CMV reactivation remains a significant cause of morbidity and mortality due to the extended period of immunodeficiency after allogeneic hematopoietic stem cell transplantation (HCT), despite great strides in management of the infection in the past two decades. Reconstitution of cytomegalovirus (CMV)-specific CD8+ T cells is essential to control of CMV infection in CMV-seropositive recipients (R+) after HCT. The CMV serologic status of the recipient before HCT has a strong influence on outcome. Key questions addressed in this study are the impact of donor CMV serostatus on the reconstitution of effective CMV immunity or risk of CMV reactivation and GCV usage in CMV R+ recipients. Betts and colleagues have reported that HIV-specific CD8+ T-cells which simultaneously degranulated and produced IFN-γ, TNF-α, MIP-1β and IL2 were associated with lower viral load and HIV long term non-progressor status. These findings in the context of HIV infection motivated us to investigate whether levels of multi-functional CMV-specific CD8+ T-cells in HCT recipients correlated with the CMV serostatus of the donor and the differentiation state of transferred CMV-specific memory T-cells. We hypothesize that a mature CD8+ T-cell functional profile leads to a lower incidence of CMV viremia in R+ recipients of T-cell replete HCT with a D+ donor. A total of 183 R+ HCT recipients were enrolled. CMV reactivation was defined as any positive CMV blood culture or plasma PCR obtained during a monitoring period that began at d40 post-HCT and continued thereafter twice weekly until d100. After d100, CMV in plasma was monitored in patients at high risk because of GVHD or immunosuppressive medication. Peripheral blood mononuclear cells from R+ HCT recipients were collected at intervals between d90 and d360 post-HCT. A subset (n=123) of the subjects limited only by sample availability were evaluated for CMV-specific CD8+ T-cells producing IFN-g (IFN-γ-CD8+). We used a well-described pp65 peptide library as a stimulatory antigen to evaluate the ex vivo functional profile of pp65-specific CD8+ T-cells from R+ recipients either receiving CMV seropositive (D+/R+) or seronegative (D/R+) T-cell replete donor grafts. D+ status was associated with higher IFN-γ-CD8+ during the sampling time-course by fitting linear generalized estimating equation models on a logarithmic scale (p=0.0004). A significant interaction was found between prior CMV reactivation and donor serostatus on IFN-γ-CD8+ levels (p=0.0001). Comparing the subset of IFN-γ-CD8+ measurements prior to reactivation, the D donor group produced lower levels of IFN-γ-CD8+ than the D+ donor group (p=0.0002), although both donor groups have similar levels of IFN-γ-CD8+ post-reactivation. Similar results were obtained when adjusting for the 9 pre-transplant covariates, and none were associated with IFN-γ-CD8+ levels, except donor serostatus and its interaction with CMV reactivation. Six-color flow cytometry was used to assess the functional profile of CMV-specific CD8+ T-cells in 62 of 183 HCT recipients prospectively followed for CMV reactivation. R+ recipients receiving grafts from D donors (D/R+) reconstituted fewer multi-functional CD8+ T-cells expressing IFN-γ, TNF-α, MIP-1β and CD107 compared to D+/R+ recipients. Unlike mono-functional CD8+ T-cells secreting IFN-g, which were abundantly generated during CMV reactivation in D/R+, the relative lack of multi-functional CD8+ T-cells persisted until at least one year post-HCT. In addition, D/R+ recipients had more CMV reactivation than D+/R+ recipients. D+/R+ transplants have elevated levels of multi-functional CD8+ T cell levels and lower hazard for CMV reactivation. Virologic and immunologic outcomes were robust to adjustment for pre-transplant factors affecting HCT, including donor type, stem cell source, recipient age, and preparative regimen. Statistical modeling to account for pre- and post-transplant factors including GVHD and its treatment by steroids had minimal effect on the contribution of serostatus to the risk of CMV reactivation and differences in multi-functional CD8+ T cell levels.

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