Background: Cytomegalovirus (CMV) infection is a common opportunistic viral infection in transplant patients. Patterns of viremia and relapse are influenced by the host immune response, specifically, CD8 T-cell responses. The use of mass cytometry (cyTOF) allows for simultaneous analysis of a large number of surface and intracellular markers that is not possible with conventional flow cytometry. We hypothesize that an in depth analysis of CD8 T-cell responses may provide novel immunological insights into the pathogenesis of CMV infection.

Methods: PBMCs were collected from 20 transplant patients at the time of CMV viremia onset. Of these, 10 were patients with positive outcomes, meaning they had spontaneous viral clearance or a rapid response to antiviral therapy with no relapse, and 10 were patients with negative outcomes in whom viremia persisted or relapsed, despite antiviral therapy. We utilized mass cytometry to simultaneously characterize cell-surface expression of CCR7, CD45RA, CD45RO, CD127, CD95, CD25, CD69, CD7, HLA-DR, CD38, CD27, PD-1, CD107a and CD57 on CD8 T-cells. Functionality was assessed by measuring intracellular effector molecules (IFN-γ, IL-2, MIP-1β, TNF-α, IL-17, IL-10, perforin, granzyme B and FOXP3) in CD8 T-cells stimulated with a pool of immunodominant CMV peptides each at 1 [mu]g/mL.

Results: As compared to patients with positive outcomes, those with negative outcomes had a greater frequency of terminally differentiated effector memory CD8 T-cells (42.2% vs 60.7%, p=0.023), and a significantly lower frequency of central memory CD8 T-cells (0.48% vs 0.15%, p=0.035). No differences were seen in total, naïve or effector memory CD8 T-cells, as well as CD8 T-cell maturation markers (eg, PD-1, CD107a, CD45RO) between the two groups. Interestingly, FOXP3⁺CD25⁺ regulatory CD8⁺ T-cells were present in greater frequency in the negative outcome group (0.26% vs 0.07%; p=0.0081), but no differences in IL-10 were observed. With respect to functionality, tri– (IFN-γ, TNF-α, IL-2) and polyfunctional (IFN-γ, TNF-α, IL-2, MIP-1β) CD8 T-cell frequencies were consistently greater in the positive outcome group (p=0.0005 and 0.048, respectively), as were perforin- and granzyme B-expressing IFN-γ⁺ TNF-α⁺ CD8 T-cells (p=0.0021).

Conclusion: We show that positive CMV outcomes are associated with higher frequencies of polyfunctional T cells but also lower frequencies of regulatory CD8 T-cells. A comprehensive assessment of CD8 T-cell immunity at onset of viremia could be used to predict CMV outcomes.

CITATION INFORMATION: Ferreira V, Kumar D, Husain S, Bhat M, Ashton P, Humar A. Cytometric Deep Profiling of the CD8+ T-Cell Compartment in Transplant Patients with CMV Viremia. Am J Transplant. 2016;16 (suppl 3).

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