Detection of a potent humoral response associated with immune-induced remission of chronic myelogenous leukemia

Abstract

The effectiveness of donor-lymphocyte infusion (DLI) for treatment of relapsed chronic myelogenous leukemia (CML) after allogeneic bone marrow transplantation is a clear demonstration of the graft-versus-leukemia (GVL) effect. T cells are critical mediators of GVL, but the antigenic targets of this response are unknown. To determine whether patients who respond to DLI also develop B-cell immunity to CML-associated antigens, we analyzed sera from three patients with relapsed CML who achieved a complete molecular remission after infusion of donor T cells. Sera from these individuals recognized 13 distinct gene products represented in a CML-derived cDNA library. Two proteins, Jkappa-recombination signal-binding protein (RBP-Jkappa) and related adhesion focal tyrosine kinase (RAFTK), were recognized by sera from three of 19 DLI responders. None of these antigens were recognized by sera from healthy donors or patients with chronic graft-versus-host disease. Four gene products were recognized by sera from CML patients treated with hydroxyurea and nine were detected by sera from CML patients who responded to IFN-alpha. Antibody titers specific for RAFTK, but not for RBP-Jkappa, were found to be temporally associated with the response to DLI. These results demonstrate that patients who respond to DLI generate potent antibody responses to CML-associated antigens, suggesting the development of coordinated T- and B-cell immunity. The characterization of B cell-defined antigens may help identify clinically relevant targets of the GVL response in vivo.

Figure 1

Quantitative serum reactivity against RAFTK and C57(RBP-Jκ) measured using ELISA. Normal sera (n = 13), sera from CML patients (n = 12) receiving hydroxyurea, and pre-BMT sera from DLI responders (n = 13) demonstrate no reactivity to RAFTK or C57 (RBP-Jκ) compared with reactivity against GST negative control. One patient with CML demonstrated significant reactivity against RAFTK. All sera were diluted at 1:200.

Figure 2

Competitive ELISA to confirm specificity of the antigen-antibody interaction in patient sera. Patient A serum was reactive with C57 (RBP-Jκ) and patient B serum was reactive with RAFTK. Patient serum samples were preincubated for 3 hours at room temperature with increasing amounts of RAFTK-GST (open diamonds, dotted line), C57-GST (open squares, dashed line), GST alone (open circles, dashed line), or with PBS (filled triangles, straight line), and then tested using ELISA for reactivity with C57 (a) or with RAFTK (b). Only preincubation with specific target protein resulted in inhibition of reactivity measured by ELISA.

Figure 3

Quantitative antibody reactivity to RAFTK (a) and C57(RBP-Jκ) (b) was measured using ELISA in responders at various times before and after DLI. Serial serum samples from three DLI responders (1:200 dilution) were tested for specific reactivity against RAFTK, and serial serum samples from four DLI responders were tested for reactivity against C57(RBP-Jκ). The dashed line in each part indicates the upper limit of the negative control (two SD above the mean ratio of OD [test antigen]/OD GST for 13 normal donor sera).