent a common GvL-associated immunogen between these two disorders.
RAB38, TBCE and DUSP12, and many other target antigens identified by our serologic screens are intracellular proteins. Antibody responses against these antigens presumably arise as a result of cell lysis, providing access of intracellular contents to the immune system. Recently, several transplant and vaccination studies have shown that development of antigen-specific B cell responses strongly correlated with clinical response.31–33
Because immunity can develop against extruded cellular contents following a cytotoxic response, antibody responses against particular antigens can be reliable biomarkers of effective tumor destruction. This notion is supported by the results of our immunoprecipitation studies (), since CML patients who developed remissions after receiving both immune and pharmacologic therapy commonly developed antibody responses to DUSP12, RAB38 and TBCE, with the greatest frequency and intensity in patients with GvL responses. Alternatively, several studies have supported an active role for antibodies against intracellular antigens in enhancing cellular immunity by facilitating cross-presentation of antigen to T cells.34–37
In the setting of GvL-induced clinically evident immunity, it is likely that coordinated humoral and cellular immunity against leukemia-associated antigens occurs. Therefore, our future studies will explore whether RAB38, DUSP12 and TBCE can elicit T cell responses that are lytic to CML CD34+ cells.
Similar to the previously identified DLI-associated antigens CML66 and CML28,3
most newly identified antigens showed no significant difference in relative gene expression between normal and malignant CD34+ cells. BCR-ABL kinase activity appears to be crucial for the enhancement of immunogenicity of CML cells,38, 39
and the BCR-ABL-positive K562 cell line possesses both standard and immunoproteasomal processing capability.40
Since we are identifying antigen-specific immune responses in patients who have developed GvL but not GvHD, we speculate that CML CD34+ cells potentially process and present antigen differently than normal CD34+ cells, which in turn may enable T cells to discriminate between normal and malignant cells. In support of this concept, cytolytic T lymphocytes specific for the CML-associated antigen proteinase 3 are cytotoxic against CML CD34+ cells but not normal bone marrow-derived CD34+ cells, despite high expression in both malignant and normal progenitor cell populations.41
In the presence of high-titer antigen-specific antibody responses developing following DLI, it is likely that CD4+ T cell responses against CML antigens are also present. However, we have not addressed this question to date.
Our discovery of a large panel of immunogenic antigens present on CML CD34+ cells opens the door for immune monitoring of therapies designed to immunologically target CML progenitor cells, such as stem cell transplantation and whole tumor cell-based vaccines.42
Since the precise immunogens targeted by cell-based treatment strategies are unknown, monitoring immune responses with a large collection of immunogenic progenitor cell-associated antigens would be informative. Finally, with further testing to determine the cytolytic potential and helper function of T cells directed against our identified antigens, our panel of targets may provide a starting point for studies to explore strategies to stimulate polyvalent antigen-specific immunity against malignant CML progenitor cells.