Figure 4. Lenalidomide maintenance therapy results in an improved CD8⁺ T cell functionality, which is hampered by non-CD8⁺ T cells present in the periphery.

(A) PBMCs obtained at the pre LEN and the LEN timepoint were examined by IFN-γ ELISPOT to measure the CD8⁺ T-cell response to viral recall epitopes derived for CMV, EBV and influenza (CEF) (n = 6). The number of spot forming units (SFU) as well as the activity in each well was determined. Bar graphs show the mean number of spots per million PBMCs or the mean activity of four replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. (B–E) CD8⁺ T cells purified from PBMC samples obtained at the pre LEN and LEN timepoint were examined by IFN-γ/IL-2/TNF-α FLUOROSPOT to characterize the CD8⁺ T-cell response to the CEF peptide pool. (B–C) Bar graphs show the mean number of spots per million CD8⁺ T cells of five replicate wells and the error bars show the standard deviation. Spot numbers and activity measured upon culture in the absence of peptide were subtracted. (D) Raw data showing three-color FLUOROSPOT results. One representative well is shown for each patient and timepoint. Cytokine-specific spots are shown in green (IFN-γ), red (TNF-α), blue (IL-2) or overlays of these colors. (E) The pie charts indicate the portion of mono- and polyfunctional CEF-specific CD8⁺ T cells. A two-way ANOVA with Sidak’s multiple comparisons test was performed to detect differences between the pre LEN and the LEN timepoint, ns: not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 and ^****p < 0.0001.