Induction of antitumor immunity by CTL epitopes genetically linked to membrane-anchored beta2-microglobulin

Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing beta2-microglobulin (beta2m) as an integral membrane protein. In this study, we explored membrane-anchored beta2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257-264, and TRP-2181-188, each genetically fused with the N terminus of membranal beta2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257-264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257-264/beta2m were significantly superior to OVA257-264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal beta2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.     

Influence of xenogeneic beta2-microglobulin on functional recognition of H-2Kb by the NK cell inhibitory receptor Ly49C

NK cells maintain self-tolerance through expression of inhibitory receptors that bind MHC class I (MHC-I) molecules. MHC-I can exist on the cell surface in several different forms, including "peptide-receptive" or PR-MHC-I that can bind exogenous peptide. PR-MHC-I molecules are short lived and, for H-2K(b), comprise approximately 10% of total MHC-I. In the present study, we confirm that signaling through the mouse NK inhibitory receptor Ly49C requires the presence of PR-K(b) and that this signaling is prevented when PR-K(b) is ablated by pulsing with a peptide that can bind to it with high affinity. Although crystallographic data indicate that Ly49C can engage H-2K(b) loaded with high-affinity peptide, our data suggest that this interaction does not generate an inhibitory signal. We also show that no signaling occurs when the PR-K(b) complex has mouse beta(2)-microglobulin (beta(2)m) replaced with human beta(2)m, although replacement with bovine beta(2)m has no effect. Furthermore, we show that beta(2)m exchange occurs preferentially in the PR-K(b) component of total H-2K(b). These conclusions were reached in studies modulating the sensitivity to lysis of both NK-resistant syngeneic lymphoblasts and NK-sensitive RMA-S tumor cells. We also show, using an in vivo model of lymphocyte recirculation, that engrafted lymphocytes are unable to survive NK attack when otherwise syngeneic lymphocytes express human beta(2)m. These findings suggest a qualitative extension of the "missing self" hypothesis to include NK inhibitory receptors that are restricted to the recognition of unstable forms of MHC-I, thus enabling NK cells to respond more quickly to events that decrease MHC-I synthesis.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.     

Xenogeneic beta 2-microglobulin substitution affects functional binding of MHC class I molecules by CD8+ T cells

NK cells and CD8+ T cells bind MHC-I molecules using distinct topological interactions. Specifically, murine NK inhibitory receptors bind MHC-I molecules at both the MHC-I H chain regions and beta2-microglobulin (beta2m) while TCR engages MHC-I molecules at a region defined solely by the class I H chain and bound peptide. As such, alterations in beta2m are not predicted to influence functional recognition of MHC-I by TCR. We have tested this hypothesis by assessing the capability of xenogeneic beta2m to modify the interaction between TCR and MHC-I. Using a human beta2m-transgenic C57BL/6 mouse model, we show that human beta2m supports formation and expression of H-2K(b) and peptide:H-2K(b) complexes at levels nearly equivalent to those in wild-type mice. Despite this finding, the frequencies of CD8+ single-positive thymocytes in the thymus and mature CD8+ T cells in the periphery were significantly reduced and the TCR Vbeta repertoire of peripheral CD8+ T cells was skewed in the human beta2m-transgenic mice. Furthermore, the ability of mouse beta2m-restricted CTL to functionally recognize human beta2m+ target cells was diminished compared with their ability to recognize mouse beta2m+ target cells. Finally, we provide evidence that this effect is achieved through subtle conformational changes occurring in the distal, peptide-binding region of the MHC-I molecule. Our results indicate that alterations in beta2m influence the ability of TCR to engage MHC-I during normal T cell physiology.     

Phosphorylation-dependent functional coupling of hSlo calcium-dependent potassium channel and its hbeta 4 subunit.

