Identification of Low Frequency Multiple Novel Aspergillus Fumigatus Specific MHC Class II Epitopes and Rapid Generation of An Aspergillus-Specific T-Cell Product Based On Activation Dependent Expression of CD154.

Abstract 1170

Poster Board I-192

Despite new antifungal drugs, invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients undergoing myeloablative chemotherapy and allogeneic stem cell transplantation. Invasion of Aspergillus sp. in immuncompetent individuals is primarily controlled by neutrophils, phagocytes and pathogen-specific T_H1 CD4⁺ cells. Therefore, adoptive transfer of Aspergillus-specific CD4⁺ T-cells could protect patients at risk from IA. Favorable candidates for such an approach are GPI-anchored antigens, which have demonstrated induction of protective adaptive immunity in murine studies.

To implement Aspergillus-specific CD4⁺ T-cells into the clinical setting, we aimed (i) to define which GPI-anchored antigen induces T_H1 response, (ii) to map several epitopes and (iii) to generate large amounts of Aspergillus-specific T_H1 cells within a short period.

Several recombinant proteins were either synthesized or were received by J-P Latge. Amongst all tested proteins, only CRF-1 induced repeatedly T_H1 response in healthy individuals.

To identify Aspergillus-specific MHC class II epitopes, we mapped peripheral blood mononuclear cells (PBMC) of healthy individuals with a custom made peptide library of CRF-1 consisting of 95 overlapping 15mer peptides. The library was divided into a complete pool, subpools of 10 and 95 single peptides. No precursor frequencies were detected in interferon-gamma (IFN-g)-ELISPOT using the complete and sub-pools. After 7 days of stimulation with subpools, 9 peptides were identified producing high amount of IFN-g in at least 3 donors with different MHC class II alleles (n=6). CD4+ T-cells clones of one peptide were then established by limited dilution cloning. Restriction to DRB1*0401 was identified using LCLs and a tetramer was generated. The peptide-specific T-cell clones showed functional activity against ethanol-inactivated fungus or fungal extracts presented by dendritic cells.

To generate Aspergillus-specific T_H1 cell lines, we stimulated PBMC with the MHC class II DRB1*0401 restricted peptide. After 7 days of in vitro stimulation (IVS) with interleukin (IL)-2 5U/ml added every other day, tetramer staining was between 0.3 and 11% of CD4⁺ cells (mean 4.2%, n=5). After 14 days of IVS with 1 restimulation of autologous peptide-pulsed monocytes at a responder: stimulator ratio of 5:1 and IL-7 and IL-15 10ng/ml added after day 7, mean percentages of tetramer staining increased to 37% (range 20-57%, n=4) and absolute cell counts were duplicated. The expansion process was further optimized using IFN-g capture assay and CD154⁺ MicroBead Kit (Miltenyi). In both assays, PBMC were stimulated for 16 hours, separated by magnetic beads and co-cultured with irradiated autologous PBMC for 14 days using IL-2 5U/ml till day 7 and IL-7 and -15 10ng/ml thereafter. We were unable to expand specific CD4⁺ cells by IFN-g capture assay in 2 of 3 donors. In contrast, we found that Aspergillus-specific CD4⁺ cells selected by CD154⁺ showed comparable tetramer specificity and expansion but higher functional activity in intracellular cytokine assay and IFN-g ELISA than CD4⁺ cell lines generated from the same donor using the restimulation protocol. The CD4⁺ cell lines generated by CD154⁺ expression showed proliferative capacity in CFSE when restimulated with peptides and functional activity in IFN-g ELISA against fungal extracts (n=4).

To generate Aspergillus-specific T_H1 cell lines recognizing multiple MHC class II epitopes we stimulated PBMC with the previously identified 9 peptides of CRF-1 protein. After 14 days of expansion using CD154⁺ MicroBead Kit, we measured high IFN-g response towards 4 of 9 peptides (n=3). We are generating T-cell clones and will characterize their HLA-restriction.

In summary, we have identified an immunodominant Aspergillus fumigatus protein including 9 MHC class II epitopes. One epitope was characterized specific for an abundant MHC class II allele. CD4⁺ T_H1 cells specific for this epitope can be activated by dendritic cells after uptake of whole fungi or fungal extracts. Furthermore, we have established a GMP-applicable protocol for rapid generation (<14d) of CD4⁺ T-cell lines specific for Aspergillus fumigatus with low precursor frequency using CD154⁺ separation. These CD4⁺ T-cell lines demonstrate functional activity against peptides and fungal extracts that could be prophylactically administered to high risk patients.