Optimization of CD4+ T Lymphocyte Response to Human Cytomegalovirus Nuclear IE1 Protein through Modifications of Both Size and Cellular Localization

Abstract

We have previously reported that the CD4+ T lymphocyte response against nuclear human CMV IE1 protein depends in part on endogenous MHC class II presentation. To optimize presentation by HLA-DR of the nuclear IE1 protein and increase the response by CD4+ T cells, we have constructed two different adenovirus vectors containing mutant versions of IE1, containing a HLA-DR3 epitope, fused to GFP. The first construct consisted of a sequence of 46 aa encoded by exon 4, called GFP-IE1 (86–131). The second construct consisted of the whole IE1 mutated on exon 4 nuclear localization signals, identified in this study, and deleted of already known exon 2 nuclear localization signals (GFP-IE1M). Both of these IE1 vectors expressed proteins with cytoplasmic localization, as evidenced by GFP expression, as opposed to control GFP-IE1, which was nuclear. GFP-IE1 (86–131) induced IE1-specific CD4+ T cell clone response that was >30-fold more potent than that against GFP-IE1 and GFP-IE1M. The CD4+ T cell response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. GFP-IE1 (86–131) was rapidly degraded by the APC, which may account for better presentation. Our data show potentiation of the CD4+ T cell response to a specific epitope through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination.

An optimized IFN-γ ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

“…Mechanisms of immune evasion inhibiting MHC-I-dependent IE-1 antigen processing and presentation might also explain the lower frequency of IE-1-specific IFN-γ-producing cells detected in our ELISpot assay [65]. In addition, reduced processing and presentation efficiency due to protein stability, size and nuclear localization might play a role in the reduced reactivity to IE-1, as opposed to pp65 [64, 66, 67]. This proposition is supported by the observation that higher concentrations of IE-1 were required in our ELISpot assay, in comparison to pp65, to trigger a significant response.…”

Validation of T-Track® CMV to assess the functionality of cytomegalovirus-reactive cell-mediated immunity in hemodialysis patients

“…Moreover, mechanisms of immune evasion involving CMV-encoded unique short (US) proteins and resulting in the inhibition of the MHC-I-dependent antigen presentation pathway appear to be responsible for impaired IE-1 antigen processing and presentation, and thus in the low frequency of IE-1-reactive CD8 + T cells [55–57]. On the other hand, differential antigen uptake, processing and presentation by APC, possibly influenced by pp65 and IE-1 intrinsic properties [54, 58, 59], might explain inter-individual differences in the frequency of CMV antigen-specific T cells. Accordingly, comparable CD8 + T cell response to IE-1 and pp65 has also been described in some CMV-seropositive healthy donors [47, 60].…”

Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1

“…The main IE1 isoform (herein referred to as IE1) appears as a 72-kDa species in protein gels and is composed of four structurally and functionally distinct regions. A short N-terminal domain (amino acids 1 to 24) is predicted to be intrinsically disordered and contains one of at least two nuclear localization signals [45][46][47][48][49]. The central region downstream from the N-terminal part has been termed the core domain (amino acids 25 to 378).…”