Rapid and sustained T cell-based immunotherapy against invasive fungal disease via a combined two step procedure

Abstract

Introduction: Aspergillus fumigatus (Asp) infections constitute a major cause of morbidity and mortality in patients following allogeneic hematopoietic stem cell transplantation (HSCT). In the context of insufficient host immunity, antifungal drugs show only limited efficacy. Faster and increased T-cell reconstitution correlated with a favorable outcome and a cell-based therapy approach strongly indicated successful clearance of fungal infections. Nevertheless, complex and cost- or time-intensive protocols hampered their implementation into clinical application.

Methods: To facilitate the clinical-scale manufacturing process of Aspergillus fumigatus-specific T cells (ATCs) and to enable immediate (within 24 hours) and sustained (12 days later) treatment of patients with invasive aspergillosis (IA), we adapted and combined two complementary good manufacturing practice (GMP)-compliant approaches, i) the direct magnetic enrichment of Interferon-gamma (IFN-γ) secreting ATCs using the small-scale Cytokine Secretion Assay (CSA) and ii) a short-term in vitro T-cell culture expansion (STE), respectively. We further compared stimulation with two standardized and commercially available products: Asp-lysate and a pool of overlapping peptides derived from different Asp-proteins (PepMix).

Results: For the fast CSA-based approach we detected IFN-γ⁺ ATCs after Asp-lysate- as well as PepMix-stimulation but with a significantly higher enrichment efficiency for stimulation with the Asp-lysate when compared to the PepMix. In contrast, the STE approach resulted in comparably high ATC expansion rates by using Asp-lysate or PepMix. Independent of the stimulus, predominantly CD4⁺ helper T cells with a central-memory phenotype were expanded while CD8⁺ T cells mainly showed an effector-memory phenotype. ATCs were highly functional and cytotoxic as determined by secretion of granzyme-B and IFN-γ.

Discussion: For patients with IA, the immediate adoptive transfer of IFN-γ⁺ ATCs followed by the administration of short-term in vitro expanded ATCs from the same donor, might be a promising therapeutic option to improve the clinical outcome.

Keywords: Aspergillus infections; Aspergillus-specific T cells; HSCT; T-cell expansion; T-cell immunotherapy; cytokine secretion.

Figure 1

Screening for Asp-specific T cells by IFN-γ EliSpot. ATC responses elicited by the (A) Aspergillus fumigatus (Asp) lysate alone (n=320) or with (B) Apsp-lysate (n=30/320) and overlapping peptide pools from Aspergillus (CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD) and Candida albicans (MP65) (n=30) were determined by IFN-γ EliSpot assays. 2.5 x 10⁵ cells/well of isolated PBMCs from healthy donors were stimulated with the investigated peptide pools (1µg per peptide/ml) and the Asp-lysate (50 µg/ml) for 16 hours on anti-IFN-γ precoated EliSpot plates. Results are indicated as number of spots per well (spw) after the subtraction of background spots (negative control = unstimulated cells). The cut-off value for a positive antigen-specific T-cell response was defined with ≥ 2 antigen-induced spw. Data are shown in total as individual result and mean. Statistically significant difference between MP6 or Asp-lysate to the other peptide pools, unless otherwise highlighted, is indicated by (**p < 0.01,***p<0.001 and ****p<0.0001). ns, not significant.

Figure 2

Characterization of magnetically selected ATCs. The enrichment efficiency of in vitro stimulated Asp-specific T cells was assessed by magnetic selection using either the IFN-γ cytokine secretion assay (CSA) or Microbeads kits in response to IFN-γ, TNF-α, CD137, and CD156 as specific T-cell activation marker. 1 x 10⁷ isolated PBMCs were stimulated overnight with the Asp-lysate (50 µg/ml) while unstimulated PBMCs served as the negative control. Aliquots of the respective cell fractions collected before (original fraction, Origin) and after enrichment (positive fraction, PF) were used for detailed characterization of activated T-cell subsets by multicolor flow cytometry. (A) Frequency of IFN-γ⁺ Asp-specific CD3⁺ T cells before (Origin) and after magnetic selection (PF). (B) Relative frequency of IFN-γ⁺ Asp-specific T-cell subsets (CD3, CD8 and CD4) within the PF after magnetic enrichment. (C) Total cell numbers of CD3⁺/IFN-γ⁺ Asp-specific T cells contained in the Origin and PF. (D) Total cell numbers of IFN-γ⁺ Asp-specific T-cell subsets (CD3⁺, CD8⁺ and CD4⁺) within the PF. Results are displayed as individual results (n=8) and mean ± SD. The different symbols represent the individuals in Figures 5A–D. Statistically significant difference is indicated by (**p < 0.01). (E, F) Frequency of Asp-specific (E) CD3⁺, (F) CD4⁺, and (G) CD8⁺ T cells before (Origin) and after magnetic selection (PF) using IFN-γ, TNF-α, CD137, and CD156 as specific selection marker. Findings are displayed as individual results (n=3) and as the mean percentage of Asp-specific T cells. (H-J) Phenotypic analyses regarding naïve (N), central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) CD3⁺, CD4⁺ and CD8⁺ T cells were performed for the IFN-γ⁺, TNF-α⁺, CD137⁺, and CD156⁺ T cells of both fractions (Origin, PF) and are visualized by the respective mean frequencies (n=3). The healthy individuals used belonged to our cohort of donors (n=320) who were pre-tested for the presence of Asp-specific T cells.