The auxiliary beta4 subunit of the human slowpoke calciumdependent potassium (slo) channel is expressed predominantly in the brain. Co-expression of beta4 subunit with the slo channel alpha subunit in HEK293 and Chinese hamster ovary cells slows channel activation and deactivation and also shifts the voltage dependence of the channel to more depolarized potentials. We show here that the functional interaction between the hbeta4 subunit and the slo channel is influenced by the phosphorylation state of hbeta4. Treatment of cells with okadaic acid (OA) reduces the effect of hbeta4 on slo channel activation kinetics and voltage dependence but not on slo channel deactivation kinetics. The effect of OA can be blocked by mutating three putative serine/threonine phosphorylation sites in hbeta4 (Thr-11/Ser-17/Ser-210) to alanines, suggesting that OA potentiates phosphorylation of hbeta4 and thereby suppresses its functional coupling to the slo channel. Mutation of Ser-17 alone to a negatively charged residue (S17E) can mimic the effect of OA. Mutating all three phosphorylation sites in hbeta4 to negatively charged residues (T11D/S17E/S210E) not only suppresses the effect of hbeta4 on slo channel activation kinetics and voltage dependence, it also suppresses its effect on slo channel deactivation kinetics. Co-immunoprecipitation/Western blot experiments indicate that all of these hbeta4 mutants, as well as the wild-type hbeta4, bind to the slo channel. Taken together, these data suggest that phosphorylation of the beta4 subunit dynamically regulates the functional coupling between the beta4 subunit and the pore-forming alpha subunit of the slo channel. In addition, phosphorylation of different residues in hbeta4 differentially influences its effects on slo channel activation kinetics, deactivation kinetics, and voltage dependence.     

Assembly of MHC class I molecules analyzed in vitro

Recent evidence suggests that peptide ligands take part in the assembly of class I molecules in living cells. We now describe a simple system for studying class I assembly in vitro. Detergent extracts of the mutant cells RMA-S and .174, in which class I assembly does not occur spontaneously, will support assembly in vitro when specific peptides are added. Peptides stabilize a conformational change in the class I heavy chain and association with beta 2-microglobulin, at concentrations approximately 100-fold lower than required in "peptide feeding" experiments with whole cells. We show that peptides bind class I molecules during assembly and demonstrate that the conformational change induced in the heavy chain is influenced by the concentrations of both peptide and beta 2-microglobulin.     

Recognition of class I MHC by a rat Ly49 NK cell receptor is dependent on the identity of the P2 anchor amino acid of bound peptide

Members of the rodent Ly49 receptor family control NK cell responsiveness and demonstrate allele specificity for MHC class I (MHC-I) ligands. For example, the rat Ly49i2 inhibitory NK cell receptor binds RT1-A1(c) but not other rat MHC class Ia or Ib molecules. RT1-A1(c) preferentially binds peptides with proline at the second, or P2, position, which defines it as an HLA-B7 supertype MHC-I molecule. Previously, our laboratory showed that mutations within the MHC-I supertype-defining B-pocket of RT1-A1(c) could lead to alterations in P2 anchor residues of the peptide repertoire bound by RT1-A1(c) and loss of recognition by Ly49i2. Although suggestive of peptide involvement, it was unclear whether the peptide P2 anchor residue or alteration of the RT1-A1(c) primary sequence influenced Ly49i2 recognition. Therefore, we directly investigated the role of the P2 anchor residue of RT1-A1(c)-bound peptides in Ly49i2 recognition. First, fluorescent multimers generated by refolding soluble recombinant RT1-A1(c) with individual synthetic peptides differing only at the P2 anchor residue were examined for binding to Ly49i2 NK cell transfectants. Second, cytotoxicity by Ly49i2-expressing NK cells toward RMA-S target cells expressing RT1-A1(c) bound with peptides that only differ at the P2 anchor residue was evaluated. Our results demonstrate that Ly49i2 recognizes RT1-A1(c) bound with peptides that have Pro or Val at P2, whereas little or no recognition is observed when RT1-A1(c) is complexed with peptide bearing Gln at P2. Thus, the identity of the P2 peptide anchor residue is an integral component of MHC-I recognition by Ly49i2.     

CD1d-independent NKT cells in beta 2-microglobulin-deficient mice have hybrid phenotype and function of NK and T cells

Unlike CD1d-restricted NK1.1(+)TCRalphabeta(+) (NKT) cells, which have been extensively studied, little is known about CD1d-independent NKT cells. To characterize their functions, we analyzed NKT cells in beta(2)-microglobulin (beta(2)m)-deficient B6 mice. They are similar to NK cells and expressed NK cell receptors, including Ly49, CD94/NKG2, NKG2D, and 2B4. NKT cells were found in normal numbers in mice that are deficient in beta(2)m, MHC class II, or both. They were also found in the male HY Ag-specific TCR-transgenic mice independent of positive or negative selection in the thymus. For functional analysis of CD1d-independent NKT cells, we developed a culture system in which CD1d-independent NKT cells, but not NK, T, or most CD1d-restricted NKT cells, grew in the presence of an intermediate dose of IL-2. IL-2-activated CD1d-independent NKT cells were similar to IL-2-activated NK cells and efficiently killed the TAP-mutant murine T lymphoma line RMA-S, but not the parental RMA cells. They also killed beta(2)m-deficient Con A blasts, but not normal B6 Con A blasts, indicating that the cytotoxicity is inhibited by MHC class I on target cells. IL-2-activated NKT cells expressing transgenic TCR specific for the HY peptide presented by D(b) killed RMA-S, but not RMA, cells. They also killed RMA (H-2(b)) cells that were preincubated with the HY peptide. NKT cells from beta(2)m-deficient mice, upon CD3 cross-linking, secreted IFN-gamma and IL-2, but very little IL-4. Thus, CD1d-independent NKT cells are significantly different from CD1d-restricted NKT cells. They have hybrid phenotypes and functions of NK cells and T cells.     

