Yu-Chun Lone

A mouse model of human adaptive immune functions: HLA-A2.1-/HLA-DR1-transgenic H-2 class I-/class II-knockout mice

HLA‐A2.1‐/HLA‐DR1‐transgenic H‐2 class I‐/class II‐knockout mice were created and their immunological potential evaluated in response to hepatitis B DNA vaccine. Every single immunized mouse developed hepatitis B virus‐specific antibodies, HLA‐DR1‐restricted helper, and HLA‐A2.1‐restricted cytolytic T cell responses directed at the same immunodominant epitopes as those identified in naturally infected or vaccinated humans. These mice were specifically protected against a hepatitis B‐recombinant vaccinia virus infection with a 10,000‐fold or more reduction of the virus load at day 4 post‐challenge. These mice represent a unique in vivo experimental model for human immune function studies without any interference with mouse MHC response which dwarfed the prediction of human responses. Furthermore, they enable the complete monitoring of immune adaptative responses for preclinical screening of candidate vaccines.

In vitro induction of specific cytotoxic T lymphocytes using recombinant single-chain MHC class I/peptide complexes.

We have previously described the production and purification of a murine single-chain, soluble recombinant major histocompatibility complex (MHC) class I molecule (SC-Kd). A similar strategy was devised to produce a recombinant HLA-A2.1 (SC-A2) molecule. The latter was composed of the first three domains of the HLA-A2.1 heavy chain connected to human beta 2-microglobulin through a spacer of 15 amino acids. Immunoaffinity-purified SC-A2 molecules-were correctly folded and biologically functional. They specifically bound HLA-A2-restricted peptides and induced a peptide-specific cytotoxic T lymphocyte (CTL) clone to proliferate and secrete interleukin-2. The ability of murine and human SC-MHC molecules to elicit primary CTLs in vitro was next investigated. When coated in high density onto beads, complexes of antigenic peptide and SC-Kd or SC-A2 molecules efficiently induced a specific primary CTL response in vitro. Furthermore, the structural features of these CTLs were characterized by T cell receptor-beta chain analysis, which revealed rearrangements very similar, if not identical, to those found in CTLs generated by in vivo immunization. Such single-chain, soluble recombinant MHC class I molecules should provide a useful tool in particular for peptide binding assays and for in vitro primary CTL induction to identify immunogenic peptides such as those derived from known tumor-associated antigens.

In vitro induction of naive cytotoxic T lymphocytes with complexes of peptide and recombinant MHC class I molecules coated onto beads: role of TCR/ligand density.

We previously reported that complexes of peptide with soluble single‐chain recombinant MHC (SC‐MHC) class I molecules are able to induce cytotoxic T lymphocytes (CTL) in vitro in a murine system with an efficiency comparable to that observed with peptide‐pulsed dendritic cells as antigen‐presenting cells. In this report, we have assessed the capacity of preformed peptide/SC‐Kd complexes in monomeric or dimeric form as well as of peptide/SC‐Kd‐loaded beads to generate in vitro specific CTL responses from naive DBA/2 spleen cells. Peptide/SC‐Kd‐coated beads were consistently more efficient. We evaluated the role of co‐stimulatory molecules, using monoclonal antibodies anti‐CD80 or anti‐CD86. In addition, the capacity of peptide/SC‐Kd‐coated beads to generate a CTL response from purified naive CD8+ T cells was ascertained. Taken together, the results indicate that, under our conditions, CTL priming does not require the participation of co‐stimulatory molecules and is the consequence of a direct interaction between the cognate TCR on peptide‐specific CTL precursors and the peptide/SC‐Kd‐loaded beads. Titration of the amount of preformed complexes of SC‐Kd and peptide 170 –179 of HLA‐CW3 that need to be coated onto the beads to prime CTL precursors shows an activation threshold which can be calculated to be between 25 000 and 50 000 complexes. In effect, in cultures stimulated with specific peptide CW3/SC‐Kd complexes representing less than 50 % occupancy of the total (105 ) complexes on the beads, no peptide‐specific cytolytic activity was observed. These results suggest that the efficiency of the primary CTL induction depends on the density of specific peptide/SC‐Kd complexes present on the beads.

Role of class I human leukocyte antigen molecules in early steps of echovirus infection of rhabdomyosarcoma cells.

