Use of Transgenic HLA A*0201/Kb and HHD II Mice To Evaluate Frequency of Cytomegalovirus IE1-Derived Peptide Usage in Eliciting Human CD8 Cytokine Response

Abstract

Unlike the pp65 protein of human cytomegalovirus (CMV), which has an immunodominant peptide, pp65 [495][496][497][498][499][500][501][502][503] , recognized by human CD8؉ cells in the context of HLA A‫,1020ء‬ the fine peptide specificity for CMV IE1 has shown no such immunodominance. With the use of transgenic HLA A‫/1020ء‬Kb and HHD II mice, a selected pool of IE1 peptides, including IE1 p256-264 , IE1 p297-304 , and IE1 p316-324 , were shown to stimulate cytolytic T-lymphocyte lysis in the context of HLA A‫.1020ء‬ Based on an intracellular gamma interferon response, IE1 p297-304 , a previously unrecognized CD8 epitope, triggered a prominent response to CMV IE1 in HLA A‫1020ء‬ subjects.Murine models to predict human cytomegalovirus (CMV) vaccine immunogenicity are available to detect the presence of a specific immune response, such as cytolytic T-lymphocyte (CTL) function (2, 5, 28), CD4ϩ gamma interferon (IFN-␥) helper response (15, 22), or antibody response to specific CMV proteins (18,20,21). Of interest is the use of the transgenic A2Kb mouse containing the human HLA A‫1020ء‬with the murine ␣3 chain (Kb) (9,19,29). The murine model HHD II is also a transgenic HLA A‫1020ء‬ mouse that contains a disrupted murine major histocompatibility complex (MHC) molecule, forcing the mouse to present all its immunological epitopes through the HLA pathway (10). Both have become useful models to investigate peptide recognition of specific proteins that can be extrapolated to human subjects.The immunodominant HLA A‫-1020ء‬restricted peptide (pp65 [495][496][497][498][499][500][501][502][503] ) of CMV pp65 has been well studied (3,6,12,30), whereas there is no immunodominant peptide recognized for the CMV IE1 protein, despite the prevalence of an IE1-specific CTL response (11,14,16,17). Three reports have described the stimulatory effect of peptides IE1 p315-323 , IE1 p316-324 , and IE1 p354-362 from CMV IE1 in the context of HLA A‫1020ء‬ (11, 17, 24) in a cytokine flow cytometry or CTL assay. However, not all CMV-seropositive subjects respond to these peptides (17). This report describes the use of HLA A2 transgenic mice to identify a novel IE1 peptide, IE1-297, that is recognized by murine CTLs as well as human CD8 cells by cytokine flow cytometry.T2 stabilization. T2 cells are defective for endogenous class I presentation, but the presence of peptide binding to the MHC molecule will stabilize its expression on the cell surface. The stabilized MHC molecule can be detected by flow cytometry with a monoclonal antibody to the HLA A‫1020ء‬ molecule (13). The peptide sequences used in this study were collected from two algorithms for HLA peptide predicted motifs: SYFPEITHI (23)

Immunization of HLA Class I Transgenic Mice Identifies Autoantigenic Epitopes Eliciting Dominant Responses in Type 1 Diabetes Patients

“…All the epitopes identified by this method and tested by ELISPOT scored positive in T1D patients and negative in control subjects. In the recent past, HHDII mice have been used to facilitate the generation of HLA-A*0201-restricted CTLs and the study of immunization protocols for effective treatment of human infectious diseases or cancers (42)(43)(44)(45)(46)49). In this study, we present the first report of plasmid-DNA immunization aimed at identifying CD8 ϩ T cells in human autoimmune disease.…”

Human Cytomegalovirus pp71 Stimulates Major Histocompatibility Complex Class I Presentation of IE1-Derived Peptides at Immediate Early Times of Infection

“…For fixation, CX-and-AcD (CX/AcD)-treated stimulator cells were resuspended in PBS-0.5% paraformaldehyde, incubated for 20 min at room temperature, and washed twice with PBS. CTL clones specific for the HLA-A2-restricted HCMV-derived peptides IE1 from amino acids 297 to 305 (IE1 TMY -CTL [8,37]) and pp65 from amino acids 495 to 503 (pp65 NLV -CTL [8,38,39]) were generated by peptide immunization of HLA-A2/huCD8 (hu stands for human) double-transgenic mice (a kind gift from L. Sherman, The Scripps Institute, La Jolla, CA, USA). Details of establishment and culturing of these cells were previously described (8).…”

Recombinant viruses as tools to study human cytomegalovirus immune modulation

“…Paraformaldehyde Wxation and ELISpot assays were performed as described before [16]. CTL lines speciWc for the HLA-A0201 (A2) restricted HCMV-derived peptides pp65 [495][496][497][498][499][500][501][502][503] [34,35] (pp65 NLV -CTL) and IE1 297-305 [36] (IE1 TMY -CTL) were used in these analyses. The CTL lines have been generated by immunizing HLA-A2/huCD8 double-transgenic (tg) mice [16].…”