Identification of Multiple HLA Class II Epitopes of Aspergillus Fumigatus by Generation of CD4+ T Cell Clones Recognizing the A. Fumigatus proteins Crf1 and Catalase1
Authors: Jolink Hetty , van Oorschot Els , Kuijper Ed J , Drijfhout Jan Willem , van Dissel Jaap T , Falkenburg J.H.Frederik , Heemskerk Mirjam H.M. 
Published: October 2019

Abstract
Abstract 2332
Invasive aspergillosis is a common and life-threatening complication in recipients of allogeneic stem cell transplantation. Patients are at risk in the neutropenic phase, but also after recovery of the neutrophil count there is an increased risk of developing invasive aspergillosis, probably caused by other defects in the innate immune system, or by impaired T cell mediated immunity after stem cell transplantation. In healthy individuals and in patients lymphoproliferative responses to crude Aspergillus extracts and recombinant antigens have been shown. Furthermore, patients after haploidentical stem cell transplantation were less susceptible to aspergillus infection when transferred with T cell lines generated against Aspergillus fumigatus. To facilitate the study of the role of T cell mediated immunity in aspergillus infection and develop new therapeutic strategies to prevent or treat invasive aspergillosis we aimed to identify T cell epitopes of Aspergillus fumigatus.
Peripheral blood mononuclear cells (PBMC) of healthy individuals were stimulated with overlapping 15mer peptides of the Aspergillus fumigatus proteins Crf1 and Catalase1. Directly after stimulation no antigen specific T cells could be detected, however after stimulation with the complete peptide pool, IL-2 and IL-15 for 7 days and subsequent restimulation with peptide pulsed autologous PBMC an increase of activated T cells could be detected in half of the healthy donors, based on IFNγ production, CD154 (CD40 ligand) and CD137 expression. From 6 donors antigen specific CD4+ T cells were single cell sorted 4 hours after restimulation with the complete peptide pool using the IFNγ capture assay or by sorting the CD137+ CD4+ T cells 48 hours after restimulation with the complete peptide pool and cells were clonally expanded. The generated T cell clones were tested for Aspergillus peptide specificity against the complete peptide pool using ELISA to determine the IFNy and IL-4 production. Aspergillus peptide specific clones were further analyzed with subpools of the overlapping peptides, to identify the specific T cell epitope. These subpools are organized in a matrix to enable us to identify the recognized epitope directly from this analysis. Subsequently, the T cell clones were stimulated with the single recognized peptides to confirm the identified epitopes. Five different T cell epitopes of Crf1 were identified: one epitope at position 161–171, which was previously described, and four novel epitopes. For the Catalase1 protein we identified 7 different epitopes, which have not been described before. By using HLA-blocking monoclonal antibodies and an HLA-typed EBV-LCL panel we determined the HLA-restriction of the different T cell epitopes. Two Crf1 epitopes and three Catalase1 epitopes were HLA-DR restricted, and one of the Crf1 epitopes was presented by HLA-DP. The HLA-restriction of the other 6 identified epitopes has not yet been characterized. The T cell clones showed 3 different patterns of cytokine production. Some clones only produced IFNγ, some clones only IL-4 and others produced both IFNγ and IL-4.
Twelve T cell epitopes in two different proteins of Aspergillus fumigatus, presented by various HLA class II molecules, were identified. The generated T cell clones showed a variable pattern of cytokine production. To evaluate whether all these epitopes are relevant for the immune response against aspergillosis, the specificity against Aspergillus fumigatus will be tested by incubating T cells and dendritic cells with inactivated fungus. If Aspergillus-specificity is demonstrated, these epitopes can be used to study T cell mediated immunity in patients with aspergillosis and be a first step towards new therapeutic options for invasive aspergillosis.

Disclosures:
No relevant conflicts of interest to declare.