Processing of exogenous hepatitis B surface antigen particles for Ld-restricted epitope presentation depends on exogenous β2- microglobulin

Processing of exogenous hepatitis B surface antigen (HBsAg) particles in an endolysosomal compartment generates peptides that bind to the major histocompatibility complex (MHC) class I molecule L^d and are presented to CD8⁺ cytotoxic T lymphocytes. Surface-associated ‘empty’ MHC class I molecules associated neither with peptide, nor with β2-microglobulin (β2m) are involved in this alternative processing pathway of exogenous antigen for MHC class I-restricted peptide presentation. Here, we demonstrate that internalization of exogenous β2m is required for endolysosomal generation of presentation-competent, trimeric L^d molecules in cells pulsed with exogenous HBsAg. These data point to a role of endocytosed exogenous β2m in the endolysosomal assembly of MHC class I molecules that present peptides from endosomally processed, exogenous antigen.     

MHC Class I Phenotype and Function of Human β2-Microglobulin Transgenic Murine Lymphocytes

Lymphoid cells from β₂-microglobulin (β₂m) knockout mice transgenic for human (h) β₂m (C57BL/10 mβ₂m^?/hβ₂m⁺) were compared with normal mice for their binding to exogenously added hβ₂m, binding to a H-2D^b peptide and for functional activity in a one-way allogenic MLC. Based on data from cellular binding studies, Scatchard analyses and flow cytometry, it is concluded that exogenous hβ₂m does not bind to hybrid MHC class I (MHC-I) molecules composed of mouse heavy chain/hβ₂m molecules expressed on lymphocytes of transgenic mice. Immunoprecipitation and SDS-PAGE analysis of metabolically labelled normal C57BL/6 lymph node cells showed binding of exogenous hβ₂m to MHC-I, in particular, to the H-2D^b molecule through an exchange with endogenous mouse β₂m. In contrast to normal H-2D^b molecules, hybrid H-2D^b expressed on the surface of transgenic lymphocytes binds radiolabelled peptide in the absence of exogenous added hβ₂m suggesting that a stable fraction of hybrid H-2D^b molecules is empty or contain peptides with very low affinity. In a one-way allogenic mixed lymphocyte culture, transgenic splenocytes were found to be far less stimulatory than normal splenocytes. In contrast, transgenic alloreactive cytotoxic T lymphocytes developed earlier in MLC than their non-transgenic counterparts. These data indicate that the hybrid mouse heavy chain/hβ₂m complex alters the alloantigenic repertoire and influences important aspects of T-cell activation.     

Lack of F1 anti-parental resistance in H-2b/d F1 hybrids devoid of β2-microglobulin

F₁ hybrid mice often reject parental hematopoietic grafts, a phenomenon known as hybrid resistance. Hybrid resistance is mediated by natural killer (NK) cells and although the molecular interactions responsible for this phenomenon are largely unknown, one hypothesis suggests that parental cells are rejected because they fail to express a complete set of host major histocompatibility complex (MHC) class I molecules. Inherent in this theory is that NK cells in the F₁ hybrid are instructed by self MHC class I molecules to form an NK cell repertoire capable of reacting against cells lacking these self MHC class I molecules. Here, we show that C57BL/6 x DBA/2 mice (H-2^b/d) devoid of β₂-microglobulin (β₂m) are incapable of rejecting β₂m?/? parental C57BL/6 cells (H-2^b) both in vivo and in vitro. From this, we conclude that the development of an NK cell repertoire, at least in F₁ mice of the H-2^b/d haplotype, requires expression of MHC class I molecules complexed with β₂m.     

