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.

Figure 1. Overview of screening strategy.

PBMCs were screened by ex vivo ELISpot analysis for recognition of 187 overlapping 15mer peptides spanning the entire IE1 and IE2. Pools of positively recognized peptides were used to expand the T cells for 12–14 days and subsequently analyzed for CD4⁺ and CD8⁺ T cell recognition using ICS and flow cytometric analysis. CD4 ⁺ T cell epitope deconvolution: The recognized 15mer peptide and the donor's HLA class II molecules were submitted to NetMHCIIpan to predict the HLA class II restriction element and the peptide core sequence interacting with the HLA class II molecule. The interaction was subsequently validated using a biochemical HLA class II binding assay. For a selection of the recognized epitopes, H₆-tagged peptides were produced and used to generate peptide-HLA class II tetramers, which were subsequently used for validation of T cell specificity and HLA class II restriction. CD8⁺ T cell epitope deconvolution: The 15mer peptides recognized by a given donor together with the donors HLA class I molecules were submitted to the HLArestrictor to predict the optimal size epitope and HLA-restriction. Interaction between the predicted epitope and HLA class I molecule was validated by biochemical affinity- and stability assays. The T cell specificity and HLA class I restriction was validated by peptide-HLA class I tetramer staining.

Figure 2. ELISpot and ICS analysis of donor 33.

PBMCs were initially screened by ex vivo ELISpot and the CD4⁺ and CD8⁺ T cell recognition subsequently determined by ICS analysis. Donor 33 recognized four different peptides, which are indicated above the results. A) Ex vivo IFN-γ ELISpot assay. Spot forming units (SFU) are indicated as positive spots per 10⁶ PBMCs. B) ICS analysis of T cells expanded on the identified 15mers. FACS plots show gated CD3⁺ T cells. The elicited frequency is indicated. Pink indicates responding CD8⁺ T cells. Green indicates responding CD4⁺ T cells.

Figure 3. Donor 33 - CD8⁺ T cell epitope validation.

CD8⁺ T cell responses were detected against the 15mers IE1_86–110 and IE1_196–210. In IE1_86–110 two B*08:01 binding optimal peptides, IE1_88–95 and IE1_88–96, were predicted, while in IE1_196–210 one B*08:01-restricted optimal peptide IE1_198–207, was predicted. A) ICS analysis of predicted optimal peptides: Chart showing IFN-γ responses following restimulation of in vitro cultured PBMCs with graded doses of IE1_88–95 and IE1_88–96. B) Optimal epitope and HLA class I restriction validated by ex vivo peptide-HLA class I tetramer staining with the IE1_88–96-HLA-B*08:01 tetramer. C) Optimal epitope and HLA class I restriction validated by ex vivo peptide-HLA class I tetramer staining with the IE1_199–207-HLA-B*08:01 tetramer. The plots show gated CD3⁺ T cells; frequencies of tetramer-positive CD8⁺ T cells (boxed-in and pink) are indicated.

Figure 4. Donor 33 - CD4⁺ T cell epitope validation.

Three different 15mers elicited CD4⁺ T cell responses, IE1_86–100, IE1_91–105, and IE2_356–370 in donor 33. T cells were expanded for 12 days on a mix of the three peptides. The specificity and HLA class II restriction of the CD4⁺ T cell responses were evaluated using peptide-HLA class II tetramers. The cells were stained with anti-CD3, -CD4, and the peptide-HLA class II tetramers either alone or in combination. IE1_86–100 and IE1_91–105 are overlapping peptides and bind to the same DRB1*03:01. The cells are stained in A) with the IE1_86–100-DRB1*03:01 tetramer, in B) with the IE1_91–105-DRB1*03:01 tetramer, and in C) with a combination of PE-labeled IE1_86–100-DRB1*03:01 and APC-labeled IE1_91–105-DRB1*03:01 tetramer. The IE2_356–370 peptide binds to both of the donor's HLA-DR molecules, DRB1*03:01 and DRB3*01:01. The cells are stained in D) with the IE2_356–370-DRB1*03:01 tetramer, in E) the IE2_356–370-DRB3*01:01 tetramer, and in F) with a combination of PE-labeled IE2_356–370-DRB1*03:01 and APC-labeled IE2_356–370-DRB3*01:01 tetramer. The plots show gated CD3⁺ T cells, and the frequency of tetramer-positive CD4⁺ T cells (boxed-in and green) is indicated.