Monitoring hCMV-specific T cell response in hematopoietic stem cell transplant recipients via rapid whole blood cytokine release assay

Human cytomegalovirus (hCMV) reactivations and disease frequently occur in hematopoietic stem cell transplant (HSCT) recipients as a consequence of the profound immunosuppression required to prevent graft rejection. Despite clear evidence of the role of hCMV-specific T cells in controlling hCMV replication, quantification of hCMV-specific T cells is not widely performed to inform the clinical management of hCMV reactivations after transplantation, largely due to the lack of a simple and reliable assay to assess hCMV-specific cellular immunity. Herein, we developed a simple and rapid whole blood IFN-γ/IL-2 release assay (CRA) that uses overlapping hCMV peptides to quantify the hCMV-specific T cell responses and validated it in the peripheral blood of 40 healthy donors and 20 HSCT patients post-transplant. We found that the CRA correlates well with classical IFN-γ ELISpot assays but demonstrates greater sensitivity in detecting hCMV-specific T cell responses in HSCT recipients. Notably, the CRA also captured dynamic changes in hCMV-specific T cell responses associated with hCMV reactivation. Collectively, these findings demonstrate that the CRA is a simple and rapid method for longitudinal monitoring of hCMV-specific T cell immunity in HSCT recipients, with potential to inform clinical management of hCMV reactivations. IMPORTANCE: Stem cell and organ transplant patients often experience human cytomegalovirus (hCMV) reactivations and disease due to the immunosuppression required to prevent graft rejection. While it is known that hCMV-specific T cells are effective in controlling hCMV replication, monitoring of these T cells after transplantations is not routinely done in clinics due to the lack of an easy and reliable assay to assess hCMV-specific T cells. Here, we developed a simple and rapid cytokine release assay to quantify hCMV-specific T cells directly in whole blood and validated it in the peripheral blood of healthy donors and hematopoietic stem cell transplant patients post-transplant. The assay correlated well with classical ELISPOT assays and captured the dynamic changes of hCMV-specific T cell responses associated with hCMV reactivation. Together with its simplicity, the assay is well suited for the longitudinal monitoring of hCMV-specific T cell immunity in transplant patients.