Selective Effects of mTOR Inhibitor Sirolimus on Naïve and CMV-Specific T Cells Extending Its Applicable Range Beyond Immunosuppression

Szilvia Bak, Sabine Tischer, Anna Dragon, Sarina Ravens, Lars Pape, Christian Koenecke, Mathias Oelke, Rainer Blasczyk, Britta Maecker-Kolhoff, Britta Eiz-Vesper, Szilvia Bak, Sabine Tischer, Anna Dragon, Sarina Ravens, Lars Pape, Christian Koenecke, Mathias Oelke, Rainer Blasczyk, Britta Maecker-Kolhoff, Britta Eiz-Vesper

Abstract

Cytomegalovirus (CMV) infection/reactivation remains among the most important complications of immunosuppression after transplantation. However, recent clinical observations indicate that mammalian target of rapamycin (mTOR) inhibition with sirolimus may improve the outcome of CMV complications. Underlying mechanisms of this observation, particularly the effect of sirolimus on naïve- and CMV-specific cytotoxic CD8+ T-cell (CMV-CTL) functionality is still undiscovered. Here, the influence of sirolimus on naïve and memory CMV-CTLs was determined by CD3/CD28 crosslinking and alloreactivity assays. After stimulating CMV-CTL with HLA-A*02:01-restricted CMVpp65-peptide loaded artificial antigen-presenting cells (aAPCs), we measured the effect of sirolimus on T-cell proliferation, phenotype, and functionality. Sirolimus significantly improved CMV-specific effector memory T-cell function and negatively influenced naïve T cells. This unique mechanism of action was further characterized by increased secretion of interferon-gamma (IFN-γ), granzyme B (GzB) and enhanced target-cell-dependent cytotoxic capacity of activated CMV-CTLs. Next-generation-sequencing (NGS) was applied to monitor T-cell receptor (TCR)-repertoire dynamics and to verify, that the increased functionality was not related to sirolimus-resistant CTL-clones. Instead, modulation of environmental cues during CMV-CTL development via IL-2 receptor (IL-2R)-driven signal transducer and activator of transcription-5 (STAT-5) signaling under mTOR inhibition allowed fine-tuning of T-cell programming for enhanced antiviral response with stable TCR-repertoire dynamics. We show for the first time that sirolimus acts selectively on human naïve and memory T cells and improves CMV-specific T-cell function via modulation of the environmental milieu. The data emphasize the importance to extend immune monitoring including cytokine levels and T-cell functionality which will help to identify patients who may benefit from individually tailored immunosuppression.

Keywords: HCMV; antiviral T cells; mTOR inhibitor; personalized immunosuppression; sirolimus; transplantation.

