Fine tuning of natural killer cell specificity and maintenance of self tolerance in MHC class I-deficient mice

TAP1 −/−, β2-microglobulin (β2m) −/− and TAP1/β2m −/− mice all express low but quantitatively different levels of MHC class I molecules. Using these mice, we have addressed questions relating to the fine tuning of natural killer (NK) cell specificity and maintenance of self tolerance in the NK cell system. NK cells from B6 wild-type mice killed target cells from TAP1 −/−, β2m −/− and TAP1/β2m −/− mice in vivo and rejected bone marrow grafts from the same mice in vivo at equivalent levels. NK cells from TAP1 −/−, β2m −/− mice did not kill target cells or reject bone marrow grafts from TAP1/β2m −/− mice. NK cells in all MHC class I-deficient mice were tolerant to autologous MHC class I-deficient cells, as revealed by in vitro cytotoxicity assays using NK cell effectors activated with the interferon-inducing agent Tilorone, or by in vivo bone marrow graft experiments. However, the self-tolerant state of MHC class I-deficient NK cells was broken by in vitro stimulation with IL-2 for 4 days. Under these conditions, NK cells from the MHC class I-deficient mice killed autologous MHC class I-deficient cells while MHC class I-positive targets were spared. The C-type lectin inhibitory receptor Ly49C has a specificity for H-2K^b and is expressed on a subset of NK1.1⁺ cells in B6 mice. Wild-type and all MHC class I-deficient mice had similar numbers of Ly49C-positive NK1.1⁺ cells. However, Ly49C expression was markedly down-regulated on NK1.1⁺ cells from B6 mice, as compared to TAP1 −/−, β2m −/− and TAP1/β2m −/− mice. In vitro stimulation of NK cells with IL-2 for 4 days did not significantly change this pattern. The present results are discussed in relation to the role of MHC class I molecules and Ly49 receptors in shaping the NK cell repertoire and raise new questions about maintenance of self tolerance in the NK cell system.     

Reduced expression of major histocompatibility complex class I free heavy chains and enhanced sensitivity to natural killer cells after incubation of human lymphoid lines with β2-microglobulin

Enhancement of major histocompatibility complex (MHC) class I expression leads to protection from recognition by natural killer (NK) cells in several systems. MHC class I gene products can be expressed in different forms at the cell surface - for example as “empty” β₂-microglobulin (β₂m)-associated heterodimers or free heavy chains. To study the role of different class I heavy chain forms in NK target interactions, we have used lymphoblastoid target cell lines preincubated with β₂m. This was found to shift the equilibrium between β₂m-associated and nonassociated - heavy chains in favor of the former. In parallel, there was a significant increase in NK sensitivity. The recognition of MHC class I-deficient cell lines was not affected by β₂m, arguing against a general nonspecific effect of fern on NK sensitivity. Our data indicate that protection against NK recognition correlates with target cell expression of free heavy chains (i.e. devoid of β₂m) rather than with expression of complexes.     

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.     

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.     

Functional cell surface expression by a recombinant single-chain class I major histocompatibility complex molecule with a cis-active β2-microglobulin domain

As a preliminary step towards the use of cell surface single-chain class I major histocompatibility complex (MHC) molecules as T cell immunogens, we have engineered a recombinant gene encoding a full-length cell surface single-chain version of the H-2D^d class I MHC molecule (SCβD^dm) which has β₂-microglobulin (β₂m) covalently linked to the amino terminus of a full-length H-2D^d heavy chain via a peptide spacer. The single-chain protein is correctly folded and stably expressed on the surface of transfected L cells. It can present an antigenic peptide to an H-2D^d-restricted antigen-specific T cell hybridoma. When expressed in peptide-transport-deficient cells, SCβD^dm can be stabilized and pulsed for antigen presentation by incubation with extracellular peptide at 27° or 37 °C, allowing the preparation of cells with single-chain molecules that are loaded with a single chosen antigenic peptide. SCβD^dm can be stably expressed in β₂m-negative cells, showing that the single-chain molecule uses its own β₂m domain to achieve correct folding and surface expression. Furthermore, the β₂m domain of SCβD^dm, unlike transfected free β₂m, does not rescue surface expression of endogenous class I MHC in the β₂m-negative cells. This strict cis activity of the β₂m domain of SCβD^dm makes possible the investigation of class I MHC function in cells, and potentially in animals, that express but a single type of class I MHC molecule.     

