Novel Immunodominant CD4+ T Cell Epitopes in the CMV Proteins IE1 and IE2

Background: Cytomegalovirus (CMV) continues to be an important human pathogen in allogeneic hematopoietic cell transplant (allo-HCT) recipients. Both CD4+ and CD8+ T cells are important for long-term control of the virus. Identification of CMV-specific T cell epitopes has primarily focused on CD8+ T cell epitopes. Adoptive transfer of CMV-specific CD4+ T cells has previously been associated with prolonged protection from reactivation and CMV disease.

Aims: To characterize the repertoire and restriction of CMV-specific CD4+ T cell responses against immediate early antigen 1 and 2 (IE1 and IE2) in 16 healthy, HLA-typed, CMV+ donors.

Methods: We performed IFN-γ ELISPOT assays ex vivo on PBMCs from CMV+ donors using 187 peptides (15 amino acids long and overlapping 10 amino acids) spanning the entire IE1 and IE2. These peptides were distributed into individual microtiter wells, PBMCs from each donor were added and incubated, and the resulting responses were measured by IFN-γ ELISPOT analysis. To validate the peptide-specificity and to establish the phenotype of the responding cells, peptides eliciting IFN-γ release in the ELISPOT analysis were subsequently used to stimulate PBMCs in vitro for 12-14 days followed by flow cytometric intracellular cytokine secretion assay. For the identified CD4+ T cell responses, peptide-MHC class II (MHCII) affinity measurements were used to suggest the most likely restricting element(s) among the donors MHCII molecules. To validate MHCII restriction element(s), MHCII tetramers (TMR) were generated and used to label in vitro stimulated PBMCs.

Results: We have identified 27 CD4+ T cell epitopes in IE1 and IE2; 19 of these are novel. Several CD4+ T cell epitopes appeared to be immunodominant. Thus, one DRB1*0101-restricted IE2 epitope was recognized by 5/5 DRB1*0101-positive donors, two DRB1*0301-restricted epitopes, one in IE2 and one in IE1, were recognized by 4/4 and 4/4 DRB1*0301-positive donors, respectively, and one DRB5*0101-restricted IE1 epitope was recognized by 4/4 DRB5*0101-positive donors. Additional epitopes appear to be immunodominant by functional analysis, but have not yet been validated with MHCII TMRs.

Conclusions: We have found multiple novel CD4+ epitopes in CMV IE1 and IE2 of which several have been validated with MHCII TMRs. These epitopes could be used to explore the reconstitution of CMV-specific CD4+ T cells following allo-HCT, and they could represent promising candidates for adoptive CD4+ T cell transfer.