Research Article: A multiparameter whole blood platform for assessment of anti-CMV immunity in allogeneic stem cell transplantation recipients
Abstract:
Human cytomegalovirus (HCMV) infection remains a major challenge in allogeneic stem cell recipients. Current immune monitoring largely relies on IFN-?-based readouts following ex vivo stimulation, which cannot fully capture the complex anti-HCMV immune defense. Therefore, we established a multiparameter HCMV-reactive whole blood (WB) assay that can be initiated at the bedside, requires minimal blood volume, preserves physiological cellular interactions, and enables integrated cellular and cytokine readouts within a closed-tube workflow. Using enhanced ?-CD40 co-stimulation for robust detection of T-cell activation markers without widely used exocytosis inhibitors, our assay facilitates parallel flow cytometric quantification of activation-induced HCMV-reactive CD4 + and CD8 + T-cell responses and multiplexed detection of secreted cytokines. In parallel, our WB platform allows multimer-based detection of HCMV-specific CD8 + T cells and quantification of additional cell populations implicated in anti-HCMV protection, including V?1 + ?? T cells, “memory-like” natural killer (NK) cells, and HCMV-specific B cells. Across matched samples, readouts in WB and isolated peripheral blood mononuclear cells showed strong correlation. High intra-assay precision was achieved for key readouts across several immune cell compartments. Overall, our WB platform offers a simple, modular framework to advance post-transplant HCMV immune profiling while also being adaptable to other pathogens and immunological applications.
Introduction:
Letermovir prophylaxis through day 100 after allogeneic stem cell transplantation (alloSCT) has markedly reduced early clinically significant cytomegalovirus infection (csCMVi) and related complications in HCMV-seropositive alloSCT recipients ( 1 ). However, late-onset csCMVi after prophylaxis discontinuation remains a major clinical challenge ( 2 ). Assessment of HCMV-reactive cell-mediated immunity (CMI) has shown promise for risk stratification and may provide an avenue toward individualized management ( 3 , 4…
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