Tapasin-/- and TAP1-/- macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH

Alternate class I MHC (MHC-I) Ag processing via cytosolic or vacuolar pathways leads to cross-presentation of exogenous Ag to CD8 T cells. Vacuolar alternate MHC-I processing involves phagolysosomal Ag proteolysis and peptide binding to MHC-I in post-Golgi compartments. We report the first study of alternate MHC-I Ag processing in tapasin(-/-) cells and experiments with tapasin(-/-) and TAP1(-/-) macrophages that characterize alternate MHC-I processing. Tapasin promotes retention of MHC-I in the endoplasmic reticulum (ER) for loading with high affinity peptides, whereas tapasin(-/-) cells allow poorly loaded MHC-I molecules to exit the ER. Hypothetically, we considered that a large proportion of post-Golgi MHC-I on tapasin(-/-) cells might be peptide-receptive, enhancing alternate MHC-I processing. In contrast, alternate MHC-I processing was diminished in both tapasin(-/-) and TAP1(-/-) macrophages. Nonetheless, these cells efficiently presented exogenous peptide, suggesting a loss of MHC-I stability or function specific to vacuolar processing compartments. Tapasin(-/-) and TAP1(-/-) macrophages had decreased MHC-I stability and increased susceptibility of MHC-I to inactivation by acidic conditions (correlating with vacuolar pH). Incubation of tapasin(-/-) or TAP1(-/-) cells at 26 degrees C decreased susceptibility of MHC-I to acid pH and reversed the deficiency in alternate MHC-I processing. Thus, tapasin and TAP are required for MHC-I to bind ER-derived stabilizing peptides to achieve the stability needed for alternate MHC-I processing via peptide exchange in acidic vacuolar processing compartments. Acidic pH destabilizes MHC-I, but also promotes peptide exchange, thereby enhancing alternate MHC-I Ag processing. These results are consistent with alternate MHC-I Ag processing mechanisms that involve binding of peptides to MHC-I within acidic vacuolar compartments.     

TAP association influences the conformation of nascent MHC class I molecules

The influence of TAP-MHC class I interactions on peptide binding to the class I heavy chain is assessed during TAP-dependent assembly using Kb-specific Abs that recognize conformational changes induced by assembly with beta2-microglobulin (beta2m) and by peptide binding. A significant portion (45%) of Kb molecules in TAP+, RMA-derived microsomes are associated with the TAP complex as measured by coimmunoisolation of Kb using anti-TAP1 Abs, while only 20% of the Kb heavy chain molecules are isolated as Kbbeta2m complexes with the alpha-Kb-specific Abs, Y-3 or K-10-56. The amount of Kb isolated with Y-3 and K-10-56 increases in proportion to transport and binding of peptide to the Kb molecules within the RMA microsomes. In contrast, less than 5% of the Kb within TAP2-RMA-S microsomes associated with the remaining TAP1 subunit. However, greater than 60% of Kb heavy chain is isolated as K-10-56- and Y-3-reactive Kbbeta2m complexes. We propose that a TAP-MHC class I interaction serves to stabilize the MHC class I:beta2m complex in an immature conformation (Y-3 and K-10-56 nonreactive) prior to high affinity peptide binding, preventing the export of class I molecules complexed with low affinity peptide ligands from the ER.     

Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH

Vacuolar alternate class I MHC (MHC-I) Ag processing allows presentation of exogenous Ag by MHC-I molecules with binding of antigenic peptides to post-Golgi MHC-I molecules. We investigated the role of previously bound peptides and their dissociation in generating peptide-receptive MHC-I molecules. TAP1-knockout macrophages were incubated overnight with an initial exogenous peptide, producing a large cohort of peptide-K(b) complexes that could influence subsequent peptide dissociation/exchange. Initial incubation with FAPGNYPAL, KVVRFDKL, or RGYVYQGL enhanced rather than reduced subsequent binding and presentation of a readout peptide (SIINFEKL or FAPGNYPAL) to T cells. Thus, K(b) molecules may be stabilized by an initial (stabilizing) peptide, enhancing their ability to bind readout peptide and implicating peptide dissociation/exchange. In contrast, incubation with SIINFEKL as stabilizing peptide reduced presentation of readout peptide. SIINFEKL-K(b) complexes were more stable than other peptide-K(b) complexes, which may limit their contribution to peptide exchange. Stabilizing peptides (FAPGNYPAL, KVVRFDKL, or RGYVYQGL) enhanced alternate MHC-I processing of HB101.Crl-OVA (Escherichia coli expressing an OVA fusion protein), indicating that alternate MHC-I Ag processing involves peptide dissociation/exchange. Stabilizing peptide enhanced processing of HB101.Crl-OVA more than presentation of exogenous OVA peptide (SIINFEKL), suggesting that peptide dissociation/exchange may be enhanced in the acidic phagosomal processing environment. Furthermore, exposure of cells to acidic pH increased subsequent binding and presentation of readout peptide. Thus, peptide dissociation/exchange contributes to alternate MHC-I Ag processing and may be influenced by both stability of peptide-MHC-I complexes and pH.     

HLA-E: strong association with beta2-microglobulin and surface expression in the absence of HLA class I signal sequence-derived peptides

The nonclassical class I HLA-E molecule folds in the presence of peptide ligands donated by the signal sequences of permissive class I HLA alleles, with the aid of TAP and tapasin. To identify HLA-E-specific Abs, four monoclonals of the previously described MEM series were screened by isoelectric focusing (IEF) blot and immunoprecipitation/IEF on >30 single-allele class I transfectants and HLA-homozygous B lymphoid cells coexpressing HLA-E and HLA-A, -B, -C, -F, or -G. Despite their HLA-E-restricted reactivity patterns (MEM-E/02 in IEF blot; MEM-E/07 and MEM-E/08 in immunoprecipitation), all of the MEM Abs unexpectedly reacted with beta(2)-microglobulin (beta(2)m)-free and denatured (but not beta(2)m-associated and folded) HLA-E H chains. Remarkably, other HLA-E-restricted Abs were also reactive with free H chains. Immunodepletion, in vitro assembly, flow cytometry, and three distinct surface-labeling methods, including a modified (conformation-independent) biotin-labeling assay, revealed the coexistence of HLA-E conformers with unusual and drastically antithetic features. MEM-reactive conformers were thermally unstable and poorly surface expressed, as expected, whereas beta(2)m-associated conformers were either unstable and weakly reactive with the prototypic conformational Ab W6/32, or exceptionally stable and strongly reactive with Abs to beta(2)m even in cells lacking permissive alleles (721.221), TAP (T2), or tapasin (721.220). Noncanonical, immature (endoglycosidase H-sensitive) HLA-E glycoforms were surface expressed in these cells, whereas mature glycoforms were exclusively expressed (and at much lower levels) in cells carrying permissive alleles. Thus, HLA-E is a good, and not a poor, beta(2)m assembler, and TAP/tapasin-assisted ligand donation is only one, and possibly not even the major, pathway leading to its stabilization and surface expression.     

Crystal structure of the murine cytomegalovirus MHC-I homolog m144

Large DNA viruses of the herpesvirus family produce proteins that mimic host MHC-I molecules as part of their immunoevasive strategy. The m144 glycoprotein, expressed by murine cytomegalovirus, is thought to be an MHC-I homolog whose expression prolongs viral survival in vivo by preventing natural killer cell activation. To explore the structural basis of this m144 function, we have determined the three-dimensional structure of an m144/beta2-microglobulin (beta2m) complex at 1.9A resolution. This structure reveals the canonical features of MHC-I molecules including readily identifiable alpha1, alpha2, and alpha3 domains. A unique disulfide bond links the alpha1 helix to the beta-sheet floor, explaining the known thermal stability of m144. Close juxtaposition of the alpha1 and alpha2 helices and the lack of critical residues that normally contribute to anchoring the peptide N and C termini eliminates peptide binding. A region of 13 amino acid residues, corresponding to the amino-terminal portion of the alpha2 helix, is missing in the electron density map, suggesting an area of structural flexibility that may be involved in ligand binding.     