β2-microglobulin with an endoplasmic reticulum retention signal increases the surface expression of folded class I major histocompatibility complex molecules

With β₂-microglobulin^? (β₂m^?) cell lines such as R1E/D^b, the surface expression of class I major histocompatibility complex molecules is greatly impaired, and class I molecules that are on the surface are generally misfolded. To determine whether β₂m must be continually present with the class I heavy chain for the class I molecule to reach the surface in a folded conformation, a sequence encoding an endoplasmic reticulum (ER) retention signal (KDEL) was attached onto the 3′ end of a β₂m cDNA. After this chimeric cDNA was transfected into R1E/D^b cells, β₂m-KDEL protein was detectable by an anti-β₂m serum within the cells but not at the cell surface. Interestingly, R1E/D^b cells transfected with β₂m-KDEL were found to express a high level of conformationally correct D^b molecules at the cell surface. This observation implies that β₂m has a critical and temporal role in the de novo folding of the class I heavy chain. We propose that the critical time for β₂m association is when the class I molecule is docked with the transporter associated with antigen processing (TAP) and first interacts with peptide.     

Influence of glycosylphosphatidylinositol-linked H-2Dd molecules on target cell protection and natural killer cell specificity in transgenic mice

The expression of certain major histocompatibility complex (MHC) class I ligands on target cells is one important determinate of their susceptibility to lysis by natural killer (NK) cells. NK cells express receptor molecules that bind to MHC class I. Upon binding to their MHC class I ligand, the NK cell is presumed to receive a signal through its receptor that inhibits lysis. It is unclear what role the MHC class I molecules of the effector and target cells play in signaling to the NK cell. We have investigated the role of the cytoplasmic and transmembrane domains of MHC class I molecules by producing a glycosylphosphatidylinositol (GPI)-linked H-2D^d molecule. The GPI-linked H-2D^d molecule is recognized by H-2D^d-specific antibodies and cytotoxic T lymphocytes. Expression of the GPI-linked H-2D^d molecule on H-2^b tumor cells resulted in protection of the tumor cells after transplantation into D8 mice (H-2^b, H-2D^d) from rejection by NK cells. In addition, NK cells from mice expressing the GPI-linked H-2D^d molecule as a transgene were able to kill nontransgenic H-2^b lymphoblast target cells. The GPI-linked MHC class I molecule was able to alter NK cell specificity at the target and effector cell levels. Thus, the expression of the cytoplasmic and transmembrane domains of MHC class I molecules are not necessary for protection and alteration of NK cell specificity.     

Major histocompatibility complex class I presentation of exogenously acquired minor alloantigens initiates skin allograft rejection

Major histocompatibility complex (MHC) class I molecules present peptides from endogenous proteins. However, in some cases class I-restricted peptides can also derive from exogenous antigens. This MHC class I exogenous presentation could be involved in minor histocompatibility antigen (mHAg)-disparate allograft rejection when donor alloantigens are not expressed in graft antigen-presenting cells (APC) that initiate the rejection mechanism. Here we addressed this question by using a skin graft experimental model where donors (H-2^b or H-2^d Tgβ-gal mice) expressed the mHAg like β-galactosidase (β-gal) in keratinocytes but not in Langerhans' cells (LC) which have an APC function. Rejection of Tgβ-gal skin by a β-gal-specific CD8 cytotoxic T lymphocyte (CTL) effector mechanism should require presentation by donor and/or recipient LC of MHC class I-restricted peptides of exogenous β-gal shed by keratinocytes. Indeed, our results showed that 1) H-2^b Tgβ-gal skin was rejected by H-2^bxs and H-2^bxd recipients; 2) rejection was mediated by β-gal-specific CD8⁺ CTL effectors; and 3) H-2^bxd mice having rejected H-2^b Tgβ-gal skin generated β-gal-specific CTL restricted by H-2^b and H-2^d class I molecules and rejected subsequently grafted H-2^d Tgβ-gal skin in an accelerated fashion, demonstrating that recipient LC have presented exogenous β-gal-derived MHC class I epitopes. These results lead to the conclusion that MHC class I exogenous presentation of donor mHAg can initiate allograft rejection.     

