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2 Abstracts / Molecular I

NA vaccination with plasmids encoding single chain ofHCI/peptide/�2 m complexes elicits robust CD8 T cell immu-

ity and pathogen protection

ojung Kim, Lijin Li, Javier Carrero, Michael Diamond, Williamillanders, Ted Hansen, Janet Connolly ∗

Washington University, St. Louis

Vaccination with plasmid DNA encoding single chain trimersSCT) of MHC class I, in which heavy chain, peptide and �2 m areovalently attached with flexible linkers, has been shown by our labnd others to elicit robust CD8 T cell responses to pathogens andumors. Indeed, we found that SCT vaccines could protect in mouse

odels of Listeria or WNV infection. To maximize SCT vaccine effi-acy, it was important to determine if there is a requirement forD4 T cell help and to understand the mechanism by which anti-en is presented. SCT constructs were generated that included aD4 helper T cell epitope and were used as DNA vaccines. Nei-her primary nor memory CD8 T cell responses were dependentpon CD4 T cell help; however the memory CD8 T cell responseas enhanced by either antigen-specific or non-cognate CD4 T cellelp. We speculate that this relative lack of helper dependency

s the result of a high level of stable Ag presentation by the SCTfter DNA vaccination. Direct presentation and cross-presentationave been implicated in antigen presentation following DNA vac-ination but the relative contribution of each of these pathwaysemains controversial. Highly relevant to the question, we showhat SCT are recognized as intact structures following DNA vacci-ation. OVAp5Y (SIINYEKL) is an altered peptide ligand of SIINFEKLhat can be recognized by T cells in the context of the SCT, but cannote directly or cross-presented by native Kb. Importantly, OVAp5YNA did not induce a response whereas DNA vaccines incorporat-

ng Kb/OVA and KbOVAp5Y stimulated CD8 T cell responses. Thusirect presentation of the SCT in the absence of cross-presentationas sufficient to elicit a robust CD8 T cell response after DNA vacci-ation. Furthermore an SCT incorporating an �3 mutant that cannote recognized by CD8 induced a negligible response compared toild type. Similarly a chimeric HLA-A2/Db �3 SCT encoding a WNV

pitope induces a much stronger CD8 T cell response than nativeLA-A2 WNV SCTs following DNA immunization in HHDII mice.hus, based on several experimental findings, SCTs are primarilyecognized as intact structures after DNA vaccination. This recog-ition could result from direct incorporation of the SCT by APC or byembrane transfer through a mechanism known as cross-dressing

r trogocytosis. In any case, the presentation of Ag by an intactCT in a relatively helper independent manner likely explains itsfficacy as a DNA vaccine.

oi:10.1016/j.molimm.2012.02.027

tructural and mechanistic aspects of the peptide loading com-lex

eter Cresswell

Howard Hughes Medical Institute, Yale University School of Medicine

High affinity peptide binding to MHC class I molecules is facil-tated by the peptide loading complex (PLC). The PLC resides inhe endoplasmic reticulum (ER) and contains the TAP transporter,

disulfide-linked heterodimer of tapasin and the thiol oxidore-uctase ERp57, calreticulin and an MHC class I-�2 m heterodimer.AP transports peptides into the ER and the concerted actions of

apasin/ERp57, calreticulin and the enzyme UDP-glucose glycopro-ein transferase (UGT-1), which maintains the single class I glycanf peptide-free human HLA class I molecules in a monoglucosylatedorm, facilitate the binding of high affinity peptides to the MHC class

ology 51 (2012) 5–41

I molecule. We are re-addressing the issue of the stoichiometry ofthe PLC using a single molecule approach and are attempting tounravel how association and dissociation of MHC class I moleculesfrom the PLC is regulated. These issues will be discussed.

doi:10.1016/j.molimm.2012.02.028

Unexpectedly high prevalence of TAP-independent MHC class Iepitopes from vaccinia virus and their contribution to protec-tion in vivo

Margarita Del Val ∗, Silvia Lázaro, Salvador Iborra, Manuel Ramos,Daniel López

CD8+ T lymphocytes screen peptides displayed at the plasmamembrane by major histocompatibility complex (MHC) class Imolecules. It is accepted that most of these peptides result fromcytosolic proteolysis. TAP transporters associated with antigen pre-sentation are crucial to deliver these peptides into the endoplasmicreticulum, where they meet nascent MHC class I molecules. Indi-vidual TAP-independent antigen processing pathways have beendescribed, but the extent to which they globally contribute to CD8+T-cell responses is still unclear. In this report, we study vacciniavirus (VACV), a virus successfully used to eradicate smallpox andcurrently considered as a vaccine vector. About 13% of the primaryCD8+ T-lymphocyte response of infected mice was directed againstviral epitopes presented by MHC class I molecules independentlyof TAP in infected dendritic cells. Analysis of 30 individual VACVepitopes revealed that as many as 12 epitopes were presented inde-pendently of TAP. Interestingly, the primary immunodominancehierarchy was radically different in TAP1-deficient and in wildtype animals. CD8+ T-lymphocyte lines mono-specific for most ofthese epitopes were established from TAP1-deficient animals. Halfof these TAP-independent peptides derived from membrane viralproteins. The remaining 6 derived from cytosolic viral proteins, andyet all gained access to vesicular or secretory antigen presentationpathways in the absence of TAP. Notably, TAP-independent epi-topes were more frequently derived from viral structural proteinsexpressed at the late phase of VACV infection. Finally, CD8+ T lym-phocytes primed by two of these peptides were equally efficientin providing protection against viral infection in TAP-deficient andin wild type mice. These findings underline the remarkable contri-bution of TAP-independent antigen processing pathways to CD8+T-cell priming and to protection from infection with VACV.

doi:10.1016/j.molimm.2012.02.029

Antigen targeting to splenic CD169+ macrophages inducesstrong humoral immune responses and CD4+ T cell activation

Henrike Veninga, Ellen Borg, Hakan Kalay, Yvette van Kooyk,Georg Kraal, Joke M.M. den Haan ∗

VU University Medical Center, Amsterdam

Immune responses against blood-borne pathogens are raisedin the spleen. The blood enters the spleen in the marginal zoneand here resident macrophages are strategically located to captureantigens. We have recently shown a previously unappreciated rolefor marginal metallophilic CD169+ macrophages in the inductionof cytotoxic T cell responses.

To investigate the role of CD169+ macrophages in the inductionof humoral immune responses, we conjugated ovalbumin (OVA) to

antibodies specific for CD169 and DEC205 and compared antigentargeting to CD169+ macrophages to targeting to DEC205+ den-dritic cells (DC). Both targeting strategies induced robust anti-OVAantibody responses at early time points after immunization. How-