Activation of CXCR4 triggers ubiquitination and down-regulation of major histocompatibility complex class I (MHC-I) on epithelioid carcinoma HeLa cells

Many cancer cells display down-regulated major histocompatibility complex (MHC) class I antigen (MHC-I), which seems to enable them to evade immune surveillance, whereas the underlying mechanisms remain incompletely understood. Here, we demonstrate that ligand (CXCL12) stimulation of CXCR4, a major chemokine receptor expressed in many malignant cancer cells, induced MHC-I heavy chain down-regulation from the cell surface of the human epithelioid carcinoma HeLa cells, the human U251 and U87 glioblastoma cells, the human MDA-MD 231 breast cancer cells, and the human SK-N-BE (2) neuroblastoma cells. Activation of CXCR4 also induced MHC-I down-regulation in human peripheral blood mononuclear cells. The internalized MHC-I heavy chain molecules were partially co-localized with Rab7, a later endosomal marker. Activation of CXCR4 induced ubiquitination of MHC-I heavy chain, and mutation of the C-terminal two lysine residues (Lys-332, Lys-337) on one of the MHC-I alleles, HLA.B7, blocked CXCR4-evoked ubiquitination and down-regulation of HLA.B7. Moreover, purified GST-conjugated CXCR4 C terminus directly associated with the purified His-tagged beta2-microglobulin (beta2M), and MHC-I heavy chain was co-immunoprecipitated with CXCR4 in a beta2M-dependent manner. This interaction appears to be critical for CXCR4-evoked down-regulation of MHC-I heavy chain as evidenced by the data that MHC-I heavy chain down-regulation was inhibited by either truncation of the CXCR4 C terminus or knockdown of beta2M. All together, these findings shed new light on the role of CXCR4 in tumor evasion of immune surveillance via inducing MHC-I down-regulation from the cell surface.     

Stability of surface H-2K(b), H-2D(b), and peptide-receptive H-2K(b) on splenocytes.

We have used flow cytometry to study the stability and peptide-binding capability of MHC class I (MHC-I) on the surface of normal C57BL/6 mouse T lymphoblasts. The MHC-I molecules on each cell are nearly evenly divided into two populations with mean half-life values of approximately 1 and 20 h. Our observations suggest that members of the later contain peptide bound with medium to high affinity. Cell surface MHC-I molecules capable of binding exogenous peptide (thus, "peptide-receptive") belong almost entirely to the less stable population. Before exogenous peptide can bind, MHC-I must undergo a change, probably loss of a very low affinity peptide. For MHC-I-K(b), we found that the maximum rate for binding of exogenous peptide corresponds to a t(1/2) value of 12 min. To maintain the 50:50 steady-state distribution of long- vs short-lived MHC-I molecules on the cell surface, approximately 20 short-lived molecules must be exported to the cell surface for each long-lived molecule.     

Expression of the human beta3 integrin subunit in mouse smooth muscle cells enhances IGF-I-stimulated signaling and proliferation

Optimal stimulation of signal transduction and biological functions by IGF-I in porcine smooth muscle cells (pSMC) requires ligand occupancy of the alphaVbeta3 integrin. Binding of heparin-binding domain (HBD) of vitronectin (VN) to the cysteine loop (C-loop) region of beta3 is required for pSMC to respond optimally to IGF-I stimulation. Mouse smooth muscle cells (mSMC), which express a form of beta3 whose sequence within the C-loop region is different than porcine or human beta3, do not respond optimally to IGF-I, and IGF-I stimulated beta3 and SHPS-1 phosphorylation which are necessary for optimal IGF-I signaling were undetectable. VN also had no effect on IGF-I stimulated the cell proliferation. In contrast, when human beta3 (hbeta3) was introduced into mSMC, there was an enhanced VN binding in spite of an equivalent amount of total beta3 expression, and IGF-I-dependent beta3, and SHPS-1 phosphorylation were detected. In addition, there was enhanced IGF-I-stimulated Shc association with SHPS-1, Shc tyrosine phosphorylation, Shc and Grb2 association, and MAP kinase activation leading to increased cell proliferation. These enhancements could be further augmented by adding a peptide containing the HBD of VN. To determine if these changes were mediated by the C-loop region of beta3, an antibody that reacts with that region of beta3 was utilized. The addition of the hbeta3 C-loop antibody abolished VN-induced enhancement of IGF-I signaling and IGF-I-stimulated cell proliferation. These results strongly support the conclusion that optimal SMC responsiveness to IGF-I requires ligand interaction with the C-loop domain of hbeta3.     