Control of thymus-independent intestinal intraepithelial lymphocytes by β2-microglobulin

Murine intestinal intraepithelial lymphocytes (i-IEL) comprise thymusdependent cells such as T cell receptor (TcR) α/β CD8α/β⁺ i-IEL, as well as thymus-independent ones such as TcRα/β CD8α/α⁺ and TcRγ/δ CD8α/α⁺ i-IEL. Whilst the development of the CD8α/β expressing i-IEL is strictly contingent on major histocompatibility complex (MHC) class I surface expression, that of CD8α/α i-IEL appears largely MHC class I independent. We have used β2-microglobulin (β2m)^?/? mutant mice lacking surface-expressed MHC class I and TcRα/β CD8α/β⁺ i-IEL to analyze the potential impact of MHC class I on regional activation of thymus-independent i-IEL. To analyze the role of TcRγ/δ i-IEL in regional cell interactions, these mice were treated with the anti-TcRγ/δ mAb, GL3. Whilst numbers of TcRα/β CD8α/α i-IEL were markedly reduced in βm^+/? mice, those of TcRγ/δ i-IEL were elevated. Administration of GL3 in vivo caused TcR down-modulation and functional inactivation of TcRγ/δ i-IEL in β2m^+/? mice. In contrast, TcR expression and functional activities of TcRγ/δ i-IEL from β2m^?/? mice were not impaired by GL3 treatment. The TcRα/β CD8β i-IEL from β2m^?/? mice were expanded and functionally activated as a consequence of TcRγ/δ engagement. The TcRγ/δ i-IEL and TcRα/β CD8α/α⁺ i-IEL from athymic nu/nu mice which express MHC class I, but lack TcRα/β CD8α/β⁺ i-IEL, responded to TcRγ/δ engagement as those from the β2m^+/? controls. In addition, the TcRγ/δ i-IEL from TcRβ^?/? and TcRβ^+/? mutants were equally affected by GL3. We conclude that the absence of β2m renders TcRγ/δ i-IEL resistant to TcR-mediated inactivation and promotes activation of TcRα/β CD8β^? i-IEL. The activation of TcRγ/δ i-IEL seems to be directly controlled by β2m/MHC class I expression and independent from TcRα/β CD8β⁺ i-IEL. Regulation of self-reactive thymus-independent i-IEL through β2m/MHC class I may contribute to control of autoreactive immune responses in the intestine.     

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.     

Analysis of graft-versus-host disease (GVHD) and graft rejection using MHC class I-deficient mice

GVHD is a major complication in allogeneic bone marrow transplantation (BMT). MHC class I mismatching increases GVHD, but in MHC-matched BMT minor histocompatibility antigens (mH) presented by MHC class I result in significant GVHD. To examine the modification of GVHD in the absence of cell surface MHC class I molecules, β₂-microglobulin-deficient mice (β₂m-/-) were used as allogeneic BMT recipients in MHC- and mH-mismatched transplants. β₂m-/- mice accepted MHC class I-expressing BM grafts and developed significant GVHD. MHC (H-2)-mismatched recipients developed acute lethal GVHD. In contrast, animals transplanted across mH barriers developed indolent chronic disease that was eventually fatal. Engrafted splenic T cells in all β₂m-/- recipients were predominantly CD3⁺ αβ TCR⁺ CD4⁺ cells (15–20% of all splenocytes). In contrast, CD8⁺ cells engrafted in very small numbers (1–5%) irrespective of the degree of MHC mismatching. T cells proliferated against recipient strain antigens and recognized recipient strain targets in cytolytic assays. Cytolysis was blocked by anti-MHC class II but not anti-CD8 or anti-MHC class I monoclonal antibodies (MoAbs). Cytolytic CD4⁺ T cells induced and maintained GVHD in mH-mismatched β₂m-/- mice, supporting endogenous mH presentation solely by MHC class II. Conversely, haematopoietic β₂m-/- cells were unable to engraft in normal MHC-matched recipients, presumably due to natural killer (NK)-mediated rejection of class I-negative cells. Donor-derived lymphokine-activated killer cells (LAK) were unable to overcome graft rejection (GR) and support engraftment.     

