Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Abstract

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.

Accurate pan-specific prediction of peptide-MHC class II binding affinity with improved binding core identification

“…To illustrate how accurate identification of the binding core of MHC class II ligands can aid the interpretation of Tcell cross-reactivity, we generated a set of five variants of the CMV epitope IE1 211–225 (NIEFFTKNSAFPKTT) restricted to HLA-DRB5*01:01 (Braendstrup et al 2014). The peptide-binding core of the WT peptide was identified using the NetMHCIIpan-3.1 method.…”

“…PBMC's from a donor previously determined to recognize the DRB5*01:01 restricted CMV IE1 211–225 epitope (Braendstrup et al 2014) was expanded on this epitope. The cells were double-stained with PE-labeled wild-type IE1 211–225 -DRB5*01:01 tetramer and APC-labeled mutant peptide-DRB5*01:01 tetramer as previously described (Braendstrup et al 2013).…”

Cytomegalovirus (CMV) Epitope–Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects

“…CDR3 sequences within DYS − CD4 samples were generally polyclonal with a few exceptions. This noteworthy observation might be driven by clonal expansions induced by epitopes that overlap with DYS as observed in HCMV IE1 epitopes (84), or to other inflation-inducing epitopes of HCMV or other pathogens. The presence of the dominant DYS + CDR3s within the DYS − repertoire at relatively lower magnitudes reflected potential loss of tetramer binding, while the reverse could reflect non-specific binding to the DYS tetramer.…”

Uncovering the Peptide-Binding Specificities of HLA-C: A General Strategy To Determine the Specificity of Any MHC Class I Molecule

“…Finally, the polymorphism of the HLA-C isotype indicates that it is capable of binding a diverse repertoire of peptides in keeping with it also having an important role in Ag presentation and generation of CTL responses. Indeed, HLA-C molecules are known to bind peptides of microbial origin and present them to CTLs [e.g., HIV (12)(13)(14) and CMV (15)]. The recent discovery of the ability of HIV-1 Nef proteins to selectively downregulate HLA-A and HLA-B, but not HLA-C, expression (16) suggests that HLA-C may play a unique role in Ag presentation.…”