Haruo Kozono

Detection of Antigen-Specific T Cells with Multivalent Soluble Class II MHC Covalent Peptide Complexes

Multimeric soluble MHC class II molecules stably occupied with covalently attached peptides bind with appropriate specificity to T cell hybridomas and T cells from T cell receptor transgenic mice. There is a direct correlation between soluble T cell receptor affinity for monomeric MHC/peptide and level of binding of multimeric MHC/peptide to T cells. While binding of the multimeric MHC/peptide complex is proportional to T cell receptor affinity and expression level, there is little influence of T cell CD4.

Differential binding properties of B7-H1 and B7-DC to programmed death-1.

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.

Triggering receptor expressed on myeloid cell-like transcript 2 (TLT-2) is a counter-receptor for B7-H3 and enhances T cell responses

The B7 family member B7-H3 (CD276) plays important roles in immune responses. However, the function of B7-H3 remains controversial. We found that murine B7-H3 specifically bound to Triggering receptor expressed on myeloid cells (TREM)-like transcript 2 (TLT-2, TREML2). TLT-2 was expressed on CD8+ T cells constitutively and on activated CD4+ T cells. Stimulation with B7-H3 transfectants preferentially up-regulated the proliferation and IFN-γ production of CD8+ T cells. Transduction of TLT-2 into T cells resulted in enhanced IL-2 and IFN-γ production via interactions with B7-H3. Blockade of the B7-H3:TLT-2 pathway with a mAb against B7-H3 or TLT-2 efficiently inhibited contact hypersensitivity responses. Our results demonstrate a direct interaction between B7-H3 and TLT-2 that preferentially enhances CD8+ T cell activation.

Multiple binding sites for bacterial superantigens on soluble class II MHC molecules

We used surface plasmon resonance to study the binding of a set of soluble mouse I-E class II major histocompatibility molecules, each occupied by a different single peptide, to the staphylococcal enterotoxin superantigens, SEA and SEB. The rates of association and dissociation to SEA varied greatly depending on the I-E-bound peptide. By contrast, binding to SEB yielded fast association and dissociation rates, which were relatively peptide independent. The results also indicated nonoverlapping binding sites for SEB and SEA on class II and raised the possibility of enhanced SAg presentation to T cells by cross-linking of cell surface class II.

Biophysical studies of T-cell receptors and their ligands

Recently developed methodologies for the production of the soluble extracellular domains of alpha beta TCRs have allowed several biophysical characterizations. The thermodynamic and kinetic parameters associated with specific ligand interactions between the TCR and MHC-peptide complexes, as well as superantigens, are now being established. Crystallographic studies of isolated TCR fragments have yielded the structures of a V alpha domain and the two extracellular domains of a beta-chain. These investigations are beginning to allow a new visualization of antigen recognition and T-cell activation processes.

Ovalbumin-Protein σ1 M-Cell Targeting Facilitates Oral Tolerance With Reduction of Antigen-Specific CD4+ T Cells

BACKGROUND & AIMS The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. METHODS Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-psigma1), psigma1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined. RESULTS A low dose of OVA-psigma1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-psigma1 than those fed psigma1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyers patches of orally tolerized mice showed increased transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) production compared with nontolerized mice. CONCLUSIONS These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.

A role for the P1 anchor residue in the thermal stability of MHC class II molecule I-Ab

The thermal stability of the murine MHC class II molecule, I-A(b), in complex with invariant chain-derived peptide (CLIP) and an antigenic peptide derived from the alpha subunit of the I-E molecule (Ealpha) at mildly acidic and neutral pH were analyzed using circular dichroism (CD). The stability of I-A(b)-CLIP was increased by a single amino acid substitution in the P1 anchor residue, from Met of CLIP to Phe of Ealpha, similar, in this respect, to I-A(b)-Ealpha. This indicates that hydrophobic interaction in the P1 pocket is critical and plays a primary role in the stability of the complex. The structural models of I-A(b)-peptides based on the crystal structure of I-A(d) might explain the increased stability and the preference for hydrophobic residues in this site. Taken together with what is known of the resident stability at a mildly acidic pH, the difference in stability would closely correlate with the ability of MHC class II to exchange peptides from CLIP to antigenic peptides in the endosome.

Contribution of a single hydrogen bond between βHis81 of MHC class II I-Ek and the bound peptide to the pH-dependent thermal stability

To determine the energetic contribution of the hydrogen bond between βHis81 of the major histocompatibility complex class II (MHC II) molecule, I‐Ek, and the bound hemoglobin peptide (Hb), we analyzed the thermal stability of the hydrogen bond‐disrupted mutant, I‐Ek‐Hb βH81Y, in which the βHis81 residue was replaced with Tyr, by differential scanning calorimetry. The thermal stability of the I‐Ek‐Hb βH81Y mutant was lower than that of the I‐Ek‐Hb wild‐type, mainly due to the decreased enthalpy change. The difference in the denaturation temperature of the I‐Ek‐Hb βH81Y mutant as compared with that of the I‐Ek‐Hb wild‐type at pH 5.5 was only slightly smaller than that at pH 7.4, in agreement with the increased stability at an acidic pH, a unique characteristic of MHC II. Thus, the hydrogen bond contributed by βHis81 is critical for peptide binding, and is independent of pH, which can alter the hydrophilicity of the His residue.

Single-Molecule Motions of MHC Class II Rely on Bound Peptides

The major histocompatibility complex (MHC) class II protein can bind peptides of different lengths in the region outside the peptide-binding groove. Peptide-flanking residues (PFRs) contribute to the binding affinity of the peptide for MHC and change the immunogenicity of the peptide/MHC complex with regard to T cell receptor (TCR). The mechanisms underlying these phenomena are currently unknown. The molecular flexibility of the peptide/MHC complex may be an important determinant of the structures recognized by certain T cells. We used single-molecule x-ray analysis (diffracted x-ray tracking (DXT)) and fluorescence anisotropy to investigate these mechanisms. DXT enabled us to monitor the real-time Brownian motion of the peptide/MHC complex and revealed that peptides without PFRs undergo larger rotational motions than peptides with PFRs. Fluorescence anisotropy further revealed that peptides without PFRs exhibit slightly larger motions on the nanosecond timescale. These results demonstrate that peptides without PFRs undergo dynamic motions in the groove of MHC and consequently are able to assume diverse structures that can be recognized by T cells.