The interaction of beta 2-microglobulin (β2m) with mouse class I major histocompatibility antigens and its ability to support peptide binding. A comparison of human and mouse β2m

The function of major histocompatibility complex (MHC) class I molecules is to sample peptides derived from intracellular proteins and to present these peptides to CD8⁺ cytotoxic T lymphocytes. In this paper, biochemical assays addressing MHC class I binding of both peptide and β₂-microglobulin (β₂m) have been used to examine the assembly of the trimolecular MHC class I/β₂m/peptide complex. Recombinant human β₂m and mouse β₂m² have been generated to compare the binding of the two β₂m to mouse class I. It is frequently assumed that human β₂m binds to mouse class I heavy chain with a much higher affinity than mouse β₂m itself. We find that human β₂m only binds to mouse class I heavy chain with slightly (about 3-fold) higher affinity than mouse β₂m. In addition, we compared the effect of the two β₂m upon peptide binding to mouse class I. The ability of human β₂m to support peptide binding correlated well with its ability to saturate mouse class I heavy chains. Surprisingly, mouse β₂m only facilitated peptide binding when mouse β₂m was used in excess (about 20-fold) of what was needed to saturate the class I heavy chains. The inefficiency of mouse β₂m to support peptide binding could not be attributed to a reduced affinity of mouse β₂m/MHC class I complexes for peptides or to a reduction in the fraction of mouse β₂m/MHC class I molecules participating in peptide binding. We have previously shown that only a minor fraction of class I molecules are involved in peptide binding, whereas most of class I molecules are involved in β₂m binding. We propose that mouse β₂m interacts with the minor peptide binding (i.e. the “empty”) fraction with a lower affinity than human β₂m does, whereas mouse and human β₂m interact with the major peptide-occupied fraction with almost similar affinities. This would explain why mouse β₂m is less efficient than human β₂m in generating the peptide binding moiety, and identifies the empty MHC class I heavy chain as the molecule that binds human β₂m preferentially.     

Conformational changes induced in the MHC class I molecule by peptide and β2-microglobulin

Assembly of the class I MHC molecule is inextricably linked to the antigen presentation function of the class I molecule. Association of the class I MHC molecule with β₂-microglobulin (β₂m) is a prerequisite for association with the heterodimeric protein TAP, and once peptide is acquired, the class I molecule folds and begins its sojourn to the cell surface. To maintain its folded conformation, class I MHC requires peptide but not β₂m, and the sequence of the peptide bound exercises a subtle influence on the structure of the class I molecule that is likely to be a factor in T cell receptor discrimination of MHC/peptide complexes.     

Inhibition of natural killer cell-mediated bone marrow graft rejection by allogeneic major histocompatibility complex class I,but not class II molecules

The role of major histocompatibility complex (MHC) class I and class II molecules in natural killer (NK) cell-mediated rejection of allogeneic, semi-syngeneic and MHC-matched bone marrow grafts was investigated. The use of β₂-microglobulin (β₂m) -/- and β₂m +/- mice as bone marrow donors to MHC-mismatched recipients allowed an analysis of whether the presence of semi-syngeneic and allogeneic MHC class I gene products would be triggering, protective or neutral, in relation to NK cell-mediated rejection. Loss of β₂m did not allow H-2^b bone marrow cells to escape from NK cell-mediated rejection in allogeneic (BALB/c) or semi-allogeneic (H-2D^d transgenic C57BL/6) mice. On the contrary, it led to stronger rejection, as reflected by the inability of a larger bone marrow cell inoculum to overcome rejection by the H-2-mismatched recipients. In H-2-matched recipients, loss of β₂m in the graft led to a switch from engraftment to rejection. At the recipient level, loss of β₂m led to loss of the capability to reject H-2-matched β₂m-deficient as well as allogeneic grafts. When MHC class II-deficient mice were used as donors, the response was the same as that against donors of normal MHC phenotype: allogeneic and semi-syngeneic grafts were rejected by NK cells, while syngeneic grafts were accepted. These data suggest a model in which allogeneic class I molecules on the target cell offer partial protection, while certain syngeneic class I molecules give full protection from NK cell-mediated rejection of bone marrow cells. There was no evidence for a role of MHC class II molecules in this system.     

