Anti-Interleukin-23 Autoantibodies in Adult-Onset Immunodeficiency

Abstract

Background: Autoantibodies against interleukin-12 (anti-interleukin-12) are often identified in patients with thymoma, but opportunistic infections develop in only some of these patients. Interleukin-12 (with subunits p40 and p35) shares a common subunit with interleukin-23 (subunits p40 and p19). In a patient with disseminated Burkholderia gladioli infection, the identification of both anti-interleukin-23 and anti-interleukin-12 prompted further investigation.

Methods: Among the patients (most of whom had thymoma) who were known to have anti-interleukin-12, we screened for autoantibodies against interleukin-23 (anti-interleukin-23). To validate the potential role of anti-interleukin-23 with respect to opportunistic infection, we tested a second cohort of patients with thymoma as well as patients without either thymoma or known anti-interleukin-12 who had unusual infections.

Results: Among 30 patients with anti-interleukin-12 who had severe mycobacterial, bacterial, or fungal infections, 15 (50%) also had autoantibodies that neutralized interleukin-23. The potency of such neutralization was correlated with the severity of these infections. The neutralizing activity of anti-interleukin-12 alone was not associated with infection. In the validation cohort of 91 patients with thymoma, the presence of anti-interleukin-23 was associated with infection status in 74 patients (81%). Overall, neutralizing anti-interleukin-23 was detected in 30 of 116 patients (26%) with thymoma and in 30 of 36 patients (83%) with disseminated, cerebral, or pulmonary infections. Anti-interleukin-23 was present in 6 of 32 patients (19%) with severe intracellular infections and in 2 of 16 patients (12%) with unusual intracranial infections, including Cladophialophora bantiana and Mycobacterium avium complex.

Conclusions: Among patients with a variety of mycobacterial, bacterial, or fungal infections, the presence of neutralizing anti-interleukin-23 was associated with severe, persistent opportunistic infections. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Figure 1.. Discovery, Validation, and Expansion Cohorts in the Study.

Three cohorts of patients were evaluated for the presence of autoantibodies against interleukin-23 (anti–interleukin-23). In the discovery cohort, 30 patients with known autoantibodies against interleukin-12 (anti–interleukin-12) and 30 healthy controls were tested for the presence of anti–interleukin-23. In 17 patients with anti–interleukin-12 who had opportunistic infections, the activity of anti–interleukin-23 was compared with that in 13 such patients without infections. In the validation cohort, banked plasma or serum samples were collected from 91 patients with thymoma, in whom anti–interleukin-12 is often identified. Status with respect to anticytokine autoantibodies was determined to calculate the prognostic accuracy of the presence of neutralizing anti–interleukin-23. In the expansion cohort, screening for anti–interleukin-23 was performed in 128 patients with infections that were similar to those seen in patients with thymoma. Screening was also performed in 971 patients who were healthy or had unrelated infections to establish the specificity of anti–interleukin-23. CMC denotes chronic mucocutaneous candidiasis, CNS central nervous system, Covid-19 coronavirus disease 2019, and MSMD mendelian susceptibility to mycobacterial diseases.

Figure 2.. Activity of Interleukin-12, Interleukin-23, and p40 in the Index Patient and in Healthy Controls.

Panel A shows the results of magnetic bead–based assays indicating human IgG binding of interleukin-12, interleukin-23, and p40 in the plasma of the index patient with disseminated Burkholderia gladioli infection in whom both anti–interleukin-12 and anti–interleukin-23 had been identified. These findings were absent in plasma samples obtained from 30 healthy controls. The fluorescence intensity of anti–interleukin-12 and anti–interleukin-23, along with p40 IgG (as measured by optical density [OD]), was increased in the index patient’s IgG-purified fraction after total IgG had been captured and eluted from a protein G column (GE Healthcare). Panel B shows dose-inhibition curves representing substantial inhibition of phosphorylation of signal transducer and activator of transcription 3 (STAT3) by anti–interleukin-23 IgG in the index patient’s plasma, which markedly inhibited STAT3 phosphorylation at 1 ng per milliliter of interleukin-23. The plasma IgG fraction contains all the interleukin-23–neutralizing activity. In contrast to the replete plasma, which inhibited 100% of STAT3 at 1 ng per milliliter of interleukin-23 after removal of total IgG by means of a protein G column, the index patient’s plasma no longer inhibited interleukin-23 signaling and had a dose responsiveness to interleukin-23 similar to that in control plasma. The I bars indicate the standard deviation. Panel C shows representative flow cytometry plots indicating the percentage of interferon-γ–positive mucosal associated invariant T (MAIT) cells at rest and after costimulation with interleukin-23 and interleukin-18 in the presence of healthy control plasma (two plots at left) as compared with the index patient’s plasma (two plots at right). The synergistic induction of interferon-γ after costimulation with interleukin-23 and interleukin-18 is completely inhibited by the index patient’s plasma containing anti–interleukin-23 IgG (far right). Comp-B710-A denotes compensation matrix for blue laser–excitable 710/40 nm bandpass filter acquisition.

