Immunomonitoring via ELISPOT Assay Reveals Attenuated T-Cell Immunity to CMV in Immunocompromised Liver-Transplant Patients

Abstract

Assessing immune responses to cytomegalovirus (CMV) after liver transplant in patients on immunosuppressive therapy remains challenging. In this study, employing ELISPOT assays, 52 liver-transplant recipients were evaluated for antiviral T-cell activity in peripheral blood mononuclear cells (PBMCs), measuring interferon-γ (IFN-γ) secretion upon stimulation with CMV-specific peptides (CMV peptide pool, CMV IE-1, and pp65 antigens). Parameters such as stimulation index, mean spot size, and mean spot count were measured. The study found that heightened immunosuppression, especially with prednisolone in triple therapy, significantly dampened CMV-specific immune responses. This was demonstrated by decreased IFN-γ production by CMV-specific T-cells (CMV peptide pool: p = 0.036; OR = 0.065 [95% CI: 0.005-0.840], pp65 antigen: p = 0.026; OR = 0.048 [95% CI: 0.003-0.699]). Increased immunosuppression correlated with reduced IFN-γ secretion per cell, reflected in smaller mean spot sizes for the CMV peptide pool (p = 0.019). Notably, shorter post-transplant intervals correlated with diminished antiviral T-cell IFN-γ release at two years (CMV peptide pool: p = 0.019; IE antigen: p = 0.010) and five years (CMV peptide pool: p = 0.0001; IE antigen: p = 0.002; pp65 antigen: p = 0.047), as did advancing age (pp65 antigen: p = 0.016, OR = 0.932, 95% CI: 0.881-0.987). Patients with undetectable CMV antigens had a notably higher risk of CMV reactivation within six months from blood collection, closely linked with triple immunosuppression and prednisolone use. These findings highlight the intricate interplay between immunosuppression, immune response dynamics, and CMV reactivation risk, emphasizing the necessity for tailored immunosuppressive strategies to mitigate CMV reactivation in liver-transplant recipients. It can be concluded that, particularly in the early months post-transplantation, the use of prednisolone as a third immunosuppressant should be critically reconsidered. Additionally, the use of prophylactic antiviral therapy effective against CMV in this context holds significant importance.

Keywords: CMV; cytomegalovirus; immunosuppressive therapy; liver transplantation; opportunistic infection.

Figure 1

Stimulation Index (SI) of the tested antigens based on the number of administered immunosuppressants (x-axis) (A–C); IFN-γ release from antiviral T-cells, represented as mean spot size (MSS), in relation to the CMV peptide pool, corresponding to an escalation in the number of administered immunosuppressants (D). CMV = Cytomegalovirus, Ag = Antigen; ** p < 0.01; * p < 0.05.

Figure 2

Photographic depiction of individual wells from the Human Interferon-γ Single Color Immunospot Assay, showcasing diverse levels of Interferon-γ activity as determined by the ImmunoSpot^® Reader from CTL (Cellular Technology Limited, Shaker Heights, OH, USA). The patients corresponding to the depicted wells received three (A), two (B), and one (C) immunosuppressive agent(s), respectively.

Figure 3

Scatter plot showing the mean spot size (MSS) of the individual antigens ((A) CMV peptide pool; (B) IE Antigen; (C) pp65) over time after transplantation; CMV = Cytomegalovirus, Ag = Antigen.