Antigen specificity and cross-reactivity drive functionally diverse anti–Aspergillus fumigatus T cell responses in cystic fibrosis
Carsten Schwarz, … , Alexander Scheffold, Petra Bacher
Carsten Schwarz, … , Alexander Scheffold, Petra Bacher
Published January 26, 2023
Citation Information: J Clin Invest. 2023;133(5):e161593. https://doi.org/10.1172/JCI161593.
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Clinical Research and Public Health Immunology Pulmonology

Antigen specificity and cross-reactivity drive functionally diverse anti–Aspergillus fumigatus T cell responses in cystic fibrosis

Abstract

BACKGROUND The fungus Aspergillus fumigatus causes a variety of clinical phenotypes in patients with cystic fibrosis (pwCF). Th cells orchestrate immune responses against fungi, but the types of A. fumigatus–specific Th cells in pwCF and their contribution to protective immunity or inflammation remain poorly characterized.METHODS We used antigen-reactive T cell enrichment (ARTE) to investigate fungus-reactive Th cells in peripheral blood of pwCF and healthy controls.RESULTS We show that clonally expanded, high-avidity A. fumigatus–specific effector Th cells, which were absent in healthy donors, developed in pwCF. Individual patients were characterized by distinct Th1-, Th2-, or Th17-dominated responses that remained stable over several years. These different Th subsets target different A. fumigatus proteins, indicating that differential antigen uptake and presentation directs Th cell subset development. Patients with allergic bronchopulmonary aspergillosis (ABPA) are characterized by high frequencies of Th2 cells that cross-recognize various filamentous fungi.CONCLUSION Our data highlight the development of heterogenous Th responses targeting different protein fractions of a single fungal pathogen and identify the development of multispecies cross-reactive Th2 cells as a potential risk factor for ABPA.FUNDING German Research Foundation (DFG), under Germany’s Excellence Strategy (EXC 2167-390884018 “Precision Medicine in Chronic Inflammation” and EXC 2051-390713860 “Balance of the Microverse”); Oskar Helene Heim Stiftung; Christiane Herzog Stiftung; Mukoviszidose Institut gGmb; German Cystic Fibrosis Association Mukoviszidose e.V; German Federal Ministry of Education and Science (BMBF) InfectControl 2020 Projects AnDiPath (BMBF 03ZZ0838A+B).

Authors

Carsten Schwarz, Patience Eschenhagen, Henrijette Schmidt, Thordis Hohnstein, Christina Iwert, Claudia Grehn, Jobst Roehmel, Eva Steinke, Mirjam Stahl, Laura Lozza, Ekaterina Tikhonova, Elisa Rosati, Ulrik Stervbo, Nina Babel, Jochen G. Mainz, Hilmar Wisplinghoff, Frank Ebel, Lei-Jie Jia, Matthew G. Blango, Peter Hortschansky, Sascha Brunke, Bernhard Hube, Axel A. Brakhage, Olaf Kniemeyer, Alexander Scheffold, Petra Bacher

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Figure 2

A. fumigatus–reactive regulatory CD4+ T cell responses in pwCF.

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A. fumigatus–reactive regulatory CD4+ T cell responses in pwCF. (A) Dot...
(A) Dot plot examples of purified CD25+CD127– Tregs from PBMCs of healthy controls or pwCF. (B) Purified CD25+CD127– Tregs were stimulated with PMA/ionomycin and analyzed for the expression of Foxp3, Helios, CD137, CTLA4, LAP, and GARP by flow cytometry. (C) In vitro suppression assay with polyclonal Tregs from healthy controls or pwCF. Ex vivo–isolated Tregs were combined with proliferation dye–labeled allogeneic CD4+ Tresp cells at different Treg to Tresp ratios. Cells were stimulated polyclonally with CD3/CD28 beads. The percentage of inhibition of Tresp proliferation is shown for healthy controls (n = 5) and pwCF (n = 6). Right: Dot plot examples, with the numbers indicating the percentage of Tresp proliferation. (D) Percentage of memory cells within polyclonal CD25+CD127– Tregs from healthy controls (n = 149) or pwCF (n = 200). (E) Representative dot plot examples for the parallel ex vivo detection of A. fumigatus–reactive CD4+ Tcons (CD154+) and Tregs (CD137+) by ARTE. PBMCs (1 × 107) were stimulated with A. fumigatus or left unstimulated, and cell counts before and after combined magnetic CD154+CD137+ enrichment are indicated. (F) Overlayed flow-cytometric analysis of A. fumigatus–specific CD154+ and CD137+ cells. Numbers indicate the percentages among CD137+CD4+ T cells (light blue) and CD154+CD4+ T cells (dark blue). (G) A. fumigatus–reactive CD137+ and CD154+ Tmem cells (n = 3) were purified by FACS and analyzed for gene expression by real-time PCR. Data were normalized to GAPDH gene expression. (H) Frequencies of A. fumigatus–reactive CD137+ Tregs from healthy donors (n = 161) and pwCF (n = 135). (I) CD45RA and Ki-67 staining of A. fumigatus–reactive CD137+ Tregs. The percentage of marker-positive cells within CD137+CD25+ Tregs is indicated. (J) Percentage of memory cells within A. fumigatus–reactive CD137+ Tregs (healthy donors, n = 161; pwCF, n = 135). (K) Ki-67 expression of A. fumigatus–reactive CD137+ Tregs (healthy donors, n = 46; pwCF, n = 25). (L and M) TCR-β sequence analysis of the top 50 expanded A. fumigatus–specific Treg clones from healthy individuals (n = 4) and pwCF (n = 3). (L) Gini index depicting the distribution of TCR sequences. (M) Rényi diversity profiles. (N) In vitro suppression assay with ex vivo–isolated A. fumigatus–reactive Tregs. A. fumigatus–reactive CD137+ Tregs were combined with proliferation dye–labeled allogeneic responder CD4+ T cells (Tresp) at a Treg to Tresp ratio of 1:4. Cells were stimulated polyclonally with CD3/CD28 beads. The percentage of inhibition of Tresp proliferation is shown for healthy controls (n = 8) and pwCF (n = 7). Each symbol in B–D, G, H, J–L, and N represents 1 donor; horizontal lines indicate the mean in B–D, H, J, K, and M. Truncated violin plots with quartiles and range are shown in G. Box-and-whisker plots display the quartiles and range in L. Data indicate the mean ± SEM in M. Statistical differences were determined by 2-tailed Mann-Whitney U test in B, D, H, J, K, and N and by 2-tailed, unpaired t test in L.