Pneumocystis Jirovecii Pneumonia: The Potential of KEX1, MSG1, and MSG2 as Key Antigens in Cytokine Release Assays

Abstract

Background/Objectives:Pneumocystis jirovecii pneumonia (PJP) is the most frequently diagnosed AIDS-defining illness in Europe, with especially high mortality in HIV-negative patients caused by delayed diagnosis and low awareness. This study aims to evaluate cytokine release assays (CRA) to facilitate a less invasive and resource-efficient PJP specific diagnostic test. We focus on the P. jirovecii antigens Kexin 1 (KEX1), MSG1, and MSG2, which were identified in prior studies as immunologically relevant. Methods: Whole blood samples from 50 participants-22 healthy individuals and 28 immunocompromised individuals, including 8 with proven PJP-were stimulated in vitro with full-length and partial KEX1, MSG1, MSG2, and a combination of all three antigens (PJ-MIX). Following 24 h incubation at 37 °C, cytokine levels of IL-2, IFN-γ, IL-17A, and IL-17F were measured. Results: Stimulation with full-length KEX1, MSG1, MSG2, and PJ-MIX antigens induced higher IL-2 concentrations in the healthy control group compared to the groups IL-2 baseline levels and to the group of proven PJP cases. Similarly, stimulation with full-length KEX1, MSG1, and PJ-MIX elevated IFN-γ levels in the healthy control group compared to baseline IFN-γ levels. Conclusions: Our findings highlight the potential of IL-2 and IFN-γ release following stimulation with PJ antigens, with PJ-MIX eliciting the strongest and most significant responses, suggesting a cumulative antigen effect. This pilot study establishes a foundation for a PJP-specific CRA, deepening our knowledge of T-cell immunity against PJP. Clinically, such a test could, among other applications, evaluate at-risk patients who should receive prophylaxis and may consequently reduce PJP-related morbidity and mortality.

Keywords: IL-17A; IL-17F; Kexin 1; MSG1; MSG2; interferon-γ release assay; interleukin-2 release assay; pneumocystis jirovecii pneumonia.

Figure 1

IFN-γ concentrations across all cohorts after stimulation with reference antigens (positive control) ¹. (a) ConA stimulation significantly increased IFN-γ in healthy controls compared to proven PJP cases (** p = 0.0055) (Mann–Whitney U test); (b) SEB stimulation significantly increased IFN-γ in healthy controls compared to proven PJP cases (** p = 0.0078) and immunocompromised control group (*** p = 0.0002) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort specified at the bottom of the figure.

Figure 2

¹ IFN-γ concentrations of healthy controls vs. proven PJP cases after full-length MSG1 stimulation compared to the baseline (NaCl). MSG1 significantly increased IFN-γ in healthy controls compared to the baseline (* p = 0.0188) (Kruskal–Wallis test with Dunn’s multiple comparisons).¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.

Figure 3

¹ IFN-γ concentrations in healthy controls vs. proven PJP cases after full-length PJ-MIX stimulation compared to the baseline (NaCl). PJ-MIX significantly increased IFN-γ in healthy controls compared to the baseline (*** p = 0.0002) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.

Figure 4

IL-2 concentrations across all cohorts following reference antigen stimulation (positive control). ¹ (a) ConA significantly increased IL-2 in healthy controls compared to proven PJP cases (** p = 0.0040) (Mann–Whitney U test); (b) SEB significantly increased IL-2 in healthy controls compared to proven PJP cases (*** p = 0.0004) and the immunocompromised controls (* p = 0.0499) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort specified at the bottom of the figure.

Figure 5

¹ IL-2 concentrations in healthy controls vs. proven PJP cases following full-length KEX1 stimulation compared to the baseline (NaCl). KEX1 significantly increased IL-2 in healthy controls compared to proven PJP cases (** p = 0.0095) and relative to its baseline (*** p < 0.0001) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.

Figure 6

¹ IL-2 concentrations in healthy controls vs. proven PJP cases After full-length MSG1 stimulation compared to the baseline (NaCl). MSG1 significantly increased IL-2 in healthy controls relative to the baseline (*** p < 0.0001) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.

Figure 7

¹ IL-2 concentrations in healthy controls vs. proven PJP cases following full-length MSG2 stimulation compared to the baseline (NaCl). MSG2 significantly increased IL-2 in healthy controls compared to both proven PJP cases (* p = 0.0175) and the baseline (** p = 0.0038) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.

Figure 8

¹ IL-2 concentrations in healthy controls vs. proven PJP cases following PJ-MIX stimulation compared to the baseline (NaCl). PJ-MIX significantly increased IL-2 in healthy controls relative to the baseline (*** p < 0.0001) (Kruskal–Wallis test with Dunn’s multiple comparisons). ¹ Circles, squares and triangles represent the respective measurement of one participant from the cohort and stimulation condition specified at the bottom of the figure.