Figures

Figure 1
Figure 1
Activation marker expression is induced on sirolimus-treated memory T cells after TCR activation and IL-2 supplementation. Surface activation marker expression was measured on naïve CD8+CD45RA+CD62L+ and memory CD8+CD45RA− T cells after treatment with (+) or without (–) sirolimus (Sirol, 10 ng/ml) in the presence (+) or absence (–) of IL-2 (50 U/ml). Shown are mean normalized percentages of activation markers relative to untreated controls (indicated as dashed lines). (A) Stimulation on allogeneic irradiated CD3− cells (n = 6). (B) Stimulation on anti-CD28-coated plates (n = 5). (C) Stimulation with anti-CD3/CD28 beads (n = 3).
Figure 2
Figure 2
Dose-dependent suppression of CMV-specific T-cell expansion by sirolimus. (A) Normalized percentages (%) of CMV-specific multimer+CD8+ T cells (n ≥ 3) on day 7, as measured by multicolor flow cytometry. The indicated sirolimus (Sirol) concentrations (0.5–1,000 ng/ml) were added on day 0, and cells were stimulated with A02pp65p-loaded aAPCs in the presence of IL-2 (50 U/ml). Cultures without sirolimus served as controls. Optimal concentrations of sirolimus were defined by inhibition of CMV-specific CD8+ T cell expansion by 25% (IC25; 5 ng/ml), 50% (IC50; 10 ng/ml), and 75% (IC75; 40 ng/ml). (B) Representative dot-plot showing the percentages of expanded CMV-specific CD8+ T cells treated with sirolimus (from one donor). (C) Percentages of expanded multimer+ CMV-specific CD8+ T cells treated with sirolimus (n = 18). (D) Percentages of proliferated CFSE+multimer+ CMV-specific CD8+ T cells. Data are shown as means plus minus (±) standard deviation (SD). The two-paired Student's t-test was used to test for statistically significant differences [**p < 0.01, ****p < 0.0001, non-significant (ns)].
Figure 3
Figure 3
Stable effector memory phenotype and increased expression of activation markers on CMV-specific T cells after sirolimus treatment. (A) CMV-specific multimer+ T-cell phenotyping was performed using CD62L and CD46RA surface markers on day 7 after A02pp65p-loaded aAPC stimulation without (control) or with IC50 sirolimus treatment (IC50) in the presence of IL-2. CM = central memory T cells; EM = effector memory T cells; TEMRA = effector memory T cells expressing CD45RA determined as percentages (%) (n = 4). (B) Percentages of expression of PD-1, CTLA-4, Tim-3, Lag-3, CD25, and CD69 on CMV-specific T cells (n ≥ 4). Data are shown as means ± SD. The two-paired Student's t-test was used to test for statistically significant differences [*p < 0.05, ****p < 0.0001, non-significant (ns)].
Figure 4
Figure 4
Improved functionality of CMV-specific T cells after sirolimus treatment. (A) Relative quantities (RQ) of IFN-γ and granzyme B (GzB) secretion were assessed using RT-qPCR following overnight A02pp65p re-stimulation on aAPC stimulated and sirolimus treated and untreated CD8+ T cells. (B) IFN-γ expression levels determined by IFN-γ ELISpot assay and expressed as the number of spot-forming units (SFU) per 1,000 CMV-specific multimer+CD8+ T cells. (C) Percentages (%) of intracellular IFN-γ, GzB, and TNF-α on CMV-specific multimer+CD8+ T cells, as determined by intracellular staining using multicolor flow cytometry. (D) Target-cell recognition assay was performed on day 7. Total CD8+ T cells were co-cultured for 5 h with A02pp65p-loaded and CFSE-labeled A*02-transduced K562 target cells (squares) at effector to target ratios of 1:1 (n = 8), 5:1 (n = 8), and 10:1 (n = 6). Unloaded K562 cells served as controls (spheres). Percentages of dead cells were detected by 7AAD staining and multicolor flow cytometry. (E) Degranulation was determined as the percentage and median fluorescence intensity (MFI) of CD107a expression on CMV-specific multimer+CD8+ T cells by multicolor flow cytometry following 4 h of re-stimulation with A02pp65p (n = 5). Values are displayed as mean (±) SD. Statistical analysis: (A–C) two-paired Student's t-test and (D,E) two-way analysis of variance [*p < 0.05, **p < 0.01, ***p < 0.001, non-significant (ns)].
Figure 5
Figure 5
