Modifications of general parameters of immune activation in the sera of Sicilian patients with Boutonneuse fever

The serum levels of β₂-microglobulin (β₂-M), soluble HLA class I antigen (sHLA-I), soluble CD4 (sCD4) and CD8 (sCD8) were studied in 98 Sicilian patients with Boutonneuse fever (BF). In different stages of infection all markers were significantly increased in sera from Sicilian patients with acute BF compared with healthy controls. sCD8 and sHLA-I reached the peak in the second week after the onset of symptoms, whereas sCD4 and β₂-M reached the peak in the first week. Afterwards sCD8 decreased to the levels of controls within the third week, the other parameters decreased later and were unmodified until the third week of infection. Significant correlations were found between sCD4 and sCD8 and the sIL-2R, as well as between serum levels of β₂-M and sCD8. The reduction of CD3⁺ and CD4⁺ and the increase of CD8⁺ T cells in the blood indicate that these cells are involved in the response to rickettsia, and their activation might be in part responsible for the release of sCD4 and sCD8. Our data suggest that these soluble markers, indexes of immune activation of T cells both in the circulation and the affected tissues, may be used in monitoring BF evolution.     

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.     

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.     

Phenotypic and functional modulation of T cells in vivo by extrathymic T cells when T cells with MHC class II disparity were injected into athymic nude mice

TCR^high cells are generated by the mainstream of T cell differentiation in the thymus, whereas TCR^int cells (or NK1.1⁺ T cells) are generated extrathymically in the liver and by an alternative intrathymic pathway. It is still unknown how these T cell populations interact in vivo with each other. To investigate the interaction of TCR^int cells with TCR^high cells, we used congenitally athymic nude (B6-nu/nu) mice which carry only TCR^int cells in all immune organs. When TCR^high cells from B6-C-H-2^bm12 (bm12) mice (i.e. I-A^bm12) were injected into B6-nu/nu mice (i.e. 1-A^b), the expanding T cell population was a mixture of TCR^high cells of donor origin and TCR^int cells of recipient origin. However, 9 Gy-irradiated nude mice permitted a full expansion of TCR^high cells which expressed the IL-2Rα⁺β⁺ phenotype, namely, they were at the most activated state. These mice died of acute graft-versus-host disease (GVHD) within 5 days. On the other hand, non-irradiated nude mice suppressed the expansion of TCR^high cells of donor origin and such TCR^high cells continued to have the IL-2Rα^±β⁺ phenotype. These mice could survive but showed signs of chronic GVHD thereafter. In both situations, CD4⁺αβ T cells expanded irrespective of donor or recipient origin. These results suggest that TCR^int cells in the recipient mice possess a regulatory function in relation to donor TCR^high cells; as a result, fully activated TCR^high cells acquired the IL-2Rα⁺β⁺ phenotype and injured the host, but TCR^high cells suppressed in vivo remained as the IL-2Rα^±β⁺ phenotype and only partially injured the host.     

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.     

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.     

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.     

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.     

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.     

Rat interleukin-2-activated natural killer (A-NK) cell-mediated lysis is determined by the presence of CD18 on A-NK cells and the absence of major histocompatibility complex class I on target cells

The precise mechanism by which target cells are recognized and subsequently lysed by interleukin-2-activated natural killer (A-NK) cells is poorly understood. In this study the role of major histocompatibility complex (MHC) class I and adhesion molecules in the recognition and lysis of tumor cells was investigated in a syngeneic Wag rat model. Preincubation of tumor cells with F(ab′)₂ fragments of anti-MHC class I monoclonal antibody (mAb) OX18 strongly enhanced the A-NK cell-mediated lysis. Also normal syngeneic cells such as T cells and A-NK cells became highly sensitive for lysis by A-NK cells after preincubation with mAb OX18. Two other mAb against MHC class I had no effect on lysis of target cells. These data indicate that masking of MHC class I on syngeneic tumor and normal cells by mAb OX18 is sufficient for A-NK cells to recognize target cells as non-self, resulting in lysis. In addition, we found that the presence of mAb against the β2 (CD18)-integrins blocked the lysis of all tumor cell lines by A-NK cells in ⁵¹Cr-release assays, also when target cells were preincubated with mAb OX18. Because of the absence of CD18 on most tumor cells we concluded that a CD18-associated integrin on A-NK cells is essential for lysis of target cells. These results show that in this syngeneic rat model CD18 on A-NK cells together with MHC class I on tumor cells determine A-NK cell-mediated lysis. Furthermore, we hypothesize that the anti-MHC class I OX18 recognizes an epitope on rat MHC class I which is, or is very close to, the restriction element determining A-NK cell-mediated lysis.     

