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.     

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.     

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.     

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.     

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.     

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.     

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.     

Alloreactive natural killer cells in the rat: complex genetics of major histocompabitility complex control

A major role for the nonclassical major histocompatibility complex (MHC) class I region, i.e. RTI.C, in controlling rat natural killer (NK) cell alloreactivity has recently been established, and several findings suggested the existence of NK-triggering alloantigens coded for by this region. Here, we have extended our studies on the MHC control of NK cell cytotoxicity against concanavalin A-activated T cell blasts by comparing semi-syngeneic and fully allogeneic combinations, and we show the following: (a) The self MHC exerted a strong influence on the NK allorecognition repertoire. (b) When anti-F₁ hybrid cytolytic activities of parental strain NK cells were measured, both recessively and non-recessively inherited susceptibility patterns emerged. (c) In most combinations parental strain cells were lysed by F₁ hybrid NK cells, thus resembling the hybrid resistance phenomenon described in mice. The cytotoxicity was lower in strain combinations where NK susceptibility was inherited non-recessively, i.e. when parent anti-F₁ reactivity was detected, than in recessive combinations. (d) LEW.1LM1 (RT1^1m1) target cells, with a deletion in the RTI.C region that includes expressed class I genes, were more sensitive to lysis by MHC matched NK cells (PVG.IL(LEW), RT1¹) than were parental LEW (RT1¹) cells. The effect of the deletion was the opposite when MHC allogeneic (RT1^c, RT1^u) as well as semi-syngeneic (RT1^1/c) NK cells were employed, i.e. sensitivity was decreased. We conclude that certain MHC-encoded antigens, depending on the haplotype combination of effector and target cells, may either trigger or inhibit rat NK cell cytotoxicity. Furthermore, the potential role of peptides bound to MHC class I molecules recognized by NK cells is discussed.     

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.     

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.     

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.     

Role of Qa-1(b)-binding receptors in the specificity of developing NK cells

NK cells acquire the ability to recognize MHC class I molecules during development. Studies with Qa-1(b) tetramers (Qa-1 tetramers) showed that nearly all NK1.1(+) cells from newborn C57BL/6 mice express Qa-1-binding receptors. Cytotoxic activity of these cells is fully inhibited by Qa-1 ligands on target cells. In contrast, neither receptors for H-2K(b) nor H-2D(b) were observed on NK1.1(+) cells from newborn mice. After birth, frequencies of Qa-1 tetramer(+)/ NK1.1(+) cells gradually decrease as the number of Ly49(+) /NK1.1(+) cells increases. Cell transfer studies showed that Qa-1 tetramer(+) cells from newborn mice do not lose expression of Qa-1 receptors, but that they further acquire expression of Ly49 molecules. Acquisition of Qa-1-binding receptors appears largely independent of host MHC class I molecules, as observed in studies using beta2-microglobulin-deficient (beta2m(-/-)) mice as well as K(b)/ D(b-/-) and K(b)/D(b)/beta2m(-/-) mice. The present results suggest that Qa-1-binding receptors play an important role in the specificity of developing NK cells, and suggest that these cells rely mainly on inhibitory receptors specific for non-classical MHC class I molecules to maintain self tolerance during the first weeks of life.     