Mouse γδ TCR+NK1.1+ thymocytes specifically produce interleukin-4, are major histocompatibility complex class I independent,and are developmentally related to αβ TCR+NK1.1+ thymocytes

Mouse T cells co-expressing an αβ T cell receptor (TCR) and the NK1.1 antigen have been shown to be major interleukin (IL)-4-producing cells and could therefore regulate cell-mediated immune responses. We have identified a related subset of thymocytes co-expressing a γδ TCR and NK1.1 which also produce IL-4. Unlike αβ⁺NK1.1⁺ thymocytes, the selection of γδ⁺NK1.1⁺ thymocytes is not dependent upon β2-microglobulin (β2m)-associated class I molecule expression because these cells are present in β2m-deficient mice. This suggests that γδ⁺NK1.1⁺ T cells may regulate immune responses to a different variety of antigens. However, the development of αβ⁺NK1.1⁺ and αβ⁺NK1.1⁺ thymocytes appears to be related. Analysis of different mutant mice lacking αβ⁺NK1.1⁺ thymocytes revealed a specific increase in γδ⁺NK1.1⁺ thymocyte production when the block in αβ⁺NK1.1⁺ thymocyte differentiation occurs after β TCR rearrangement.     

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.     

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.     

Low number of H-2Dd-negative haematopoietic cells in mixed bone marrow chimeras convey in vivo tolerance to H-2Dd-negative cells but fail to prevent resistance to H-2Dd-negative leukaemia

Natural killer (NK) cells kill cells lacking self major histocompatibility complex (MHC) class I. This missing self reactivity is beneficial in haploidentical bone marrow transplantations to cure leukaemia, in which donor-derived NK cells reject MHC disparate leukaemia cells and prevent relapse. To understand the role of NK cells in transplantation, we have studied NK cell tolerance in mice receiving mixed bone marrow transplants with limiting number of the MHC disparate component. Using an MHC class I (Dd) transgenic mouse model, we generated bone marrow chimeras carrying mixtures of Dd-positive and -negative cells. NK reactivity against Dd-negative cells (missing self) was assayed by outgrowth of lymphoma cells, stability of the chimerism in vivo and killing of Concanavalin A blasts in vitro. Up to 20% Dd-negative haematopoietic cells reduced, but did not abrogate, rejection of Dd-negative tumours and killing of Dd-negative T-cell blasts. In contrast, the ratios between Dd-positive and -negative cells were stable in vivo, suggesting tolerance to normal cells. Our data suggest that NK cell tolerance to normal cells and tumours in mixed MHC environments is differentially regulated, tolerance to normal cells being more easily induced. These results are important in relation to the role of NK cells in antileukaemic reactions after bone marrow transplantation.     

The biology of NK cells and their receptors affects clinical outcomes after hematopoietic cell transplantation (HCT)

Natural killer (NK) cells were first identified for their capacity to reject bone marrow allografts in lethally irradiated mice without prior sensitization. Subsequently, human NK cells were detected and defined by their non-major histocompatibility complex (MHC)-restricted cytotoxicity toward transformed or virally infected target cells. Karre et al. later proposed ‘the missing self hypothesis’ to explain the mechanism by which self-tolerant cells could kill targets that had lost self MHC class I. Subsequently, the receptors that recognize MHC class I to mediate tolerance in the host were identified on NK cells. These class I-recognizing receptors contribute to the acquisition of function by a dynamic process known as NK cell education or licensing. In the past, NK cells were assumed to be short lived, but more recently NK cells have been shown to mediate immunologic memory to secondary exposures to cytomegalovirus infection. Because of their ability to lyse tumors with aberrant MHC class I expression and to produce cytokines and chemokines upon activation, NK cells may be primed by many stimuli, including viruses and inflammation, to contribute to a graft-versus-tumor effect. In addition, interactions with other immune cells support the therapeutic potential of NK cells to eradicate tumor and to enhance outcomes after hematopoietic cell transplantation.     

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.     

Regulation of CR3 (CD11b/CD18)-dependent natural killer (NK) cell cytotoxicity by tumour target cell MHC class I molecules

Phagocyte and NK cell CR3 functions as both an adhesion molecule and an iC3b receptor mediating cytotoxic responses to microorganisms. Cytotoxic activation of iC3b receptor function requires ligation of both a CD11b I-domain site for iC3b and a lectin site located in the C-terminus of CD11b. Because tumours lack the CR3-binding polysaccharides of bacteria and fungi, iC3b-opsonized tumours do not stimulate CR3-dependent cytotoxicity. Previous studies showed that NK cells could be induced to kill iC3b-opsonized tumours with small soluble β-glucans that bound with high affinity to CR3, bypassing the absence of similar polysaccharides on tumour membranes. Because CR3 signalling requires several tyrosine phosphorylation events, it appeared possible that CR3-dependent killing of autologous tumour cells might be suppressed by NK cell inhibitory receptors for MHC class I (KIR and CD94/NKG2) whose action involves recruitment of SHP-1 and SHP-2 tyrosine phosphatases. In the current study, Epstein–Barr virus (EBV)-transformed B cells were used as targets following opsonization with iC3b. Soluble β-glucan primed CR3 for killing of iC3b-coated B cells, but autologous class I-bearing targets were 84% more resistant than class I-deficient Daudi cells. Blockade of target cell class I with a MoAb specific for a domain recognized by both KIR and CD94/NKG2 resulted in comparable killing of class I⁺ B cells. By contrast, another MoAb to class II had no effect on cytotoxicity. These data suggest that NK cell recognition of class I suppresses CR3/tyrosine kinase-dependent cytotoxicity in the same way as it suppresses cytotoxicity mediated by other tyrosine kinase-linked receptors such as FcγRIIIA (CD16).     