Impaired MHC class I (H-2Dd)-mediated protection against Ly-49A+ NK cells after amino acid substitutions in the antigen binding cleft

The MHC class I molecule H-2D^d (D^d) acts as a ligand for the inhibitory NK cell receptor Ly-49A. We have constructed altered D^d molecules by site-directed mutagenesis, replacing residues with the corresponding amino acids from the D^b molecule, which fails to inhibit via Ly-49A. Mutations at positions 73 and 156 (D^dS73WD156Y) impaired the protective effect of the D^d molecule, as evaluated by testing lymphoma cells transfected with the mutant gene for sensitivity to killing by Ly-49A⁺ NK cells in vitro and rejection by NK cells in vivo. The altered residues form a hydrophobic ridge across the floor of the antigen binding cleft. A mutation in the α helix of the α2 domain, facing the solvent and without direct contact with the peptide (D^dA150S) had no effect. D^d recognition by Ly-49A⁺ NK cells is considered to be peptide dependent, but not peptide specific. Our results indicate that alterations of residues buried in the antigen binding cleft can induce changes in peptide binding patterns and/or conformational changes in the D^d molecule that make the trimolecular complex less permissive for inhibition of Ly-49A⁺ NK cells.     

Induction and functional characterization of β 2-microglobulin (β2-μ)-free HLA class I heavy chains expressed by β2-μ-deficient human FO-1 melanoma cells

The frequent loss of β2-microglobulin (β₂-μ) in malignant cells has stimulated interest in the functional characteristics of β₂-μ-free HLA class I heavy chains, since this information contributes to assess the impact of β₂-μ abnormalities on the interaction of malignant cells with immune cells. Therefore, the present study has investigated the ability of β₂-μ-free HLA class I heavy chains to modulate NK cell-mediated lysis of melanoma cells and to present melanoma-associated antigen (MAA)-derived peptides to HLA class I-restricted, MAA-specific cytotoxic T lymphocytes (CTL). β₂-μ-free HLA class I heavy chains were induced on B₂m null FO-1 cells by sequential incubation with IFN-α for 48 h at 37 °C and for 24 h at 26 °C. Transfection of cells with a wild-type H-2L^d gene (FO-1L^d) enhanced the induction of β₂-μ-free HLA class I heavy chains under such experimental conditions. β₂-μ-free HLA class I heavy chains expressed on the cell membrane did not protect the B₂m null FO-1 cells from NK cell-mediated lysis. Furthermore, FO-1 cells which express β₂-μ-free HLA-A2 heavy chains following transfection with a wild-type HLA-A2 gene were not lysed by HLA-A2-restricted, MAA-specific CTL lines and clones. These results indicate that association with β₂-μ is required for interaction of HLA class I molecules with NK inhibitory receptors and for peptide presentation to CTL.     

Calnexin expression does not enhance the generation of MHC class I-peptide complexes

We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, K ^d , K^b , K^k , D ^d , D^b , and L^d . Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For K^b and K ^d , the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse β2-microglobulin. Also addressed was the effect of calnexin on the surface expression of K^b molecules bearing the immunodominant peptide from ovalbumin (OVA_{257 – 264} ). This was detected with a recently described monoclonal antibody specific for the K^b/peptide complex. Calnexin expression had no significant effect on the formation of K^b /peptide complexes generated from full-length OVA, cytosolic OVA_{257 – 264} , or endoplasmic reticulum-targeted OVA_{257 – 264} , which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of K ^d /peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.     