Major histocompatibility complex genes determine natural killer cell tolerance

Murine natural killer (NK) cell subsets, as defined by expression of members of the Ly49 gene family, discriminate target cells expressing different major histocompatibility complex (MHC) class I alleles. For example, Ly49A⁺ NK cells lyse H-2^b but not H-2^d tumor target cells. The specificity arises because D^d on target cells binds to Ly49A, transducing an inhibitory signal into the Ly49A⁺ NK cells. The capacity of NK cells to discriminate allelic class I determinants raises a key issue: are NK cells self-tolerant, and if so what are the mechanisms that lead to self-tolerance? As previously reported, potentially autoaggressive Ly49A⁺ NK cells are not clonally deleted in H-2^b mice. However, IL-2- cultured Ly49A⁺ effector cells from H-2^b mice exhibit reduced lysis of H-2^b (self) concanavalin A blast target cells, compared to Ly49A⁺ effector cells from H-2^d mice. Possible mechanisms accounting for this self-tolerance are addressed in this report. Self-tolerance was not due to anergy of the cells, because the Ly49A⁺ effector cells from both types of mice lysed β2-microglobulin-deficient target cells efficiently and equivalently. These results also suggest that tolerance results from inhibition mediated by β2m-dependent H-2^b class I molecules. Significantly, blockade of Ly49A on Ly49A⁺ effector cells from H-2^b mice did not restore lysis of H-2^b target cells, suggesting that inhibition is not mediated through the Ly49A receptor. Additional experiments suggest that inhibition is also not mediated primarily through the Ly49C receptor. These results suggest that Ly49A⁺ effector cells from H-2^b mice, unlike those from H-2^d mice, express inhibitory receptors specific for H-2^b molecules that are distinct from Ly49A and Ly49C.     

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.     

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.     

MURINE CUTANEOUS LEISHMANIASIS: RESISTANCE IN RECONSTITUTED NUDE MICE AND SEVERAL F1 HYBRIDS INFECTED WITH LEISHMANIA TROPICA MAJOR

After cutaneous injection of promastigotes of an isolate of the intramacrophage protozoan parasite, Leishmania tropica major, mouse strains develop chronic cutaneous lesions or show a resolving pattern of disease. On this basis, they can be classified as resistant (e.g. CBA/H and C57BL/6) or susceptible (e.g. BALB/c, BALB/c.H-2^b and BALB/c.H-2^k). Hypothymic nude (nu/nu) mice of either BALB/c, CBA/H or C57BL/6 genotype are susceptible to chronic disease. However, nude mice of these genotypes, including BALB/c, are resistant to chronic cutaneous leishmaniasis when injected at the time of parasite challenge with small numbers of H-2 compatible lymphoid cells from normal mice. Nude mice remain susceptible when injected with fully H-2 incompatible cells. Using cells from H-2 mutant mice for reconstitution of resistance in C57BL/6.nu/nu mice, evidence was obtained that I region compatibility is necessary for cells to mediate host-protective effects. Cells from chronically-diseased BALB/c mice do not have protective effects in BALB/c.nu/nu mice at any cell dose and will abrogate the resistance-promoting effect of lymphoid cell populations from chronically-diseased BALB/c.H-2^k and BALB/c.H-2^b mice can be demonstrated when assayed at certain cell doses in H-2 compatible CBA/H.nu/nu and C57BL/6.nu/nu mice, respectively. The data suggest that chronically-diseased (genetically-susceptible) mice contain a mixture of resistance-promoting and disease-promoting T cells in their peripheral lymphoid organs and that expression of the resistance-promoting subset can occur in nude mice of resistant genotype. Previous data have indicated that Lyl⁺2^? T cells are efficient mediators of both T cell-dependent activities. No evidence for the operation of disease-promoting or resistance-promoting antibodies in perpetuation or resolution of disease has been obtained in extensive serum transfer experiments. Some discrepancies exist in the literature on the question of the dominance of susceptibility or resistance in F₁ hybrid mice. A re-examination of susceptibility/resistance in F₁ hybrids between BALB/c and several other parental strains was undertaken using cloned pathogenic promastigotes derived from a heterogeneous L. t. major isolate in order to reduce effects of parasite heterogeneity in the analysis. Resistance was dominant in some but not all F₁ hybrids, with most showing a delayed healing pattern of disease relative to the resistant parental strain. Despite the use of genetically-homogeneous parasites, the analysis was complicated by variability within groups of F₁ hybrid mice as well as between males and females and between F₁ hybrids of reciprocal crosses. A hypothesis based on antigen and H-2 expression on infected macrophages is advanced to account for the balance between the effects of resistance-promoting and disease-promoting Lyl⁺2^? T cells in mice of various genotypes.     

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.     

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.     

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.