Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation
Michael T. Falta, … , Clemencia Pinilla, Andrew P. Fontenot
Michael T. Falta, … , Clemencia Pinilla, Andrew P. Fontenot
Published February 25, 2021
Citation Information: J Clin Invest. 2021;131(9):e144864. https://doi.org/10.1172/JCI144864.
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Research Article Pulmonology

Beryllium-specific CD4+ T cells induced by chemokine neoantigens perpetuate inflammation

Abstract

Discovering dominant epitopes for T cells, particularly CD4+ T cells, in human immune-mediated diseases remains a significant challenge. Here, we used bronchoalveolar lavage (BAL) cells from HLA-DP2–expressing patients with chronic beryllium disease (CBD), a debilitating granulomatous lung disorder characterized by accumulations of beryllium-specific (Be-specific) CD4+ T cells in the lung. We discovered lung-resident CD4+ T cells that expressed a disease-specific public CDR3β T cell receptor motif and were specific to Be-modified self-peptides derived from C-C motif ligand 4 (CCL4) and CCL3. HLA-DP2–CCL/Be tetramer staining confirmed that these chemokine-derived peptides represented major antigenic targets in CBD. Furthermore, Be induced CCL3 and CCL4 secretion in the lungs of mice and humans. In a murine model of CBD, the addition of LPS to Be oxide exposure enhanced CCL4 and CCL3 secretion in the lung and significantly increased the number and percentage of CD4+ T cells specific for the HLA-DP2–CCL/Be epitope. Thus, we demonstrate a direct link between Be-induced innate production of chemokines and the development of a robust adaptive immune response to those same chemokines presented as Be-modified self-peptides, creating a cycle of innate and adaptive immune activation.

Authors

Michael T. Falta, Jeremy C. Crawford, Alex N. Tinega, Laurie G. Landry, Frances Crawford, Douglas G. Mack, Allison K. Martin, Shaikh M. Atif, Li Li, Radleigh G. Santos, Maki Nakayama, John W. Kappler, Lisa A. Maier, Paul G. Thomas, Clemencia Pinilla, Andrew P. Fontenot

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Figure 6

Validation of Be-modified CCL4 and CCL3 as antigenic targets of CD4+ T cells in CBD.

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Validation of Be-modified CCL4 and CCL3 as antigenic targets of CD4+ T c...
(A) IL-2 response (mean ± SD pg/ml) of LKGGG CDR3β hybridomas to recombinant CCL4 (upper) and CCL3 (lower) proteins is displayed. Results are representative of 2 experiments. (B) In vitro Be-induced secretion of CCL4 (upper) and CCL3 (lower) from BAL cells isolated from CBD patients (n = 13) is shown. Chemokines were assessed by ELISA in duplicate. Dotted lines represent ELISA limits of detection. Statistical significance was determined using Wilcoxon’s rank sum test. ***P < 0.001. (C) Staining of T cell hybridomas with BeSO4-saturated HLA-DP2 tetramers. (D) Summary of HLA-DP2 tetramer staining of ex vivo BAL cells from BeS (n = 7–8) and CBD (n = 10) subjects. For HLA-DP2+ CBD patients, each subject is represented by a different color/symbol combination. Statistical significance was determined using a Mann-Whitney U test. *P < 0.05; **P < 0.01. (E) Summary of HLA-DP2 tetramer and IFN-γ ICS of ex vivo BAL cells from HLA-DP2+ CBD patients. Color/symbol combinations used for patients match those in D. Percentages (mean ± SEM) of CD4+ T cells expressing IFN-γ and the fraction of CD4+ T cells expressing IFN-γ that also bind each tetramer are shown. Gray circles represent patients with no evidence of active disease. (F) Flow cytometric analysis of HLA-DP2 tetramer and intracellular IFN-γ staining of ex vivo BAL cells from an HLA-DP2– CBD patient is displayed. (G) Flow cytometric density plots showing the frequency of CD4+ T cells from CBD patient 8845 that bind to HLA-DP2–CLIP (upper left), HLA-DP2–CCL4/Be (upper middle), and HLA-DP2–CCL3/Be tetramers. Density plots in lower panels depict tetramer staining in relation to IFN-γ expression induced in CD4+ T cells after stimulation with BeSO4 (lower left, medium control). (H) Tetramer/ICS staining of ex vivo BAL cells from CBD patients 8845 (left) and 8133 (right) 5 years after single-cell TCR studies and flow cytometric analysis of BAL cells. Cells were stained for intracellular IFN-γ (upper panels), HLA-DP2–CCL3/Be (middle panels), and HLA-DP2–PLXN/Be tetramers (lower panels).