Direct Ex Vivo Analyses of HLA-DR1 Transgenic Mice Reveal an Exceptionally Broad Pattern of Immunodominance in the Primary HLA-DR1-Restricted CD4 T-Cell Response to Influenza Virus Hemagglutinin

Abstract

The recent threat of an avian influenza pandemic has generated significant interest in enhancing our understanding of the events that dictate protective immunity to influenza and in generating vaccines that can induce heterosubtypic immunity. Although antigen-specific CD4 T cells are known to play a key role in protective immunity to influenza through the provision of help to B cells and CD8 T cells, little is known about the specificity and diversity of CD4 T cells elicited after infection, particularly those elicited in humans. In this study, we used HLA-DR transgenic mice to directly and comprehensively identify the specificities of hemagglutinin (HA)-specific CD4 T cells restricted to a human class II molecule that were elicited following intranasal infection with a strain of influenza virus that has been endemic in U.S. human populations for the last decade. Our results reveal a surprising degree of diversity among influenza virus-specific CD4 T cells. As many as 30 different peptides, spanning the entire HA protein, were recognized by CD4 T cells, including epitopes genetically conserved among H1, H2, and H5 influenza A viruses. We also compared three widely used major histocompatibility class II algorithms to predict HLA-DR binding peptides and found these as yet inadequate for identifying influenza virus-derived epitopes. The results of these studies offer key insights into the spectrum of peptides recognized by HLA-DR-restricted CD4 T cells that may be the focus of immune responses to infection or to experimental or clinical vaccines in humans.

Cutting Edge: CD4 T Cells Generated from Encounter with Seasonal Influenza Viruses and Vaccines Have Broad Protein Specificity and Can Directly Recognize Naturally Generated Epitopes Derived from the Live Pandemic H1N1 Virus

“…In our previous studies on the primary response to infection with A/New Caledonia 20/99, we found that the HA-specific CD4 T cell repertoire was broad and included .30 epitopes (8). Comparison of HA sequences expressed by the pandemic H1N1 A/California/04/09 virus and other seasonal H1N1 strains of virus (not shown) indicates that there are segments of high sequence diversity where little T cell crossreactivity is expected and regions of complete conservation where we assume full cross-recognition.…”

“…Influenza A/New Caledonia/20/99 and A/California/04/2009 E3 viruses were prepared as previously described (8) and used to infect APCs. APCs purified as described above were washed in serum-free DMEM media and infected with virus [multiplicity of infection (MOI) of 1 or 10 as indicated in the figure legends] for 1 h in serum-free media at 37˚C, followed by dilution and washing with complete culture media, described above.…”

“…As a source of APCs, PBMCs from the same donor were T depleted using CD4 and CD8 microbeads (Miltenyi Biotec). ELISPOT assays were performed as described (8,9), with some modifications for human samples. Briefly, 96-well filter plates (Millipore, Billerica, MA) were coated with 15 mg/ml purified anti-human IFN-g (clone 1-D1K, MabTech, Cincinnati, OH), washed, and incubated with RPMI 1640 containing 10% FCS to block nonspecific binding.…”

Abortive activation of CD4 T cell responses during competitive priming in vivo

“…A single cell suspension from the LN of an individual mouse was prepared and depleted of non-CD4 T cells as described previously (50). During kinetic experiments, LN from an individual mouse was enriched for CD4 T cells by negative selection (Miltenyi Biotech).…”

Infection with Seasonal Influenza Virus Elicits CD4 T Cells Specific for Genetically Conserved Epitopes That Can Be Rapidly Mobilized for Protective Immunity to Pandemic H1N1 Influenza Virus

“…Influenza virus A/New Caledonia/20/99 was prepared in the allantoic cavities of embryonated chicken eggs, as described previously (46). Influenza virus A/California/04/2009 E3 (referred to below as A/California; generously provided by David Topham at the University of Rochester) is known to contain alterations in the HA gene resulting in three amino acid differences from the original strain isolated from humans (27).…”