Using Ig as a molecular scaffold, we have developed a general approach to making soluble divalent analogs of MHC class I and II molecules (MHC-I/Ig and MHC-II/Ig). The divalent nature of these molecules effectively increased the affinity of these analogs for their cognate ligands, antigen specific T cells. Peptide loaded divalent high affinity MHC analogs (pepMHC/Ig) can be used to directly visualize antigen specific T cells by flow cytometry. The goals of the current application are to selectively modulate and monitor antigen-specific T cell responses in murine models using pepMHC/Ig complexes. Our first specific aim will focus on defining the mechanism of in vitro CTL modulation by pepMHC-I/Ig complexes. We will determine if pepMHC/Ig complexes inhibit 2C CTL by competitive inhibition, induction of anergy, or induction of apoptosis. The second specific aim will analyze the effects of pepMHC-I/Ig complexes on T cells in vivo using two model systems: peptide- specific 2C CTL and a Hepatitis B specific CTL, 6C2. The influence of pepMHC-I/Ig on both trafficking and modulation of CTL responses in vivo will be defined. Initial experiments will be aimed at optimizing platform development for homogeneously loading MHC-I/Ig complexes with the peptides of interest. Trafficking studies on 2C CTL will be studied using B6 animals injected with P815 tumor cells and suppression will be studied in a 2C-mediated model of diabetes. We will analyze the effects of HepBLd/Ig on Hepatitis B specific CTL by characterizing the ability of HepBLd/Ig complexes to: bind to HepB specific CTL, inhibit HepB specific CTL mediated lysis of target cells in vitro, and modulate hepatocellular necrosis in HBsAg transgenic mice injected with 6C2, HepB specific CTL in vivo. Should the direct modulation of specific CTL in vivo prove ineffective, the ability of pepMHC-I/Ig coupled to a fungal toxin, ricin, to specifically kill T cells in vitro and in vivo will be analyzed. To analyze the ability of pepMHC/Ig complexes to selectively regulate class II-restricted immune responses, we will study the ability of pepMHC-II/Ig compounds to modulate class II-restricted, immune responses. The model system to be analyzed will be the effects of MCCI-Ek/Ig on cytochrome-specific immune responses. Analysis of the effects of soluble divalent class II MHC molecules will reveal several interesting and unique features about regulation of class II-restricted immune responses. The first part of this specific aim we will analayze modulation of in vitro immune responses by MHC-II/Ig complexes and also address the ability of soluble divalent analogs of peptide antagonists complexed to the I-Ek/Ig molecule to modulate in vitro immune responses. In the second part of this specific aim we will study the ability of MHC-II/Ig complexes to modulate in vivo immune responses.
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