Acquired predisposition to mycobacterial disease due to autoantibodies to IFN-gamma

Abstract

Genetic defects in the IFN-gamma response pathway cause unique susceptibility to intracellular pathogens, particularly mycobacteria, but are rare and do not explain mycobacterial disease in the majority of affected patients. We postulated that acquired defects in macrophage activation by IFN-gamma may cause a similar immunological phenotype and thus explain the occurrence of disseminated intracellular infections in some patients without identifiable immune deficiency. Macrophage activation in response to IFN-gamma and IFN-gamma production were studied in whole blood and PBMCs of 3 patients with severe, unexplained nontuberculous mycobacterial infection. In all 3 patients, IFN-gamma was undetectable following mitogen stimulation of whole blood, but significant quantities were detectable in the supernatants of PBMCs when stimulated in the absence of the patients' own plasma. The patients' plasma inhibited the ability of IFN-gamma to increase production of TNF-alpha by both autologous and normal donor PBMCs, and recovery of exogenous IFN-gamma from the patients' plasma was greatly reduced. Using affinity chromatography, surface-enhanced laser desorption/ionization mass spectrometry, and sequencing, we isolated an IFN-gamma-neutralizing factor from the patients' plasma and showed it to be an autoantibody against IFN-gamma. The purified anti-IFN-gamma antibody was shown to be functional first in blocking the upregulation of TNF-alpha production in response to endotoxin; second in blocking induction of IFN-gamma-inducible genes (according to results of high-density cDNA microarrays); and third in inhibiting upregulation of HLA class II expression on PBMCs. Acquired defects in the IFN-gamma pathway may explain unusual susceptibility to intracellular pathogens in other patients without underlying, genetically determined immunological defects.

Figures

Figure 1

IFN-γ production in response to mitogen stimulation in whole blood (WB) and isolated PBMCs. IFN-γ was not detected in patients’ plasma derived from whole blood after stimulation with PHA (or PMA/ionomycin in patient 2), whereas large quantities of IFN-γ were detected when isolated PBMCs were studied in the absence of patient plasma. IFN-γ production in PBMCs is expressed as a percentage of that in blood of healthy controls studied in the same experiments. Supernatants from triplicate samples of whole blood or PBMCs were pooled for each patient and control and assayed in duplicate by ELISA. The coefficients of variation (CVs) in each assay were less than 5%.

Figure 2

IFN-γ–induced TNF-α upregulation is inhibited in the presence of patients’ sera. PBMCs from healthy donors showed marked upregulation of TNF-α production when exposed to concentrations of IFN-γ as low as 20 ng/ml in the presence of control serum (black) or fetal calf serum (blue). Addition of IFN-γ failed to upregulate TNF-α production in the presence of patients’ serum (red) at concentrations of IFN-γ from 20 to 100 ng/ml, and upregulation was only seen at very high concentrations. The figure shows a representative experiment using serum from patient 1. All samples were set up in triplicate and supernatants pooled for IFN-γ ELISA measurements in duplicate wells. The CV between duplicates was less than 5%.

Figure 3

Recovery of IFN-γ from patient or control serum. Increasing concentrations of purified IFN-γ were added to patient (red) or control serum (blue). After 1 hour incubation at room temperature, the concentration of IFN-γ in the serum was determined by ELISA. Exogenous IFN-γ was not recovered from the patients’ serum even when added at concentrations 5 logs greater than that required for recovery from normal serum. The figure shows a representative experiment using serum from patient 1. All samples were set up in triplicate and supernatants pooled for IFN-γ ELISA measurements in duplicate wells. The CVs between duplicates were less than 5%.

Figure 4

High-density cDNA microarrays showing functional activity of IFN-γ–binding antibody. PBMCs from a healthy donor were exposed to 4 different conditions — no treatment with IFN-γ or antibody (control); IFN-γ alone; IFN-γ with the patient antibody (pt Ab); and IFN-γ with a nonspecific antibody (ns Ab) — each over 4 time points (0, 2, 6, and 21 hours). After data selection and normalization, we filtered for genes demonstrating at least 2.5-fold change from baseline at any 2 of the time points surveyed. The resulting genes were ordered by agglomerative hierarchical clustering (average linkage method) using Cluster software. The genes are displayed in rows, time points in columns. Induced genes are depicted in red, repressed genes in green. Gray represents missing data. When the patient’s purified anti–IFN-γ antibody was present with IFN-γ, the gene expression profile most closely resembled that of the control subject. Cluster I shows TNF genes; cluster II, genes most affected by the presence of the antibody; and cluster III, expression of IFN-γ.

Figure 5

Inhibition of HLA class II expression by IFN-γ–binding antibody. The antibody isolated from the serum of the patient is able to neutralize the function of IFN-γ: a 1:10 dilution of the antibody in the cell culture decreases the effect of IFN-γ in upregulating HLA-DR expression on monocytes. (A) Results expressed as mean fluorescence index (MFI) of HLA-DR on the surface of the monocyte population, based on triplicate measurements. (B) Representative plot defining HLA-DR positivity. A monocyte gate was applied to the PBMCs stained for HLA-DR.