Serologic markers of effective tumor immunity against chronic lymphocytic leukemia include nonmutated B-cell antigens

Abstract

Patients with chronic lymphocytic leukemia (CLL) who relapse after allogeneic transplant may achieve durable remission following donor lymphocyte infusion (DLI), showing the potency of donor-derived immunity in eradicating tumors. We sought to elucidate the antigenic basis of the effective graft-versus-leukemia (GvL) responses associated with DLI for the treatment of CLL by analyzing the specificity of plasma antibody responses developing in two DLI-treated patients who achieved long-term remission without graft-versus-host disease. By probing high-density protein microarrays with patient plasma, we discovered 35 predominantly intracellular antigens that elicited high-titer antibody reactivity greater in post-DLI than in pre-DLI plasma. Three antigens-C6orf130, MDS032, and ZFYVE19-were identified by both patients. Along with additional candidate antigens DAPK3, SERBP1, and OGFOD1, these proteins showed higher transcript and protein expression in B cells and CLL cells compared with normal peripheral blood mononuclear cells. DAPK3 and the shared antigens do not represent minor histocompatibility antigens, as their sequences are identical in both donor and tumor. Although ZFYVE19, DAPK3, and OGFOD1 elicited minimal antibody reactivity in 12 normal subjects and 12 chemotherapy-treated CLL patients, 5 of 12 CLL patients with clinical GvL responses were serologically reactive to these antigens. Moreover, antibody reactivity against these antigens was temporally correlated with clinical disease regression. These B-cell antigens represent promising biomarkers of effective anti-CLL immunity.

Figure 1. Post-DLI plasma contains greater antigen reactivity than pre-DLI plasma

(A) Plots of the pre- vs. post-DLI median background-subtracted fluorescence signal for all microarray proteins for Patients A and B. Each point represents the average across the two replicate protein spots on the microarray. (B) The calculated significance of the measured pre- and post-DLI fluorescence, corrected for protein concentration, for each microarray protein (20). The black line denotes the cutoff function used to determine significance. ♦ significant interaction; other protein.

Figure 2. Validation studies confirm greater antigen-specific antibody reactivity in post-DLI compared to pre-DLI plasma

(A) Schematic representation of the immunoprecipation-based validation assay. (B) Validation of serologic reactivity generated by protein microarray analysis using immunoprecipitation followed by Western blot. Antigens were generated as biotinylated glutathione-S-transferase (GST) fusion proteins by in vitro transcription and translation using rabbit reticulocyte lysate, immunoprecipitated with pre- and post-DLI plasma from Patients A and B, and immunoblotted with streptavidin-HRP. Shown are ZFYVE19 (64kDa), MDS032 (44.5kDa), C6orf130 (45kDa), SERPB1 (70kDa), DAPK3 (80kDa), FAM122A (58kDa), OGFOD1 (91kDa), and EBNA1 (48kDa), with sizes including the antigen sequence on the microarray and attached GST tag. Sample full-length blots are presented in Supplemental Figure 1.

Figure 3. Gene and protein expression of the shared candidate antigens in normal PBMC, normal CD19+ B cells, and CLL cells

(A) Gene expression was measured by gene-specific quantitative real-time PCR and normalized to transcript levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Black bars denote mean values. Significance of the difference between groups by the Wilcoxon rank-sum test is shown on each graph. The number of samples in each group is given below the figures. Candidate antigens with similar relative expression in both normal and CLL B cells (C6orf130, MDS032, ZFYVE19, OGFOD1) or with increased expression in CLL cells compared to normal B cells (DAPK3, SERBP1). (B) Protein expression of candidate antigens measured by Western blot of cell lysates for PBMC (n=2), normal CD19+ B cells (n=2) and CLL cells (n=5). Samples presented are independent of the samples tested in (A). Protein lysates were loaded at 20 µg/lane. Separate Western blots were probed with commercial antibodies to DAPK3, SERBP1, OGFOD1, MDS032, ZFYVE19 and β-actin (as control).

Figure 4. Serologic reactivity against CLL candidate antigens

(A) The antibody responses of Patients A and B temporally correlate with the clinical anti-tumor response. DAPK3 (80kD), OGFOD1 (91kD), ZFYVE19 (64kD) and EBNA1 (48kD) were generated as biotinylated GST-fusion proteins by in vitro transcription and translation. These were immunoprecipitated using Protein A beads with pre- and post-DLI plasma, and detected with streptavidin-HRP. The bottom graphs depict the percent of marrow intertrabecular space filled with CLL cells following DLI and the white blood cell (WBC) counts across time. (B) Plasma reactivity of multiple patient and normal subject groups against antigens DAPK3, OGFOD1 and ZFYVE19 measured using immunoprecipitation. Interactions were graded against that of normal subjects, with light gray denoting weak interactions and dark gray strong interactions. CLL-chronic lymphocytic leukemia; CML-chronic myelogenous leukemia; GvL-Graft versus Leukemia, denoted as present (+) or absent (−); GvHD-Graft versus Host Disease, denoted as present (+) or absent. Treatments included ^*donor lymphocyte infusion (DLI), ^†non-myeloablative allogeneic hematopoietic stem cell transplant (HSCT), and ^‡myeloablative allogeneic HSCT. Numbers denote individual patients, with the first two groups having matched pre- and post-immunotherapy samples. (C) The antibody responses of Patients 8, 9 and 10 against antigens DAPK3, OGFOD1 and ZFYVE19 developed over time after allogeneic HSCT. Antibody responses were measured as in (A). The preparative regimen and salvage chemotherapy for HSCT resulted in disease remission, and precludes comparison of WBC and bone marrow timecourses. Full-length blots are presented in Supplemental Figure 2.