A human CD4 transgene rescues CD4−CD8+ cells in β2-microglobulin-deficient mice

The specificity of the αβ T cell receptor for class I or class II major histocompatibility complex (MHC) molecules determines whether a mature T cell will be of the CD4^?CD8⁺ or CD4⁺CD8^? phenotype, respectively. We show here that a human CD4 transgene can rescue a significant fraction of CD4^?CD8⁺ T cells in β2-microglobulin-deficient mice. Cells with this phenotype could be induced to become potent killers of targets expressing allogeneic MHC antigens, indicating that lineage commitment can precede the rescue of developing cells by the T cell receptor for antigen and the CD4 coreceptor.     

CD4-negative cytotoxic T cells with a T cell receptor α/βintermediate expression in CD8-deficient mice

Targeted disruption of the CD8 gene results in a profound block in cytotoxic T cell (CTL) development. Since CTL are major histocompatibility complex (MHC) class I restricted, we addressed the question of whether CD8–/– mice can reject MHC class I-disparate allografts. Studies have previously shown that skin allografts are rejected exclusively by T cells. We therefore used the skin allograft model to answer our question and grafted CD8–/– mice with skins from allogeneic mice deficient in MHC class II or in MHC class I (MHC-I or MHC-II-disparate, respectively). CD8–/– mice rejected MHC-I-disparate skin rapidly even if they were depleted of CD4⁺ cells in vivo (and were thus deficient in CD4⁺ and CD8⁺ T cells). By contrast, CD8+/+ controls depleted of CD4⁺ and CD8⁺ T cells in vivo accepted the MHC-I-disparate skin. Following MHC-I, but not MHC-II stimulation, allograft-specific cytotoxic activity was detected in CD8–/– mice even after CD4 depletion. A population expanded in both the lymph nodes and the thymus of grafted CD8–/– animals which displayed a CD4^?8^?3^intermediateTCRα/β^intermediate phenotype. Indeed its T cell receptor (TCR) density was lower than that of CD4⁺ cells in CD8–/– mice or of CD8⁺ cells in CD8+/+ mice. Our data suggest that this CD4^?8^?T cell population is responsible for the CTL function we have observed. Therefore, MHC class I-restricted CTL can be generated in CD8–/– mice following priming with MHC class I antigens in vivo. The data also suggest that CD8 is needed to up-regulate TCR density during thymic maturation. Thus, although CD8 plays a major role in the generation of CTL, it is not absolutely required.     

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c-kit⁺ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3^? IL-2Rβ⁺) and extrathymic T cells (CD3^int IL-2Rβ⁺) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8⁺ CD3^int IL-2Rβ⁺ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1⁺ and NK1.1^? subsets of CD3^int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1⁺ subset of CD3^int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1^? subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.     

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.     

Class I-deficient resistant mice intracerebrally inoculated with Theiler's virus show an increased T cell response to viral antigens and susceptibility to demyelination

Intracerebral inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelination in susceptible mouse strains. The histology of TMEV-induced demyelination is similar to that seen in patients suffering from multiple sclerosis. It was previously shown that the susceptibility of mice to TMEV-induced demyelination in certain strain combinations is closely associated with the major histocompatibility complex (MHC) class I locus. Here we examine disease susceptibility of β2-microglobulin (β2M)-deficient transgenic mice lacking class I expression and functional CD8⁺ T cells. In contrast to TMEV-infected parental C57BL/6 mice, the transgenics develop high levels of virus-specific DTH and T cell proliferation accompanied by an increased frequency of central nervous system (CNS) demyelinating lesions. However, clinical signs of demyelination were not noted. Neither antibody titer nor viral persistance were significantly affected in the β2M-deficient mice. These results suggest that in the absence of functional class I/CD8⁺ cells, the class II-restricted T cell response to TMEV is enhanced and CNS pathogenesis is heightened, although the level is not severe enough to result in clinical disease. When the TMEV-infected mice were subcutaneously immunized with virus, however, the β2M-deficient mice displayed clinical symptoms. Therefore, our results strongly suggest that CD8⁺ T cells do not directly contribute to CNS demyelination. In contrast, such T cells appear to be primarily involved in down-regulation of a potentially damaging CD4⁺ T cell response in resistant animals, although some of the T cells may play a role in clearing viral persistence in the CNS, resulting in the protection of the host from viral demyelination.     