Figure 3.. Effects of Binding and Neutralizing Anti–Interleukin-23 Activity.

In Panel A, among patients who tested positive for anti–interleukin-12, shown is the binding of anti–interleukin-23 (expressed as raw fluorescence intensity) in 17 patients with opportunistic infections as compared with 13 patients without infections, a difference that was not significant. In Panel B, anti–interleukin-23 neutralizing activity is expressed as the effective concentration of interleukin-23 that was required to induce 50% (EC₅₀) of the maximal STAT3 phosphorylation response. This response was significantly more potent in the patients with opportunistic infections than in those without infections. In Panel C, the inhibition of interferon-γ production by anti–interleukin-23 is shown after stimulation with interleukin-23 and interleukin-18. This inhibition correlates with the presence of opportunistic infections (in Group 1) or the absence of infections (in Group 2), according to the percentage of interferon-γ–positive MAIT cells with Live+/CD3+/CD8a+/CD161+/ TCR Vα7.2+ or MR1–5-OP-RU tetramer-binding MAIT cells. In Panels A, B, and C, the I bars indicate the standard deviation. In Panel D, representative fluorescence-activated cell sorter (FACS) plots show the inhibition of interleukin-23–induced interferon-γ production in MAIT cells, which correlates with the presence of opportunistic infections. These values ranged from less than 1% of interferon-γ–positive MAIT cells that were associated with the presence of anti–interleukin-23 IgG in plasma obtained from a patient with multiple opportunistic infections (including recurrent pulmonary nontuberculous mycobacterial disease, recurrent severe Klebsiella pneumoniae, Pseudomonas aeruginosa sinopulmonary infections requiring hospitalization, and CMC to more than 20% MAIT cells in patients with only CMC infections or no infections and in healthy controls.

Figure 4.. Detection of Anti–Interleukin-23 in Unusual Infection Presentations.

In Panel A, bead-based binding assays show the standardized fluorescence intensity of anti–interleukin-23 binding IgG in samples obtained from various patient cohorts along with healthy controls. The study patients included a mixture of those with similar opportunistic infections and those with nonsimilar infections. Highlighted are the findings in the index patient and in two other study patients with unusually severe opportunistic infections of the central nervous system (CNS). One of these patients had intracranial abscesses and obstructive ventriculitis caused by Cladophialophora bantiana, and the other patient had human immunodeficiency virus (HIV) infection with an abscess caused by Mycobacterium avium complex (MAC). In Panel B, magnetic resonance imaging (MRI) shows a T2-weighted sagittal view of the patient with C. bantiana infection. In Panel C, potent inhibition of STAT3 phosphorylation is shown in response to interleukin-23 stimulation in the presence of plasma from the study patient with severe C. bantiana CNS infection. In Panel D, multiplex autoantibody binding assays show isolated human IgG against interleukin-12 and interleukin-23 in the index patient and against interleukin-23 in the patients with C. bantiana and MAC infections, as compared with 30 healthy controls and with 68 HIV controls with opportunistic infections. In Panel E, MRI shows a T1-gadolinium–enhanced axial view of the midbrain of the patient with MAC infection. In Panel F, potent inhibition of STAT3 phosphorylation in response to recombinant interleukin-23 stimulation is shown in the presence of plasma from the patient with MAC infection. APECED denotes autoimmune polyendocrinopathy candidiasis ectodermal dystrophy, and GM-CSF granulocyte–macrophage colony-stimulating factor.