Figure 3

Magnetically enrichment of PepMix-stimulated ATCs. The enrichment efficiency of in vitro stimulated Asp-specific T cells was assessed by magnetic selection using the IFN-γ cytokine secretion assay (CSA). 1 x 10⁷ isolated PBMCs were stimulated overnight with the pooled overlapping peptide pools from Asp and Candida albicans-(PepMix: CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD and MP65) each pool at a final consternation of 1 µg per peptide/ml while unstimulated PBMCs served as the negative control. Aliquots of the respective cell fractions collected before (original fraction, Origin) and after enrichment (positive fraction, PF) were used for detailed characterization of activated T-cell subsets by multicolor flow cytometry. (A) Frequency of IFN-γ⁺ Asp-specific CD3⁺ T cells before (Origin) and after magnetic selection (PF) and the proportion of IFN-γ⁺ Asp-specific T-cells among CD3, CD4 and CD8 T cells after magnetic enrichment in the PF are shown. Results are displayed as individual results (n=4) and mean ± SD. (B) Absolut cell numbers of IFN-γ⁺ Asp-specific CD3⁺ T-cells and among the different subsets (CD3, CD8 and CD4) are shown for two experiments, as insufficient cell counts after enrichment did not permit further analyses visualized by the respective mean ± SD. Squares and circles represent the same individuals of our healthy donor cohort (n=320).

Figure 4

Functional activity of short-term expanded (STE) ATCs analyzed via IFN-γ EliSpot assay. PBMCs were short-term expanded (STE) for 12 days with either the (A) Asp-lysate (50 µg/ml) or the (B) combination of eight fungal peptide pools termed PepMix (1 µg per peptide/ml per peptide pool, composed of: CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD and MP65 peptide pool). A) Numbers of IFN-γ spots per 10⁵ cells/well of lysate-expanded ATCs (including mean values) after restimulation for 16 hours with either Asp-lysate (n=9), single peptide pools (n=7), or PepMix (n=7), as indicated, are shown. B) Numbers of IFN-γ spots per 10⁵ cells/well of PepMix-expanded ATCs (including mean values) after restimulation for 16 hours with either the Asp-lysate (n=6), single peptide pools (n=7), or the PepMix (n=7), as indicated, are shown. For analyses of spots per well, background values were subtracted. The cut-off criterion (red dotted line) for a clinical product were set to 80 spots/10⁵ cells. Data are shown in total as individual result and mean of healthy individuals from our cohort of donors (n=320). Representative EliSpot results are shown below the respective graph. Statistically significant difference is indicated by (***p < 0.001).

Figure 5

Intracellular cytokine staining of IFN-γ and TNF-α producing ATCs. PBMCs were short-term expanded (STE) for 12 days with either the (A) Asp-lysate (50 µg/ml) or a (B) combination of eight fungal peptide pools termed PepMix (1 µg per peptide/ml per peptide pool, composed of: CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD and MP65 peptide pool) . Representative dot plots (gated on CD3⁺ T cells) and percentage values of IFN-γ^- and TNF-α^- expressing CD3⁺ ATCs, expanded with either (A) Asp-lysate or (B) PepMix and restimulated with indicated antigens (x-axis) are shown. Diagrams include the mean values obtained from n=3-5 individual HDVs from the cohort of donors (n=320), pre-tested for the presence of Asp-specific T cells. Dot plots highlighted in grey represent cells expanded and restimulated including the same antigen. Background values, based on unstimulated cells (neg) were subtracted. Data are shown in total as individual result and mean. Statistically significant difference is indicated by (*p < 0.05), ns – not significant.