Impaired NK‐cell education diminishes resistance to murine CMV infection

Ly49G2 (G2⁺) NK cells mediate murine (M)CMV resistance in MHC D^k‐expressing mice. Bone marrow transplantation (BMT) studies revealed that G2⁺ NK cell‐mediated MCMV resistance requires D^k in both hematopoietic and nonhematopoietic cells. As a Ly49G2 ligand, D^k in both cell lineages may contribute to lysis of virus‐infected cells. Alternatively, cellular differences in self‐MHC D^k may have affected NK‐cell education, and consequently NK cell‐mediated viral clearance. We investigated the D^k‐licensing effect on BM‐derived NK cells in BMT recipients by analyzing cytokines, cytotoxicity and MCMV resistance. In BMT recipients with lineage‐restricted D^k, G2⁺ NK‐cell reactivity and cytotoxicity was diminished in comparison to BMT recipients with self‐MHC in all cells. Reduced G2⁺ NK‐mediated MCMV resistance in BMT recipients with lineage‐restricted self‐MHC indicates that licensing of G2⁺ NK cells is related to NK‐cell reactivity and viral control. Titrating donor BM with self‐MHC‐bearing hematopoietic cells, as well as adoptive transfer of mature G2⁺ NK cells into BMT recipients with self‐MHC in non‐hematopoietic cells only, enhanced NK‐cell licensing and rescued MCMV resistance. This disparate self‐MHC NK‐cell education model would suggest that inadequately licensed NK cells corresponded to inefficient viral sensing and clearance.     

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.     

Clearance of Sendai virus by CD8+ T cells requires direct targeting to virus-infected epithelium

Minimal numbers of CD8⁺ T cells are found in bronchoalveolar lavage (BAL) populations recovered from Sendai virus-infected mice that are homozygous (?/?) for β2-microglobulin (β2-m) gene disruption. The prevalence of the CD8⁺ set was substantially increased in the pneumonic lungs of 8?12-week radiation chimeras made using substantially class I major histocompatibility complex (MHC) glycoprotein-negative β2-m (?/?) recipients and normal β2-m (+/+) bone marrow. Even so, the CD8⁺ (but not the CD4⁺) lymphocyte counts were still much lower than in the (+/+)→(+/+) controls. The (+/+)→(+/+) and (+/+)→(?/?) chimeras cleared Sendai virus and potent virus-immune CD8⁺ cytotoxic T lymphocytes (CTL) specific for H-2K^b + viral nucleoprotein peptide were found in the BAL from both groups. However, following in vivo depletion of the CD4⁺ population, only the (+/+)→(+/+) mice were able to deal with the infection. Similarly, adoptively transferred, H-2K^b-restricted CD8⁺ T cells from previously-primed (+/+) mice also failed to clear virus from the lungs of (+/+)→(?/?) chimeras infected within 2 weeks of reconstitution with bone marrow, though they were effective in the (+/+)→(+/+) controls. Sendai virus-immune CD8⁺ T cells are thus unable to eliminate virus-infected β2-m (?/?) lung epithelial cells that might be thought to be expressing very small amounts of either isolated class I heavy chain, or class I MHC glycoprotein that has bound β2-m derived from β2-m (+/+) T cells or macrophages present in the pneumonic lung. Furthermore, the CD8⁺ CTL that are being exposed to β2-m (+/+) stimulators in the BAL population cannot operate in some bystander mode to clear virus from respiratory epithelium.     

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.