The ability of H-2Dd molecule to affect natural resistance to hemopoietic allografts is an intrinsic property shared by Ddm1 but not Ld

F₁ hybrid resistance (HR) to parental bone marrow grafts is mediated by natural killer (NK) cells, and thought to be controlled by the non-class I hemopoietic histocompatibility (Hh) genes linked to the major histocompatibility complex (MHC). However, as in the in vitro NK cytotoxicity against hemopoietic targets, expression of certain class I MHC molecules does affect HR, although mechanisms underlying such an effect are not understood. In this study, we examine the relevance of the “self/non-self” property of class I molecules and the molecular domains responsible for this function. H-2^b/Hh-1^b lymphoma cells were transfected with class 1 H-2D^d or L^d gene, and its effect on the Hh-1 phenotype was examined by testing the transfectant's ability to competitively inhibit the in vivo rejection of parental H-2^b/Hh-1^b bone marrow grafts by irradiated F₁ hybrid hosts. Multiple independent clones of transfectants show that the genomic or cDNA of the D^d gene, but not of L^d, renders the Hh-1^b-positive cells incapable of inhibiting HR in F₁ mice, although both genes belong to the same region of the same haplotype. The same effect could be observed not only in H-2^b/d F₁ mice for which D^d and L^d are self, but also in H-2^b/k F₁ mice for which both D^d and L^d are non-self. Thus, this function of the D^d molecule is an intrinsic property, not necessarily related to its self/non-self characteristic relative to the effector cells. Furthermore, given the nature of the assay used in this study, the results favor a “target interference” model as the underlying mechanism of the D^d effect. To locate the relevant domain(s) of the D^d molecule, mutant D^dm1 gene was tested and found to have the same effect as the non-mutant D^d. D^dm1 is a hybrid molecule between D^d and L^d, sharing with D^d only the α1 domain and a portion of the α2 domain. The two N-terminal domains of D^dm1 differ from those of D^d by three amino acid substitutions, two of which affect the molecules' peptide-binding properties.     

β2 -Microglobulin-deficient NK cells show increased sensitivity to MHC class I-mediated inhibition,but self tolerance does not depend upon target cell expression of H-2Kb and Db heavy chains

Mice lacking β2 -microglobulin (β₂ m− mice) express greatly reduced levels of MHC class I molecules, and cells from β₂ m− mice are therefore highly sensitive NK cells. However, NK cells from β₂ m− mice fail to kill β₂ m− normal cells, showing that they are self tolerant. In a first attempt to understand better the basis of this tolerance, we have analyzed more extensively the target cell specificity of β₂ m− NK cells. In a comparison between several MHC class I-deficient and positive target cell pairs for sensitivity to β₂ m− NK cells, we made the following observations: First, β₂ m− NK cells displayed a close to normal ability to kill a panel of MHC class I-deficient tumor cells, despite their nonresponsiveness to β₂ m− concanavalin A (Con A)-activated T cell blasts. Secondly, β₂ m− NK cells were highly sensitive to MHC class I-mediated inhibition, in fact more so than β₂ m+ NK cells. Third β₂ m− NK cells were not only tolerant to β₂ m− Con A blasts but also to Con A blasts from H-2Kb − /Db − double deficient mice in vitro. We conclude that NK cell tolerance against MHC class I-deficient targets is restricted to nontransformed cells and independent of target cell expression of MHC class I free heavy chains. The enhanced ability of β₂ m− NK cells to distinguish between MHC class I-negative and -positive target cells may be explained by increased expression of Ly49 receptors, as described previously. However, the mechanisms for enhanced inhibition by MHC class I molecules appear to be unrelated to self tolerance in β₂ m− mice, which may instead operate through mechanisms involving triggering pathways.     

Differential reactivity of residual CD8+ T lymphocytes in TAP1 and β2-microglobulin mutant mice

