CMV-Specific CD8+ T-CELL Function Is NOT Impaired In CLL
Authors: Raa Doreen te , Pascutti Fernanda , Garcia-Vallejo Juan J , Reinen Emilie , Remmerswaal Ester B.M. , Van Lier René A.W. , Eldering Eric , Van Oers Marinus H.J. , Tonino Sanne H. , Kater Arnon P. 
Published: October 2019

Abstract

Introduction
T-cell exhaustion is a state of acquired T-cell dysfunction that arises during chronic infections, but also in the presence of cancer. Chronic lymphocytic leukemia (CLL)-derived CD8+ T cells were recently found to exhibit features of exhaustion as shown by increased expression of inhibitory receptors (including PD-1), impaired proliferative and cytotoxic capacity and disturbed immunological synapse (IS) formation (Riches et al, Blood ’13). In contrast, we have reported a decreased expression of PD-1 on effector-type CD45RA+/-CD27- CD8+ T cells in CLL patients (Tonino et al., Leuk&Lymph ‘12). As this population is enriched for CMV-specific T cells, this implies that PD-1 expression might also be decreased on CMV-specific T cells. Importantly, CMV viral loads are generally undetectable in untreated CLL patients. Taken together these data suggest that the phenotypical and functional changes found in the total CD8+ T-cell population in CLL may not be applicable to CMV-specific CD8+ T cells. In this study, we have thoroughly analyzed phenotype and function of CMV-specific CD8+T cells in CLL.

Methods and Results
In agreement with Riches et al. we found an increased expression of the exhaustion markers PD-1, CD160 and CD244 (assessed by flow cytometry) on CLL-derived total CD8+ T cells (n=38) as compared to CD8+ T cells from age-matched healthy controls (HC; n=13). In sharp contrast, we found a significantly lower expression of these markers on CMV-tetramer+CD8+ T cells in CLL patients (n=18) as compared to HC (n=7). Next we analyzed the functionality of CMV-specific CD8+ T cells derived from CLL patients and HC. First, the sensitivity of the functional assays used was verified by treating CD8+ T cells with cyclosporine, which mimics intrinsic T-cell defects in vitro. Cytokine production was measured upon stimulation with PMA/Ionomycin. We found no differences between CLL patients and age-matched HC as to IFNγ, TNFα and IL-2 production by CMV-tetramer+CD8+ cells. Interestingly, the fraction of CMV-tetramer+CD8+ T cells producing cytokines after stimulation with CMV-pp65-peptide loaded EBV-transformed B-lymphoblastoid cell lines (EBV-LCLs) derived from normal B lymphocytes also did no differ between CLL patients and HC. Next, the cytotoxic potential of these cells was examined. CMV-specific CD8+ T cells from CLL patients were as effective as those from HC in killing CMV-peptide-loaded EBV-LCLs at varying effector:target ratios. Finally, CMV-specific IS formation was assessed by imaging flow cytometry analysis. This technique combines quantitative and qualitative data and has advantages over the current gold standard in the study of IS, i.e. confocal laser-scanning microscopy, since it is able to measure large cell populations in an automated fashion. On average 0.6x105 total cells per condition were acquired containing approximately 550 doublets. Again, percentages of CD8+T cells forming specific proper IS with CMV-peptide loaded EBV-LCLs were comparable in CLL and HC T cells (around 45%).

Conclusion
In contrast to evidence for a global disturbed T-cell functionality in CLL, characterized by T cells exhibiting phenotypic and functional signs of exhaustion, we now show that CMV-specific CD8+ T cells derived from CLL patients have a lower expression of exhaustion markers and are functionally intact. These data shed new light on the complexity of interactions between T-lymphocytes and CLL cells and indicate that changes found in the total CD8+ T-cell population in CLL cannot be extrapolated to CMV-specific CD8+ T cells.

Disclosures:
No relevant conflicts of interest to declare.