High titer autoantibodies to GM-CSF in patients with AML, CML and MDS are associated with active disease

Abstract

Antibodies to granulocyte-macrophage colony-stimulating factor (GM-CSF) can be induced when GM-CSF is used as an adjuvant to solid tumor vaccination. Neutralizing anti-GM-CSF IgG has been associated with pulmonary alveolar proteinosis (PAP), and secondary PAP has been linked to myeloid leukemia. We studied 69 patients with acute myeloid leukemia, chronic myeloid leukemia and myelodysplastic syndrome, including 19 patients who received GM-CSF with peptide antigen and incomplete Freund's adjuvant in a vaccine trial for the presence or induction of anti-GM-CSF antibodies. Anti-GM-CSF IgG were present in 36 (52%) patients with myeloid leukemia compared to only 1 of 33 (3%) healthy subjects (P=0.008) and in none of 6 patients with lymphoid leukemia (P=0.0001). Antibody titers were unaffected by vaccination. Anti-GM-CSF IgA and IgM were found in 33 and 20% of patients, respectively; IgA from two patients neutralized GM-CSF. Strikingly, while anti-GM-CSF IgG titers were higher in patients with active disease (n=52) versus those in complete remission (n=14, P=0.0009), GM-CSF expression was not increased in either group. These data are first to show that anti-GM-CSF antibodies of multiple isotypes are present in patients with active myeloid leukemia without PAP and may be useful markers of disease activity.

Figure 1

High titers of anti-GM-CSF IgG are present in myeloid leukemia patients and are unaffected by vaccination with GM-CSF as an adjuvant. (a) Serum anti-GM-CSF IgG titers were evaluated by ELISA in vaccinated (n=19) and non-vaccinated (n=50) patients with myeloid leukemia and in a control group of patients with lymphoid leukemia (n=6) and HD (n=33). The results are the means of two experiments performed in duplicate; horizontal lines are column means. (b) Serum anti-GM-CSF IgG were evaluated in vaccinated patients (n=17) before the first vaccination and 3 weeks after the third vaccination. The lines connect results at different time points for each individual. Relative unit (RU): anti-GM-CSF reactivity of 1 μg IVIg. HD, healthy donors; Igs, immunoglobulins; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 2

High titer of anti-GM-CSF IgG correlates with active disease. Anti-GM-CSF IgG titers were evaluated by ELISA in patients with active leukemia (n=52) and in patients in clinical remission (n=14). Both groups included vaccinated and non-vaccinated patients. Each sample was studied in duplicate. Boxes indicate column medians, 25 and 75th percentile; whiskers indicate the columns' largest and smallest values. Relative unit (RU): anti-GM-CSF reactivity of 1 μg IVIg. ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte-macrophage colony-stimulating factor.

Figure 3

Multiple isotypes of anti-GM-CSF antibodies are present in a subset of patients with AML and CML and are not induced by vaccine. (a) GM-CSF was resolved in 15% SDS-PAGE into three species with molecular weights of 19.5, 16.8 and 15.5 kDa. Western blotting with sera of four representative vaccinated patients (P15, P16, P18 and P19) and consequent probing with antibodies specific to human IgG, IgA and IgM revealed corresponding subclasses of anti-GM-CSF Igs. Antibody MAb 215 (anti-human GM-CSF) and mouse IgG1 were used as positive (P) and negative (N) controls, respectively. (b) Anti-GM-CSF IgA and (c) anti-GM-CSF IgM were present in patients' sera before (−) and after (+) vaccination. ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte-macrophage colony-stimulating factor; Igs, immunoglobulins; MAb, monoclonal antibody; SDS-PAGE, SDS-polyacrylamide gel electrophoresis.

Figure 4

Anti-GM-CSF IgA neutralizes GM-CSF-dependent proliferation of TF1 cells. GM-CSF-reactive Igs were purified from patients' sera by affinity chromatography, as indicated in Patients and methods. Igs from patient P3, which do not bind GM-CSF, were likewise purified for control. Cells were cultured for 48 h in the presence of GM-CSF (2.5 ng ml⁻¹) and Igs (250 μg ml⁻¹). At the end of incubation, MTT assay was performed. GM-CSF neutralizing antibody MAb 215 and murine IgG1 were used as positive (+) and negative (−) controls, respectively. The results are shown as means±s.e.m. in one of three experiments performed in triplicate. ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte-macrophage colony-stimulating factor; Igs, immunoglobulins; MAb, monoclonal antibody.

Figure 5

GM-CSF expression is reduced in non-vaccinated patients with AML. (a) GM-CSF concentration in peripheral blood (12 AML, 10 CML and 30 HD) and bone marrow (12 AML plus CML and 6 non-myeloid malignancies) was evaluated by ELISA. GM-CSF concentration in each sample was calculated as a mean of three independent measurements performed in duplicate. (b) GM-CSF mRNA expression in peripheral blood of patients with myeloid leukemia (n = 12) and HD (n = 9) was evaluated by RT-PCR and subsequent PCR with GM-CSF RNA-specific primers. For both (a) and (b), the boxes are the column medians, 25 and 75th percentiles; whiskers are the largest and smallest values in each group. AML, acute myeloid leukemia; CML, chronic myeloid leukemia; HD, healthy donors; ELISA, enzyme-linked immunosorbent assay; GM-CSF, granulocyte-macrophage colony-stimulating factor; RT-PCR, reverse transcription-PCR.