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.     

Surface expression of β2-microglobulin-associated thymus-leukemia antigen is independent of TAP2

Mouse thymus-leukemia antigen (TL), like other major histocompatibility complex (MHC) class I-b antigens, displays signs of a specialized function. It is normally expressed at high levels on immature thymocytes and at moderate levels on gut epithelium and activated mature T cells. A promoter/enhancer region unique among class I genes accounts for this narrow range of tissue distribution. Like most other class I molecules, TL is dependent upon endogenous β2-microglobulin (β2m) for transport to the surface. However, here we show that unlike most other MHC class I molecules, TL is expressed efficiently in the absence of functional transporter associated with antigen processing subunit 2 (TAP2). A putative fourth TL^a gene cloned from A.SL1 cells was expressed in RMA and RMA-S cells. In bulk transformants, TL expression is higher in TAP2^? RMA-S cells than in wild-type RMA cells, and is not elevated by incubation at reduced temperatures or exposure to exogenous β2m. Analysis of immunoprecipitasted molecules by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicates that TL is processed normally in RMA-S cells and is associated with β2m both intracellularly and at the cell surface. However, TL heavy chains expressed on the cell surface in the absence of TAP2 are cleaved to a predominant 38 kDa fragment, presumably the result of an altered conformation that renders TL more susceptible to proteolysis. These results suggest that while TL may normally acquire TAP2-dependent peptides, this class I-b molecule does not require them for efficient export to, and stable expression at the cell surface.     

Viral infection transiently reverses activation receptor‐mediated NK cell hyporesponsiveness in an MHC class I‐independent mechanism

Continuous engagement of the Ly49H activating receptor with its ligand (m157) in a transgenic mouse expressing m157 (m157‐Tg) results in hyporesponsiveness of Ly49H⁺ NK cells. The same interaction, during murine cytomegalovirus (MCMV) infection, leads to activation of Ly49H⁺ NK cells. MCMV infection results in decreased MHC class I (MHC‐I) expression on the infected cell as well as inflammatory responses, both of which do not take place in the uninfected m157‐Tg mouse, potentially allowing for activation of NK cells in the context of MCMV infection. In this study, we demonstrated that viral infection transiently reverses activation receptor‐mediated NK cell hyporesponsiveness in an MHC‐I‐independent mechanism. Furthermore, Ly49H⁺ NK cells in an MHC‐I‐deficient environment remained hyporesponsive in the context of m157 expression, even when mature WT splenocytes were transferred into m157‐Tg mice in an MHC‐I‐deficient environment. However, the administration of cytokines TNF‐α, IL‐12, and IFN‐β resulted in a partial recovery from activation receptor‐induced hyporesponsiveness. Thus, the release of the aforementioned cytokines during MCMV infection and not the downregulation of MHC‐I expression appears to be responsible for partial resolution of Ly49H receptor‐induced NK cell hyporesponsiveness.     

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.     

Increased number of cytotoxic T cells within CD4+8− T cells in β2-microglobulin,major histocompatibility complex class I-deficient mice

Targeted disruption of β₂-microgobulin gene results in deficient major histocompatibility complex class I expression and failure to develop CD4^?8⁺ T cells. Despite this, β₂M^?/? mice reject skin grafts and cope with most viral infections tested. We asked whether CD4⁺8^? cytotoxic T cells could play a role in compensating for the defect in CD4^?8⁺ cytotoxic T cell function. We found that the cytotoxic activity against class II⁺ targets is significantly higher among CD4⁺8^? T cells of β₂M^?/? than among those of β₂M^+/+ mice. In the limiting dilution experiment, we showed that the precursor frequency for the cytotoxic, CD4⁺8^?, class II-specific T cells is at least fivefold higher in β₂M ^?/?than in β₂M^+/+ mice. These results suggest that CD4+8^? cytotoxic T cells could play a major role in carrying out cytotoxic function in β₂M^?/? mice.