Assembly and dissociation of human leukocyte antigen (HLA)-A2 studied by real-time fluorescence resonance energy transfer

Class I major histocompatibility complex (MHC) heterodimer, composed of human leukocyte antigen (HLA)-A2 heavy chain and human beta(2)-microglobulin (beta(2)m), was produced by denaturation and gel filtration of the recombinant water-soluble HLA-A2/beta(2)m/peptide ternary complex in 8 M urea Tris-HCl buffer, followed by refolding of the separated chains without peptide. Peptide affinity and kinetics of the ternary complex formation and dissociation were investigated in real time by monitoring the fluorescence resonance energy transfer (FRET) from intrinsic HLA-A2 heavy-chain tryptophans to a dansyl fluorophore conjugated to the bound peptide. Peptide binding to the heterodimer was a second order process with rate constants linearly dependent upon temperature in Arrhenius coordinates over 0-20 degrees C. The binding rate constant of pRT6C-dansyl [ILKEPC(dansyl)HGV] at 37 degrees C evaluated by extrapolation of the Arrhenius plot was (2.0 +/- 0.5) x 10(6) M(-1) s(-1). Association of the heavy chain with beta(2)m was a first order process, apparently controlled by a conformational transition in the heavy chain. One of these conformations bound to beta(2)m to form the heavy chain/beta(2)m heterodimer whereas the second conformer oligomerized. Peptide dissociation from the ternary complex was a first-order reaction over the temperature range 20-37 degrees C, suggesting that the ternary complex also exists in two conformations. Taken together, the present data suggest that association of beta(2)m changes the HLA-A2 heavy-chain conformation thereby promoting peptide binding. Peptide dissociation from the ternary complex induces dissociation of the heavy-chain/beta(2)m heterodimer thereby causing oligomerization of the heavy chain. The lability of the HLA-A2/beta(2)m heterodimer and the strong tendency of the "free" heavy chain to oligomerize may provide an efficient mechanism for control of antigen presentation under physiological conditions by reducing the direct loading of HLA with exogenous peptide at the cell surface.     

Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules

MHC class I molecules present peptides derived primarily from endogenously synthesized proteins on the cell surface as ligands for CD8+ T cells. However, CD8+ T cell responses to extracellular bacteria, virus-infected, or tumor cells can also be elicited because certain professional APC can generate peptide/MHC class I (MHC-I) complexes from exogenous sources. Whether the peptide/MHC-I complexes are generated because the exogenous proteins enter the classical cytosolic, TAP-dependent MHC-I processing pathway or an alternate pathway is controversial. Here we analyze the generation of peptide/MHC-I complexes from recombinant Escherichia coli as an exogenous Ag source that could be delivered to the phagosomes or directly into the cytosol. We show that peritoneal and bone marrow macrophages generate peptide/MHC-I complexes by the classical as well as an alternate, but relatively less efficient, TAP-independent pathway. Using a novel method to detect proteolytic intermediates we show that the generation of the optimal MHC-I binding peptide in the alternate pathway requires cysteine as well as other protease(s). This alternate TAP-independent pathway also operates in vivo and provides a potential mechanism for eliciting CD8+ T cell responses to exogenous Ags.     

The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.

Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.     

Human major histocompatibility complex (MHC) class I molecules with disulfide traps secure disease-related antigenic peptides and exclude competitor peptides

The ongoing discovery of disease-associated epitopes detected by CD8 T cells greatly facilitates peptide-based vaccine approaches and the construction of multimeric soluble recombinant proteins (e.g. tetramers) for isolation and enumeration of antigen-specific CD8 T cells. Related to these outcomes of epitope discovery is the recent demonstration that MHC class I/peptide complexes can be expressed as single chain trimers (SCTs) with peptide, beta(2)m and heavy chain connected by linkers to form a single polypeptide chain. Studies using clinically relevant mouse models of human disease have shown that SCTs expressed by DNA vaccination are potent stimulators of cytotoxic T lymphocytes. Their vaccine efficacy has been attributed to the fact that SCTs contain a preprocessed and preloaded peptide that is stably displayed on the cell surface. Although SCTs of HLA class I/peptide complexes have been previously reported, they have not been characterized for biochemical stability or susceptibility to exogenous peptide binding. Here we demonstrate that human SCTs remain almost exclusively intact when expressed in cells and can incorporate a disulfide trap that dramatically excludes the binding of exogenous peptides. The mechanistic and practical applications of these findings for vaccine development and T cell isolation/enumeration are discussed.