Multicytokine and Polyfunctional CMV-Specific T Cells Are Associated with Stem Cell Transplant Donor CMV Serostatus.
Authors: Zhou Wendi , Longmate Jeffrey , Palmer Joycelynne , Lacey Simon F. , Hawkins Ghislaine A. , Spielberger Ricardo , Forman Stephen J. , Zaia John A. , Diamond Don J. 
Published: October 2019

Abstract
Recently, a phenotype of CD8+ T cells was discovered that distinguishes long-term non-progressors from rapid progressors in HIV infection. The phenotype consists of a combination of cytokines and the CD107 surface marker, associated with competence for cytotoxicity, all expressed from the same CD8+ T cell. Our published work highlighted a significant difference in frequency of IFN-γ and TNF-α double cytokine expressing T cells, which are more frequently associated with CMV-positive than negative donors of stem cell transplants (HCT). Both clinical and basic studies have shown that TNF-α expression is associated with mature immunity, and its proper regulation is associated with a favorable outcome of HCT. We investigated the quality of T cell immunity targeting the CMV-pp65 antigen in a longitudinal study of HCT recipients up to 1 year post-Tx. HCT recipients with both CMV-positive and negative donors were accrued, and differences were compared between the two cohorts based on single and multi-cytokine expression (IFN-γ, MIP1-β, TNF-α) and the cytotoxicity marker, CD107. We tested whether the length of time these phenotypic immune differences persisted in the HCT recipient was associated with donor CMV status. A total of 61 donor-recipient pairs were compared. Using an ex vivo approach, multiparameter flow cytometry was used to obtain percentages for each individual T cell cytokine producing population, as well as comparisons between populations after CMV-pp65 full-length peptide library stimulation. We present results at 90–360d post-HCT. Comparisons of levels of multi-functional (4 parameter) T cells showed significant differences from CMV-positive and negative donors reflecting an increased frequency of 4-function T cells between 90–360d post-Tx. These levels were statistically significant (p<0.01) between the groups (student T-test). In contrast, removal of TNF-α from the panel resulted in the elimination of the difference between the 2 cohorts. The strong association of CMV status with inclusion of TNF-α, a maturation marker in the multi-cytokine phenotype, forecasts the key role of the memory T cell phenotype in the early development of protective immunity in the HCT recipient. Similar to the case in HIV progression studies, the preponderance of multifunctional (3 or 4 function) CD8+ T cells were found in recipients with CMV-positive donors versus those with CMV-negative donors, who were biased towards the double or single cytokine function phenotype between 90–360d post-HCT. The direct impact focuses on memory development, while insight on the relationship of these T cell populations to the frequency of CMV reactivation, infection, and overall survival were also investigated. Comparing HCT recipients who had CMV reactivation from those who did not, as well as stratification by donor CMV status clearly shows the greater frequency of the memory T cell population in recipients with CMV-positive donors. CMV reactivation contributes to the development of a CMV-specific T cell population that is intermediate between the low levels of those with no reactivation and a CMV-negative donor, versus having a CMV-positive donor. This data also reflects the influence of prior memory status in contributing to the more rapid development of a mature CMV-specific T cell population in the HCT recipient.