Sirolimus has no effect on the dynamics of TCR repertoires. Isolated CD8+ T cells were expanded for 7 days on aAPCs and treated with or without sirolimus in the presence of IL-2. On day 7, CMV-specific CD8+ cells were sorted based on their multimer-specificity (A02pp65p) by high speed flow cytometry sorters and subjected to high throughput TCR repertoire analysis. (A) The highest expanded CDR3 clones in two donors are shown as percentages (%) of productive reads. (B) Numbers of the CDR3 clones were normalized to 5,000 reads. (C) The Shannon index indicates the diversity of the repertoires, shown as normalized values. Values are displayed as mean (±) SD. Statistical analysis: two-way analysis of variance [non-significant (ns)].
Figure 6
Figure 6
Incomplete mTORC1 inhibition has differential effects on the phosphorylation of signaling molecules. Total CD8+ T cells were re-stimulated with 10 μg/ml A02pp65p for 1 h following 7 days of aAPC stimulation with or without sirolimus in the presence of IL-2, and the percentages (%) of phosphorylated (A) S6, (B) ERK1/2, (C) AktThr308, (D) AktSer473, and (E) STAT-5 were determined by phospho-flow cytometry. Values are displayed as mean (±) SD. The two-paired Student's t-test was used to test for statistically significant differences [***p < 0.001, non-significant (ns)].
Figure 7
Figure 7
Functional improvement of CMV-specific T cells treated with sirolimus strongly depends on IL-2R-driven STAT-5 signaling. (A) Percentages (%) of CMV-specific multimer+CD8+ T cells after 7 days of aAPC stimulation with or without sirolimus in the presence of the following cytokines: IL-7, IL-12, IL-15, IL-21 (each 10 ng/ml) which were added independently as different sets of cell cultures. Results for supplemental IL-2 (50 U/ml) without sirolimus (black dashed lines) and with sirolimus (gray dashed lines) are shown (n = 4). (B) Percentages of IFN-γ following 5 h peptide re-stimulation, as determined by intracellular cytokine staining on CMV-specific multimer+CD8+ T cells (n ≥ 3). (C) Percentage of expanded CMV-specific multimer+CD8+ T cells following overnight STAT-5 inhibition (STAT-5i) on day 7. (D) Percentages of pS6 and pSTAT-5, as determined by phospho-flow cytometry or (E) fluorescence microscopy on CMV-specific CD8+ T cells after 1 h of peptide re-stimulation. (F) Intracellular expression of IFN-γ (%) following 5 h of peptide re-stimulation, as measured by multicolor flow cytometry. Values are displayed as mean (±) SD. Statistical analysis: (A,B) Student's t-test and (C,D,F) two-way analysis of variance (*p < 0.05, **p < 0.01, ***p < 0.001).
Figure 8
Figure 8
Sirolimus differentially affects the expression of target and effector genes and miRNAs. Expression levels of (A) miR-21, miR-155, miR-181a, perforin, SHIP-1, SOCS1, MAPK1, Bcl-1, Ki-67, T-bet and EOMES, as determined by RT-qPCR following overnight A02pp65p re-stimulation on 7 day aAPC stimulated CD8+ T cells treated with or without sirolimus in the presence of IL-2. (B) Selected results from global gene expression analysis in multimer-sorted CMV-specific CD8+ T cells on day 7 obtained from untreated (Control 1 and 2) and sirolimus-treated cells (IC50 1 and 2) of two donors (n = 2). CD8+ T cells were sorted based on their multimer specificity using high-speed flow cytometry sorters. Following overnight A02pp65p re-stimulation, total RNA was isolated and investigated by microarray analysis. Clustering and heat map analyses were performed using the Morpheus web-based tool. The data are means ± SD. The two-paired Student's t-test was used to test for statistically significant differences [non-significant (ns)].
Figure 9
Figure 9
In vitro IL-2 treatment with TCR activation increases CD8+ T-cell responses after in vivo sirolimus treatment. PBMCs from patients (n ≥ 4) treated with sirolimus in vivo, PBMCs from untreated healthy donors (n ≥ 5) treated overnight with or without sirolimus (IC50) in vitro were stimulated with (+) or without (–) IL-2 (100U/ml) in the presence (+) or absence (–) of CD3/CD28 beads. (A) Following 15 min of stimulation, the percentages (%) of pS6 and pSTAT-5 were determined by phospho-flow cytometry. (B) Percentages of intracellular expression of IFN-γ, GzB and TNF-α in CD8+ T cells were measured by multicolor flow cytometry after 5 h of stimulation. The data are shown as mean ± SD. Statistical analysis was done by two-way analysis of variance. No significant differences were determined.

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