Anti-IFN-γ autoantibodies underlie disseminated Talaromyces marneffei infections

Abstract

Talaromyces marneffei causes life-threatening opportunistic infections, mainly in Southeast Asia and South China. T. marneffei mainly infects patients with human immunodeficiency virus (HIV) but also infects individuals without known immunosuppression. Here we investigated the involvement of anti-IFN-γ autoantibodies in severe T. marneffei infections in HIV-negative patients. We enrolled 58 HIV-negative adults with severe T. marneffei infections who were otherwise healthy. We found a high prevalence of neutralizing anti-IFN-γ autoantibodies (94.8%) in this cohort. The presence of anti-IFN-γ autoantibodies was strongly associated with HLA-DRB1*16:02 and -DQB1*05:02 alleles in these patients. We demonstrated that adult-onset acquired immunodeficiency due to autoantibodies against IFN-γ is the major cause of severe T. marneffei infections in HIV-negative patients in regions where this fungus is endemic. The high prevalence of anti-IFN-γ autoantibody-associated HLA class II DRB1*16:02 and DQB1*05:02 alleles may account for severe T. marneffei infections in Southeast Asia. Our findings clarify the pathogenesis of T. marneffei infection and pave the way for developing novel treatments.

Figure 1.

Anti–IFN-γ autoantibodies in plasma of patients with severe T. marneffei infection. (A) An IFN-γ–immobilized plate was used to detect antibodies against IFN-γ in plasma. After applying serially diluted patient plasma, peroxidase-conjugated anti-human IgG was added to detect and quantify the presence of human autoantibodies against IFN-γ. Low OD values were obtained with plasma from healthy donors (107 cases, filled square) and three antibody-negative patients (Ab [−] patients, filled triangle), while high values (OD > 0.5 in 1:100 dilution) were detected in 55 antibody-positive patients (Ab [+] patients, filled circle). (B) Plasma from anti–IFN-γ autoantibody–positive patients interfered with the detection of human IFN-γ. Serially diluted patient plasma samples were respectively incubated with a fixed concentration (100 pg/ml) of IFN-γ. The amount of remaining unbound IFN-γ was detected by peroxidase-conjugated anti–IFN-γ antibodies. (C–E) Anti–IFN-γ autoantibodies from T. marneffei–infected patients showed neutralizing activity in vitro. IFN-γ–neutralizing activity was addressed by detecting the impact of patient plasma on IFN-γ–induced HLA-DR expression of THP-1 cells (C and D) and STAT-1 phosphorylation (pSTAT-1) in THP-1 cells (E). (C) THP-1 cells without IFN-γ were used as a negative control (blue peaks) as compared with those with IFN-γ induction (red peaks). (D) A scatter plot summarizing the result of HLA-DR expression with all the patient plasma samples in this cohort is shown. (E) STAT-1 phosphorylation in THP-1 cells was detected by flow cytometry. The result was shown as fold induction measured by the ratio of mean fluorescence intensity relative to those of cells without IFN-γ activation. All results are representative of at least two independent experiments. (D and E) Values represent median with interquartile range. The statistical analysis was performed by Mann–Whitney test. ****, P < 0.0001. NA, not activated.

Figure S1.

Determination of anti–IFN-γ autoantibody IgG subtypes. Representative bar graph shows the IgG subclass of IFN-γ–reactive antibodies from selected patient plasma (1:1,000 diluted) as determined by indirect ELISA. The assays were performed in duplicate independently. HC, healthy control.

Figure S2.

Plasma from patients with anti–IFN-γ autoantibodies inhibited the IFN-γ–mediated clearance of T. marneffei in THP-1 cells. THP-1 cells were stimulated with 20 ng/ml PMA for 48 h followed by resting for 12 h in refreshed medium. The differentiated cells were incubated with plasma in the absence or presence of IFN-γ (50 ng/ml) for 24 h. The cells were reseeded and co-cultured with T. marneffei yeasts (multiplicity of infection, 0.05) for 2 h. The cells were washed twice, then further cultured in RPMI-1640 medium containing 10% FBS, 1% penicillin/streptomycin, and 0.03 µg/ml amphotericin B for 48 h, followed by cell lysis and fungal CFUs counting. The results were shown as mean with SD obtained from two independent experiments. Statistical analysis was performed with the Student t test. **, P < 0.01; ***, P < 0.001. HC, healthy control; NA, not activated; ns, not significant.

Figure S3.

The plasma of case 28 contains nonneutralizing autoantibodies against GM-CSF. (A) The autoantibodies against GM-CSF were detected by indirect ELISA with serially diluted plasma. C.B., coating buffer only; HC, healthy control. (B) GM-CSF neutralizing activity of the plasma was performed by STAT-5 phosphorylation (pSTAT-5) assay with peripheral blood mononuclear cells from healthy volunteers. IL-3 treatment was used as a positive control for pSTAT-5. Plasma from one anti–GM-CSF autoantibody patient served as positive control (Kuo et al., 2017). The assays were performed in duplicate independently. NA, not activated.