Figure 6

Cytotoxic potential of csaATCs and seATCs. Asp-specific T cells obtained by IFN-γ Cytokine Secretion Assay (csaATCs) and after short-term expansion (STE) for 12 days (seATCs) were investigated for their cytotoxic functionality. To generate csaATCs and seATCs, PBMCs were stimulated with either the Asp lysate (50 µg/ml) or a combination of eight fungal peptide pools called PepMix (1 µg per peptide/ml per peptide pool consisting of: CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD and MP65 peptide pools). To obtain a sufficient number of csaATCs for the analyses, cells were additionally expanded for 12 days. With expanded ATCs, cytotoxicity tests were performed with unloaded and Asp-lysate- or PepMix-loaded autologous PBMCs as target cells. (A) Frequencies of cells with 7-AAD⁺ cells among target cells (CellTrace Proliferation dye positive). Light bars: unloaded target cells. Dark bars: loaded target cells. Results are displayed as individual results and as means (n=3). Statistical significance was calculated using Two-way ANOVA and multiple comparisons test (*p < 0.05). (B) Cell culture supernatants from cytotoxicity assays from day 12 for effector to target ratios of 1:1 and 10:1were analyzed with respect to presence of cytotoxic effector molecules by LEGENDPlex Assay. Results are displayed as heatmap. The results are expressed as the mean of the duplicates for samples of three tested donors, pre-tested for the presence of Asp-specific T cells (n=3/320). The values are represented by different colors, as referenced in the bar. As control, unloaded, Asp-lysate or PepMix-loaded target cells only as well as Aps-lysate-specific and PepMix-specific T cells only were used.

Figure 7

Functional and cytotoxic analyses of seATCs via FluroSpot assay and ELISA. PBMCs were short-term expanded (STE) for 12 days with Asp-lysate following Asp-restimulation for 16 hours with Asp-lysate (n=5). The cytotoxic activity of Asp-specific T cells was investigated by means of IFN-γ and granzyme B. (A) Numbers of IFN-γ and granzyme B positive spots per 2.5 x 10⁵ cells/well of ATCs with mean values represented via Box blots of five individual HDVs including representative images of IFN-γ (green) and granzyme B (red) secreting ATCs are shown. Results are given as the number of spots per well (spw), representing the number of spots in the antigen well after subtracting those of the respective negative control well. (B) The diagram shows individual values of granzyme B secretion (in pg/ml) in the supernatant of 12 days expanded ATCs, detected by ELISA (n=5). The different symbols represent the individuals ATCs from healthy donors who were pre-tested for the presence of Asp-specific T cells. (C, D). Aspergillus fumigatus hyphae were either left untreated (Afu only) or incubated for six hours with CMVpp65 peptide pool expanded T cells (+TC_CMVpp65), as additional control, or Asp-lysate (+ATC_Asp-lysate) and PepMix-(+ATC_PepMix) specific seATCs. Viability was analyzed by XTT reduction assay. The healthy individuals used belonged to our cohort of donors (n=320) who were pre-tested for the presence of Asp-specific T cells. Results are displayed as mean ± SD for samples from one representative healthy donor. Statistically significant difference is indicated by (*p < 0.05 and **p<0.01). A.fu, Aspergillus fumigatus; TC, T cells.

Figure 8

Immunophenotyping of short-term expanded seATCs. PBMCs were short-term expanded (STE) for 12 days with either the (A, C, E, G) Asp-lysate (50 µg/ml, n=10) or the (B, D, F, H) PepMix (1 µg per peptide/ml per peptide pool, composed of: CatB, Crf1, f22, Gel1, pmp20, SHMT, SOD and MP65 peptide pool, n=7). Absolute cell numbers per well before (day 0) and after expansion (day 12) for seATCs expanded with (A) Asp-lysate- or (B) PepMix are shown. Statistically significant difference is indicated by (**p < 0.01 and ****p<0.0001). (C, D) Mean proportion of CD3⁺, CD4⁺ and CD8⁺ seATCs, NK cells, B-cells, and monocytes within CD45⁺ cells after expansion with (C) Asp-lysate or (D) PepMix are shown as mean ± SD. (E, F) Immune phenotypes of CD8⁺ and CD4⁺ seATCs and (G, H) CD4⁺ T-cell subsets, expanded with Apsp-lysate or PepMix are shown as mean ± SD. The healthy individuals used belonged to our cohort of donors (n=320) who were pre-tested for the presence of Asp-specific T cells.