TAP1 -/- and β2-microglobulin (β2m) -/- mice (H-2^b background) express very low levels of major histocompatibility complex (MHC) class I molecules on the cell surface. Consequently these mice have low numbers of mature CD8⁺ T lymphocytes. However, TAP1 -/- mice have significantly higher numbers of CD8⁺ T cells than β2m -/- mice. Alloreactive CD8⁺ cytotoxic T lymphocyte (CTL) responses were also stronger in TAP1 -/- mice than in β2m -/- mice. Alloreactive CTL generated in TAP1 -/- and β2m -/- mice cross-react with H-2^b-expressing cells. Surprisingly, such cross-reactivity was stronger with alloreactive CTL from β2m -/- mice than with similar cells from TAP1 -/- mice. The β2m -/- mice also responded more strongly when primed with and tested against cells expressing normal levels of H-2^b MHC class I molecules. Such H-2^b-reactive CD8⁺ CTL from β2m -/- mice but not from TAP1 -/- mice also reacted with TAP1 -/- and TAP2-deficient RMA-S cells. In contrast, H-2^b-reactive CD8⁺ CTL from neither β2m -/- mice nor TAP1 -/- mice killed β2m -/- cells. In line with these results, β2m -/- mice also responded when primed and tested against TAP1 -/- cells. We conclude that the reactivity of residual CD8⁺ T cells differs between TAP1 -/- and β2m -/- mice. The MHC class I-deficient phenotype of TAP1 -/- and β2m -/- mice is not equivalent: class I expression differs between the two mouse lines with regard to quality as well as quantity. We propose that the differences observed in numbers of CD8⁺ T cells, their ability to react with alloantigens and their cross-reactivity with normal H-2^b class I are caused by differences in the expression of MHC class I ligands on selecting cells in the thymus.     

α1 / α2 domains of H-2Dd,but not H-2Ld,induce “missing self” reactivity in vivo – No effect of H-2Ld on protection against NK cells expressing the inhibitory receptor Ly49G2

Introduction of the MHC class I transgene H-2D^d on C57BL / 6 (B6) background conveys NK cell-mediated “missing self” reactivity against transgene-negative cells, and down-regulates expression of the inhibitory receptors Ly49A and Ly49G2 in NK cells. We here present an analysis of transgenic mice expressing chimeric H-2D^d / L^d MHC class I transgenes, and show that the α1 / α2 domains of H-2D^d were necessary and sufficient to induce “missing self” recognition and to down-modulate Ly49A and Ly49G2 receptors. In contrast, transgenes containing the α1 / α2 domains of H-2L^d induced none of these changes, suggesting that not all MHC class I alleles in a host necessarily take part in NK cell education. The lack of effect of the α1 / α2 domains of H-2L^d on NK cell specificity was surprising, considering that both H-2L^d and H-2D^d have been reported to interact with Ly49G2. Therefore, the role of H-2L^d for protection against NK cells expressing Ly49G2 was re-investigated in a transfection system. In contradiction to earlier reports, we show that H-2D^d, but not H-2L^d, abolished killing by sorted Ly49G2⁺ NK cells, indicating that H-2L^d does not inhibit NK cells via the Ly49G2 receptor.     

Qa-1 interaction and T cell recognition of the Qa-1 determinant modifier peptide

The peptide-binding properties of the nonclassical major histocompatibility complex (MHC) class 1b molecule Qa-1 were investigated using a transfected hybrid molecule composed of the α1 and α2 domains of Qa-1^b and the α3 domain of H-2D^b. This allowed the use of a monoclonal antibody directed against H-2D^b whilst retaining the peptide-binding groove of Qa-1^b. By comparison with classical MHC class I molecules, intracellular maturation of the chimeric molecule was inefficient with weak intracellular association with β2-microglobulin. However, at the cell surface the hybrid molecules were stably associated with β2-microglobulin and were recognized by cytotoxic T lymphocyte (CTL) clones specific for the Qa-1^b -presented peptide Qdm (AMAPRTLLL). A whole-cell binding assay was used to determine which residues of Qdm were important for binding to Qa-1^b and CTL clones served to identify residues important for T cell recognition. Substitutions at position 1 and 5 did not reduce the efficiency of binding and had little effect on CTL recognition. In contrast, substitutions at position 9 resulted in loss of MHC class I binding. Mass spectrometric analysis of peptides eluted from immunopurified Qa-1^b/D^b molecules indicated that Qdm was the dominant peptide. The closely related peptide, AMVPRTLLL, which is derived from the signal sequence of H-2D^k, was also present, although it was considerably less abundant. The mass profile suggested the presence of additional peptides the majority of which consisted of eight to ten amino acid residues. Finally, the finding that a peptide derived from Klebsiella pneumoniae can bind raises the possibility that this non-classical MHC class I molecule may play a role in the presentation of peptides of microorganisms.     

