Occurrence of T Cells Specific for the Aspergillus Proteins Crf1 and Catalase1 in Patients Recovering From Invasive Aspergillosis
Authors: Jolink Hetty , van Dissel Jaap T , Falkenburg J.H. Frederik , Heemskerk Mirjam H.M. 
Published: October 2019

Abstract
Abstract 3008
Invasive aspergillosis is a common and life-threatening complication in recipients of allogeneic stem cell transplantation (SCT). Patients are most severely at risk in the neutropenic phase, but there is increasing evidence that impaired T cell mediated immunity also increases the risk of invasive aspergillosis. In healthy individuals we identified Aspergillus -specific T cells by measuring IFN γ production in response to stimulation with overlapping peptides of the A. fumigatus proteins Crf1 and Catalase1. Antigen specific CD4+ T cells were single cell sorted to identify the recognized epitope and Aspergillus specificity was confirmed, based on reactivity to dendritic cells loaded with Aspergillus crude extract. Further characterization of the T cell mediated immune response against A. fumigatus is crucial for the development of new therapeutic strategies including adoptive transfer of antigen specific T cells.
To investigate the role of Aspergillus -specific T cells in the clearance of aspergillus infection, we analyzed the T cell mediated immune response against A. fumigatus in patients who recovered from invasive aspergillosis. All patients had received an allogeneic SCT because of a hematological malignancy and aspergillus infection was diagnosed 4 to 5 months after SCT. At 4 time points after the diagnosis of invasive aspergillosis the T cell mediated immune response was analyzed. Peripheral blood mononuclear cells (PBMC) of patients who recovered from invasive aspergillosis were stimulated with the overlapping peptides of Crf1 and Catalase1. Directly ex vivo no Aspergillus -specific T cells could be detected by intracellular staining for IFN γ and CD154. However, when PBMC were stimulated with the peptide mixtures of Crf1 and Catalase1, cultured for 7 days in the presence of IL-2 and restimulated with Crf1 and Catalase1 we detected clear populations of Aspergillus -specific T cells in 3 of the 4 analyzed patients.
In one patient 3% Crf1-specific and 1.7% Catalase1-specific CD154-expressing CD4+ T cells were shown 1 week after aspergillosis was diagnosed, coinciding with a rapid improvement of the infection with a clear regression of the pulmonary lesions. Up to 45% of the activated T cells produced IFN γ. After resolution of the infection, a decline in the percentage of activated and IFN γ producing T cells was observed, with a low percentage of 0.2% Aspergillus -specific CD4+ T cells persisting 3 years after the clearance of invasive aspergillosis. Two other patients were treated for aspergillosis for a long time, because of a prolonged period of leucopenia. Both were treated with prednisolone, one for GvHD, the other for auto-immune hemolytic anemia and granulocytopenia. The first patient had a mixed radiological response with a decline in the number of peribronchial infiltrates and a newly arisen nodular lesion 3 months after diagnosis, which was stable in size in the following 6 months. During this period no Aspergillus -specific T cells could be detected. After 1 year, 3 months after recovery of the leukocyte count, low numbers of Catalase1-specific CD4+ T cells were present coinciding with regression of the nodular lesion. Both Catalase1-specific (1%) and Crf1-specific (0.3%) CD4+ T cells could be shown 21 months after initial diagnosis, when the nodular lesion was still present, but declining in size. In the other patient 0.1 % Crf1-specific CD4+ T cells were detected during the whole period of Voriconazole treatment. One year after restoration of normal leukocyte counts and clearance of the aspergillus infection 0.65% Crf1-specific CD4+ T cells were present, but no Catalase1-specific T cells could be shown.
In conclusion, using only 2 A. fumigatus proteins as target antigens, we have demonstrated the development of Aspergillus -specific T cells in 3 out of 4 patients, coinciding with the decline of aspergillus lesions. These data indicate that an immune response directed against A. fumigatus proteins helps to clear an aspergillus infection. Therefore, Aspergillus -specific T cells generated in vitro by stimulating with A. fumigatus proteins like Crf1 and Catalase1, may be used for adoptive T cell therapy for invasive aspergillosis.

Disclosures:
No relevant conflicts of interest to declare.