ULBP2-expressing HCMV mutants modulate NKG2D expression and function on NK and CD8+ T cells
MEYER G. 1,4, SIEMES R. 2,4, KÜHNE J. 1,3,4, BASZCZOK V. 1, BEUSHAUSEN K. 1, KEIL J. 1, WAGNER K. 2, STEINBRÜCK L. 2, BORST E. 2, MESSERLE M. 2,3,4, FALK C. 1,3,4
1 Institute of Transplant Immunology, Hannover Medical School, Hannover, Germany; 2 Institute of Virology, Hannover Medical School, Hannover, Germany; 3 German Center for Infection Research (DZIF), TTU-IICH (Immunocompromised host), Braunschweig/hannover, Germany; 4 FOR 2830 Advanced Concepts in Cellular Immune Control of Cytomegalovirus, DFG, Würzburg, Germany
Introduction: Solid organ transplant recipients with insufficient HCMV memory T-cell responses are at high risk of viral reactivation and life-threatening complications due to immunosuppression. We hypothesize that HCMV-specific NK and CD8+ T cell responses can be boosted by viral expression of NKG2D ligands (NKG2D-L, i.e. ULBP2) in mutants that lack or retain immune-evasins (US2-6) for HLA molecules.
Material & Methods: ULBP2-expressing mutants of the parental TB40 strain (ΔUS2-6) and the repaired TB40R strain containing US2-6 were constructed by replacing UL16 with ULBP2 driven by a weak or strong promoter. Fibroblasts (HFF) infected with TB40, TB40R or the mutants were phenotypically characterized with respect to HLA class I and NK ligands, and secretion of soluble ULBP2 was quantified by ELISA. In co-cultures of infected HFF with allogeneic PBMCs, T/NK cell activation and degranulation via CD107a surface expression were analyzed.
Results: Downregulation of HLA class I expression occurred in TB40-infected cells with stronger downregulation by TB40R due to US2-6 expression. Fine-tuned, increased surface expression of ULBP2 was observed for the weak- and strong ULBP2-expressing mutants, but not in parental strains due to UL16-mediated retention. High amounts of soluble ULBP2 (sULBP2) were shed into supernatants also reflecting the promoter strength of ULBP2. However, sULPB2 did not affect NK cell cytotoxicity. ULBP2 expression in HCMV-infected HFF resulted in a fine-tuned NKG2D downregulation depending on ULBP2 cell surface expression levels, activation (CD69+) as well as DNAM-1- and TIGIT subset composition of CD8+ T and NK cells. Degranulation was markedly reduced upon contact to TB40/TB40R-infected HFF, and slightly increased upon contact to the ULBP2-expressing variants though not reaching degranulation levels comparable to uninfected HFF.
Conclusion: Expression of NKG2D ligands like ULBP2 to achieve NK cell attenuation while simultaneously deleting HLA class I immune-evasins to induce HCMV-specific T cells represents a promising approach for HCMV vaccine development.