Several echoviruses use decay accelerating factor (DAF) as a cell surface receptor. However, most of them require additional cell surface coreceptors. We investigated the respective roles of DAF and class I human leukocyte antigen (HLA) molecules in the early steps of the echovirus 11 (EV11) lifecycle in rhabdomyosarcoma (RD) cells. EV11 infection was inhibited at an early stage by anti-beta2-microglobulin (beta2m) and anti-HLA monoclonal antibodies and by a soluble monochain HLA class I molecule. Expression of class I HLA molecules restored the early steps of the EV11 lifecycle, but its expression was not sufficient for EV11 replication and particle production. Expression of HLA class I molecules was associated with leukocyte cell line permissiveness to EV11 infection. In conclusion, HLA class I molecules are involved in the early steps of EV11 infection of RD cells and appear to participate in a complex interplay of surface molecules acting as coreceptors, including DAF.

The Th1 immune response against HIV-1 Gag p24-derived peptides in mice expressing HLA-A02.01 and HLA-DR1

Using HLA‐DR1‐transgenic H‐2 class II knockout mice, we identified two new HLA‐DR1‐restricted HIV‐1 Gag p24‐derived epitopes (Gag321–340 and Gag331–350) and confirmed the immunogenicity of seven that have been previously described. The human relevance was confirmed for the two new ones (Gag321–340 and Gag331–350) assaying peripheral blood mononuclear cells from HLA‐DR1+ HIV‐1‐infected long‐term asymptomatic subjects and showing that Gag331–350 could prime CD4+ T cells from two HLA‐DR1+ HIV‐1 seronegative donors in vitro. Seven of these epitopes, structurally conserved among HIV‐1 clade B isolates, were selected for a comparative evaluation of their Th1 helper potential by immunizing HLA‐A02.01/HLA‐DR1‐transgenic, H‐2 class I/class II knockout mice with recombinant mouse invariant chain constructs in which each helper epitope was inserted in association with two reporter HIV‐1‐derived HLA‐A02.01‐restricted CD8+ T cell epitopes. A T helper effect was demonstrated in all cases, and was particularly strong with epitopes Gag301–320, Gag321–340 and Gag271–290, which should, therefore, be considered in the design of new vaccines.

Mutational analysis and molecular modeling of the binding of Staphylococcus aureus enterotoxin C2 to a murine T cell receptor Vβ10 chain

We investigated the role of the β chain HV4 region in the binding of a Vβ10 T cell receptor to superantigen S. aureus enterotoxin C2 (SEC2)/MHC class II complexes. Residues 6971 of the Cw3/1.1 TCR Vβ10 chain, derived from an H‐2Kd‐restricted cytotoxic clone, were individually changed to alanine using site‐directed mutagenesis, and mutated TCR β chains were transfected along with the wild‐type TCR α chain into a TCR α–β– T hybridoma. SEC2/MHC recognition was measured by IL‐2 production. Alanine substitutions in the HV4β region, either did not affect (Ser69 and Lys71), or increased the recognition of SEC2/HLA‐DR1 complex (Asp70), arguing against a general and direct role for the HV4β region in superantigenrecognition. A theoretical‐computational model of the SEC2/TCR Vβ10 chain complex was constructed and predicted the presence of a unique salt bridge between Vβ Asp30 and SEC2 Lys103. A perfect correlation was found between the likely presence of this salt bridge and the capacity of the HV4β and previously obtained CDR1β alanine mutants to induce an equal or greater response than the wild‐type TCR.

Identification of novel HLA-A11-restricted T-cell epitopes in the Ebola virus nucleoprotein

The Ebola virus (EBOV) is a very contagious virus that is highly fatal in humans and animals. The largest epidemic was in West Africa in 2014, in which over 11,000 people died. However, to date, there are no licensed vaccines against it. Studies show that CD4+ and CD8+ T-cell responses, especially cytotoxic T-lymphocyte (CTL) responses, play key roles in protecting individuals from EBOV infection. Since HLA-restricted epitope vaccines are likely to be effective and safe immunization strategies for infectious diseases, the present study screened for CTL epitopes in the EBOV-nucleoprotein that are restricted by HLA-A11 (a common allele in Chinese people). Predictive computer analysis of the amino-acid sequence of EBOV-nucleoprotein identified ten putative HLA-A11-restricted epitopes. ELISPOT assay of immunized HLA-A11/DR1 transgenic mice showed that five (GR-9, VR-9, EK-9, PK-9, and RK-9) induced effective CTL responses. Additional epitope analyses will aid the design of epitope vaccines against EBOV.