Antibodies Directed Against Monomorphic and Evolutionary Conserved Self Epitopes may be Generated in 'Knock-Out' Mice. Development of Monoclonal Antibodies Directed Against Monomorphic MHC Class I Determinants

Beta-2 microglobulin (β₂m) gene ‘knock-out’ mice (C1D) were primed wilh purified H-2K^b and H-2D^b molecules and spleen cells from immunized mice were used to generate monoclonal antibody secreting B-cell hybridomas. Approximately 0.2% of the Ig-secreting primary microcultures contained H-2^b binding antibodies. Three stable anti-MHC class I (MHC-I) antibody secreting hybridoma clones were established and subcloned. All three MoAbs precipitated radiolabelled H-2 molecules as analysed by SDS PAGE, and all three MoAbs stained H-2^b, H-2^d, as well as H-2^k cells by FACS analysis. The MoAbs stained to two β₂m loss mutant cell lines, C4.4-25- and R1E, suggesting that some MHC-I heavy chain is exported to the cell surface even in the absence of endogenous β₂m. Staining of murine cell lines kept under serum-free culture conditions was strongly influenced by the addition of bovine or human serum as a source of exogenous β₂m suggesting that xenogeneic β₂m affects the conformation of class I molecules. Furthermore, all three MoAbs strongly stained the peptide transporter deficient cell line, RMA-S, when cultured at 26°C, however, staining was reduced five-fold when RMA-S cells were cultured at 37°C. In total, these observations suggest that the MoAbs recognize conformational, presumably β₂m and peptide dependent, self epitopes on MHC-class I. One of the three MoAbs stained rat blood mononuclear blood cells (BMC), all three MoAbs stained hamster BMC, whereas two of the MoAbs stained human cells. These data suggest that the MoAbs recognize determinants which are conserved between species. All three antibodies strongly inhibited the development of CTLs generated in an allogeneic one-way MLC, provided that the MoAbs were present during the first 24 h of culture. It is concluded that MoAbs reacting with monomorphic self epilopes may be generated using animals deleted of the gene of interest. The implications may be far reaching since such MoAbs potentially identify evolutionary conserved and physiologically important epitopes.     

Modified Human Beta 2‐Microglobulin (desLys58) Displays Decreased Affinity for the Heavy Chain of MHC Class I and Induces Nitric Oxide Production and Apoptosis

Beta2‐microglobulin (β2m) is the light chain of major histocompatibility complex class I (MHC‐I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8⁺ T cells. β2m can be modified in vivo and in vitro by proteolytic cleavage by complement C1 and subsequent carboxypeptidase B‐like activity – processes that lead to the generation of desLys⁵⁸β2m (dβ2m). This work aims to study the effect of dβ2m on peptide binding to MHC‐I, the influence of dβ2m on the binding of β2m to the MHC‐I heavy chain and the biological activity of dβ2m. Both β2m and dβ2m are able to support the generation of MHC‐I/peptide complexes at 18 °C, but complexes formed in the presence of dβ2m destabilize at 37 °C. Moreover, a 250 times higher concentration of dβ2m than of β2m is needed to displace MHC‐I associated β2m from the cell surface. In addition, only β2m is able to restore MHC‐I/peptide complex formation on acid‐treated cells whereas dβ2m appears to bind preferentially to denatured MHC‐I heavy chains. In cell cultures, exogenously added dβ2m, but not β2m, induces apoptotic cell death in monocytic leukaemic cell lines but spares other kinds of leukaemic cells. Additionally, the presence of dβ2m, and to a lesser extent β2m, enhances IFN‐γ‐induced NO production by monocytic leukaemic cells. In conclusion, these data show that dβ2m is not able to support the formation of a stable tri‐molecular MHC‐I complex at physiological temperature and that dβ2m exerts other biological functions compared to β2m when bound to cells.