EBV-Specific CD8⁺ T-Cells Are Not Functionally Impaired in Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia (CLL) is characterized by a tumor induced T-cell dysfunction, which leads to increased susceptibility to infections and a decreased immunosurveillance (Görgün et al. JCI, 2005). Furthermore, T-cell dysfunction impairs novel treatment strategies that rely on T-cell mediated effects. The dysfunction of T-cells in CLL is characterized by an inability to form immune synapses, increased expression of exhaustion markers and impaired cytotoxicity and proliferative capacity (Ramsay et al. JCI 2008; Ramsay et al. Blood 2012; Riches et al. Blood 2013). However, we recently found that CMV-specific CD8⁺ T-cells from CLL patients are functionally intact with respect to cytokine production, cytotoxicity and immune synapse formation when compared to age-matched healthy controls (HC)(te Raa et al. Blood 2014). The finding that specific subsets of T-cells in CLL patients are functionally intact challenges the concept of a global T-cell dysfunction in CLL. Whether intact functionality of CMV-specific T-cells is a rare exception or whether T-cell functionality is indeed more heterogeneous is currently unknown.

Aim

To analyze T-cell function heterogeneity in CLL, we studied the immunophenotype and functionality of CD8⁺ T-cells specific for Epstein-Barr-virus (EBV), another widely common chronic latent viral infection.

Methods

EBV-specific CD8⁺ T-cells were analyzed using EBV tetramers and 14-color flow cytometry in 42 untreated CLL patients and 23 age-matched HC. We studied T-cell differentiation based on surface markers CD45RA, CCR7, CD27 and CD28 and 2 master regulators of T-cell differentiation, the transcription factors T-bet and Eomes. We also measured expression of exhaustion markers (PD-1, CD244 and CD160), functional markers (such as KLRG1, CD127, granzyme B, granzyme K and Ki-67) and homing markers (CXCR3 and CX3CR1). To study the functionality of EBV-specific CD8⁺ T-cells, we determined cytokine production and polyfunctionality after stimulation with EBV-derived peptides.

Results

Using a comprehensive T-cell differentiation staining we found that when compared to HC, EBV-specific T-cells in CLL patients are further differentiated with a significantly smaller percentage of "early" effector memory cells (also called EM1, CD45RA^- CCR7^- CD27⁺ CD28⁺; CLL=39.6% vs HC=57.68%). These results are mirrored by the expression patterns of the transcription factors T-bet and Eomes; 25.79% EBV-specific T-cells of CLL patients display a T-bet^high Eomes^high phenotype vs 17.44% in HC. In comparison with HC, EBV-specific T-cells in CLL patients show higher expression of exhaustion markers CD244 and CD160 (MFI 4896.42 vs 3130.56 and 2320.09 vs 1097.38, respectively), but not PD-1. However, there were no significant differences in granzyme B and K expression in EBV-specific T-cells, suggesting an unaltered cytotoxic potential.

On a functional level, no differences between CLL and HC were found with respect to production of the cytokines TNFα, IFNγ, IL-2 and MIP-1β of EBV-specific T-cells after peptide stimulation. Also, degranulation (measured as CD107a⁺ cells) was similar between CLL patients and healthy controls after peptide stimulation. Finally, polyfunctionality of EBV-specific T-cells of CLL patients was comparable with HC. We are currently determining cytotoxicity and immune synapse formation.

Conclusion

So far, although the phenotype may suggest an increased exhaustive state, we have not observed signs of dysfunction of EBV-specific T-cells in CLL patients when compared to HC. We are currently performing experiments to test cytotoxicity and ability to produce immune synapses of EBV-specific T-cells (which we will be able to present during the ASH meeting). Based on these results, we will be able to conclude if EBV-specific CD8⁺ T-cells are also functionally intact in CLL patients, and whether this population joins CMV-specific T-cells as a subset that eludes CLL induced T-cell dysfunction. T-cell dysfunction in CLL needs to be better understood in order to improve anti-tumor immunotherapies that rely on T-cell mediated effects. T-cell populations that escape suppression may be good targets for future therapies to build around.