In Vitro Profiling of Commonly Used Post-transplant Immunosuppressants Reveals Distinct Impact on Antiviral T-cell Immunity Towards CMV

Abstract

Infectious complications, including widespread human cytomegalovirus (CMV) disease, frequently occur after hematopoietic stem cell and solid organ transplantation due to immunosuppressive treatment causing impairment of T-cell immunity. Therefore, in-depth analysis of the impact of immunosuppressants on antiviral T cells is needed. We analyzed the impact of mTOR inhibitors sirolimus (SIR/S) and everolimus (EVR/E), calcineurin inhibitor tacrolimus (TAC/T), purine synthesis inhibitor mycophenolic acid (MPA/M), glucocorticoid prednisolone (PRE/P) and common double (T+S/E/M/P) and triple (T+S/E/M+P) combinations on antiviral T-cell functionality. T-cell activation and effector molecule production upon antigenic stimulation was impaired in presence of T+P and triple combinations. SIR, EVR and MPA exclusively inhibited T-cell proliferation, TAC inhibited activation and cytokine production and PRE inhibited various aspects of T-cell functionality including cytotoxicity. This was reflected in an in vitro infection model, where elimination of CMV-infected human fibroblasts by CMV-specific T cells was reduced in presence of PRE and all triple combinations. CMV-specific memory T cells were inhibited by TAC and PRE, which was also reflected with double (T+P) and triple combinations. EBV- and SARS-CoV-2-specific T cells were similarly affected. These results highlight the need to optimize immune monitoring to identify patients who may benefit from individually tailored immunosuppression.

Keywords: CMV-specific T cells; adoptive T-cell therapy; hematopoietic stem cell transplantation; immunosuppression; solid organ transplantation.

FIGURE 1

IFN-γ ELISpot, activation and cytokine secretion of CMV_pp65-stimulated PBMCs under immunosuppression. (A) PBMCs were isolated from CMV+ donors, rested overnight and stimulated with CMV_pp65 on day 1 in presence and absence of indicated immunosuppressants on IFN-γ ELISpot plates. After 24h, secreted IFN-γ was detected. Representative and summarized IFN-γ ELISpot results shown as spots per well (spw)/2.5 × 10⁵ PBMCs, spot intensity and spot size, normalized to untreated control (UT). (B–D) PBMCs were isolated from CMV+ donors, rested overnight and stimulated with CMV_pp65 on day 1 in presence and absence of indicated immunosuppressants. After 24 h cells were harvested for flow cytometric analysis and cell culture supernatants were collected for multiplex analysis. (B) Frequencies of CD69⁺ cells among CD4⁺ T cells (bar graph) and memory CD4⁺ T-cell subsets (heat map), normalized to UT. (C) Summarized frequencies of CD69⁺ cells among CD8⁺ T cells and memory CD8⁺ T-cell subsets (heat map), normalized to UT. (B–C) Bar graphs show median and interquartile range Q1-Q3, each symbol represents data from one donor (n = 12). (B–D) Heat maps show median values, normalized to UT (n = 12). Statistical significance (in comparison to UT) was calculated using (A–C) Friedman test followed by Dunn’s multiple comparison and (D) 2way ANOVA followed by Dunnett’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC negative control (unstimulated), UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone, TN naïve T cells (CD45RA⁺/CD62L⁺), TCM central memory T cells (CD45RA⁻/CD62L⁺), TEM effector memory T cell (CD45RA⁻/CD62L⁻), TEMRA effector memory T cell re-expressing CD45RA (CD45RA⁺/CD62L⁻).

FIGURE 2

Cytokine profiling of CMV_pp65 stimulated PBMCs under immunosuppression. PBMCs were isolated from CMV+ donors, rested overnight and stimulated with CMV_pp65 on day 1 in presence and absence of indicated immunosuppressants. After 24h, intracellular cytokine production was detected using multicolor flow cytometry and secreted cytotoxic mediators were measured using a flow cytometry-based multiplex assay (LEGENDplex). (A,B) Bar graphs summarize frequencies of IFN-γ⁺, TNF-α⁺ and IL-2⁺ cells among (A) CD4⁺ and (B) CD8⁺ T cells. The data are shown as median and interquartile range Q1-Q3 (n = 12). (C) Heat maps summarize frequencies of IFN-γ⁺, TNF-α⁺ and IL-2⁺ cells among CD4⁺ (left) and CD8⁺ (right) memory T-cell subsets, normalized to untreated control (UT). Data are shown as median (n = 12). Statistical significance (in comparison to UT) was calculated using Friedman test followed by Dunn’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. NC negative control (unstimulated), UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone, TN naïve T cells (CD45RA⁺/CD62L⁺), TCM central memory T cells (CD45RA⁻/CD62L⁺), TEM effector memory T cell (CD45RA⁻/CD62L⁻), TEMRA effector memory T cell re-expressing CD45RA (CD45RA⁺/CD62L⁻).

FIGURE 3

Proliferation analysis of purified CMV-specific T cells under immunosuppression. PBMCs were isolated from CMV+ donors, labeled with CellTrace™ Violet (CTV) and rested overnight, followed by magnetic enrichment of CMV-specific T cells using Cytokine Secretion Assay and CMV_pp65 stimulation. Afterwards, the T cells were expanded on irradiated autologous PBMCs in presence or absence of indicated immunosuppressants, followed by flow cytometric analysis. (A) Histograms showing CTV signals of CMV-specific T cells from a representative donor after 5 days of expansion (upper graph). Bar graph shows summarized mean fluorescent intensities (MFIs) of CTV from proliferating T cells on day 5, normalized to untreated control (UT) (lower graph). (B) Bar graphs show summarized MFIs of CTV from proliferating CD4⁺ (left) and CD8⁺ (right) T cells on day 5, normalized to untreated control (UT) (upper). Heat maps show summarized MFIs of CTV from proliferating CD4⁺ (left) and CD8⁺ (right) memory T-cell subsets on day 5, normalized to untreated control (UT) (lower). (A,B) Bar graphs show median and interquartile range Q1-Q3, each symbol represents data from one donor (n = 5). Heat maps show data as median values (n = 5). Statistical significance (in comparison to UT) was calculated for each T-cell subset using Friedman test followed by Dunn’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001. UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone, TN naïve T cells (CD45RA⁺/CD62L⁺), TCM central memory T cells (CD45RA⁻/CD62L⁺), TEM effector memory T cell (CD45RA⁻/CD62L⁻), TEMRA effector memory T cell re-expressing CD45RA (CD45RA⁺/CD62L⁻).

FIGURE 4

Cytotoxic capacity and activation of CMV-specific T cells under immunosuppression. PBMCs were isolated from CMV+ donors and rested overnight, followed by magnetic enrichment of CMV-specific T cells using Cytokine Secretion Assay and CMV_pp65 stimulation. The T cells were expanded on irradiated autologous PBMCs for 11 days and subsequently co-cultured with CTV-labeled autologous CMV_pp65-loaded PBMCs in different effector-to-target ratios and in presence or absence of indicated immunosuppressants. After 4 h their cytotoxic capacity was analyzed using flow cytometry. Unloaded PBMCs served as negative control. (A) Bar graphs show the frequencies of dead (7-AAD⁺) target cells, normalized to untreated control (UT). (B) Bar graphs show the CD69 expression (MFI) among CD8⁺ T cells, normalized to untreated control (UT) (upper). Heat maps show the CD69 expression (MFI) among CD8⁺ memory T-cell subsets, normalized to untreated control (UT) (lower). (A,B) Bar graphs show median and interquartile range Q1-Q3, each symbol represents data from one donor (n = 4). Heat maps show data as median values (n = 5). Statistical significance (in comparison to UT) was calculated using Friedman test followed by Dunn’s multiple comparison. *p < 0.05, **p < 0.01, ***p < 0.001. UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone, TN naïve T cells (CD45RA⁺/CD62L⁺), TCM central memory T cells (CD45RA⁻/CD62L⁺), TEM effector memory T cell (CD45RA⁻/CD62L⁻), TEMRA effector memory T cell re-expressing CD45RA (CD45RA⁺/CD62L⁻).

FIGURE 5

Cytotoxic capacity of CMV-specific T cells towards CMV-infected fibroblasts under immunosuppression. PBMCs were isolated from CMV+ donors and rested overnight, followed by magnetic enrichment of CMV-specific T cells using Cytokine Secretion Assay and CMV_pp65 stimulation. The T cells were expanded on irradiated autologous PBMCs for 11 days and subsequently co-cultured with uninfected, CMV-infected or CMV_pp65-loaded Human Foreskin Fibroblasts (HFF) in an effector-to-target ratio of 1:1 and in presence or absence of indicated immunosuppressants for 7 days using an xCELLigence RTCA S16 Real Time Cell Analyzer. (A) Microscopic image of the different target cells prior to co-culture. (B) Realtime impedance-based growth curves of HFF cells cultured alone (HFF cells only) or together with CMV-specific T cells in presence or absence of indicated immunosuppressants. Black arrows indicate time of T-cell addition. (C) Bar graphs display the AUC of growth curves shown in (B), normalized to untreated control (UT). UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone.

FIGURE 6

In vitro profiles of commonly used post-transplant immunosuppressants in context of antiviral T-cell immunity. Spider web graphs summarizing the impact of the respective immunosuppressants on CMV-specific T cells as measured by the indicated assays and in comparison to untreated controls. Values used for the diagrams are (clockwise starting from the top): IFN-γ ELISpot (spot numbers), activation status (frequencies of CD69⁺ CD4⁺ and CD8⁺ T cells), intracellular cytokine staining (cumulative frequencies of IFN-γ⁺/TNF-α⁺/IL-2⁺ CD4⁺ and CD8⁺ T cells), multiplex cytokine profiling (concentrations of pro-inflammatory molecules), proliferation (CD3⁺ T cells), cytotoxicity (4 h) (frequencies of dead target cells), activation (4 h) (CD69-MFIs of CD8⁺ T cells), realtime cytotoxicity (area under curve). UT untreated, SIR/S sirolimus, EVR/E everolimus, TAC/T tacrolimus, MPA/M mycophenolic acid, PRE/P prednisolone.