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Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.

Intracranial (i.c.) infection of susceptible C57BL/6 mice with the neurotropic JHM strain of mouse hepatitis virus (JHMV) (a member of the Coronaviridae family) results in acute encephalomyelitis and viral persistence associated with an immune-mediated demyelinating disease. The present study was undertaken to better understand the molecular pathways evoked during innate and adaptive immune responses as well as the chronic demyelinating stage of disease in response to JHMV infection of the central nervous system (CNS). Using single-cell RNA sequencing analysis (scRNAseq) on flow-sorted CD45-positive (CD45+) cells enriched from brains and spinal cords of experimental mice, we demonstrate the heterogeneity of the immune response as determined by the presence of unique molecular signatures and pathways involved in effective antiviral host defense. Furthermore, we identify potential genes involved in contributing to demyelination as well as remyelination being expressed by both microglia and macrophages. Collectively, these findings emphasize the diversity of the immune responses and molecular networks at defined stages following viral infection of the CNS.IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the molecular signatures of immune cells within the CNS at defined times following infection with a neuroadapted murine coronavirus using scRNAseq. This approach has revealed that the immunological landscape is diverse, with numerous immune cell subsets expressing distinct mRNA expression profiles that are, in part, dictated by the stage of infection. In addition, these findings reveal new insight into cellular pathways contributing to control of viral replication as well as to neurologic disease.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

Experimental autoimmune encephalomyelitis is a model for multiple sclerosis. Here we show that induction generates successive waves of clonally expanded CD4+, CD8+ and γδ+ T cells in the blood and central nervous system, similar to gluten-challenge studies of patients with coeliac disease. We also find major expansions of CD8+ T cells in patients with multiple sclerosis. In autoimmune encephalomyelitis, we find that most expanded CD4+ T cells are specific for the inducing myelin peptide MOG35-55. By contrast, surrogate peptides derived from a yeast peptide major histocompatibility complex library of some of the clonally expanded CD8+ T cells inhibit disease by suppressing the proliferation of MOG-specific CD4+ T cells. These results suggest that the induction of autoreactive CD4+ T cells triggers an opposing mobilization of regulatory CD8+ T cells.

T cells are found in atherosclerotic plaques, with evidence supporting a potential role for CD8+ T cells in atherogenesis. Prior studies provide evidence of low-density lipoprotein and apoB-100 reactive T cells, yet specific epitopes relevant to the disease remain to be defined. The current study was undertaken to identify and characterize endogenous, antigen-specific CD8+ T cells in atherosclerosis. A peptide fragment of apoB-100 that tested positive for binding to the mouse MHC-I allele H2Kb was used to generate a fluorescent-labeled H2Kb pentamer and tested in apoE-/- mice. H2Kb pentamer(+)CD8+ T cells were higher in apoE-/- mice fed an atherogenic diet compared with those fed a normal chow. H2Kb pentamer (+)CD8+ T cells in atherogenic diet-fed mice had significantly increased effector memory phenotype with a shift in Vβ profile. H2Kb pentamer blocked lytic activity of CD8+ T cells from atherogenic diet-fed mice. Immunization of age-matched apoE-/- mice with the apoB-100 peptide altered the immune-dominant epitope of CD8+ T cells and reduced atherosclerosis. Our study provides evidence of a self-reactive, antigen-specific CD8+ T-cell population in apoE-/- mice. Immune modulation using the peptide antigen reduced atherosclerosis in apoE-/- mice.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons.

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons.

Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8(+) T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell-mediated killing via the "missing self" axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the "self" signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell- and MHC I-dependent manner. NK cell-mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self-dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8(+) T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell-mediated killing via the "missing self" axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the "self" signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell- and MHC I-dependent manner. NK cell-mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self-dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease.

Some mature natural killer (NK) cells cannot be inhibited by major histocompatibility complex (MHC) I molecules, either because they lack corresponding inhibitory receptors or because the host lacks the corresponding MHC I ligands for the receptors. Such NK cells nevertheless remain self-tolerant and exhibit a generalized hyporesponsiveness to stimulation through activating receptors. To address whether NK cell responsiveness is set only during the NK cell differentiation process, we transferred mature NK cells from wild-type (WT) to MHC I-deficient hosts or vice versa. Remarkably, mature responsive NK cells from WT mice became hyporesponsive after transfer to MHC I-deficient mice, whereas mature hyporesponsive NK cells from MHC I-deficient mice became responsive after transfer to WT mice. Altered responsiveness was evident among mature NK cells that had not divided in the recipient animals, indicating that the cells were mature before transfer and that alterations in activity did not require cell division. Furthermore, the percentages of NK cells expressing KLRG1, CD11b, CD27, and Ly49 receptors specific for H-2(b) were not markedly altered after transfer. Thus, the functional activity of mature NK cells can be reset when the cells are exposed to a changed MHC environment. These findings have important implications for how NK cell functions may be curtailed or enhanced in the context of disease.

The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-D(d). To compare Ly-49D and Ly-49A interactions with H2-D(d), we created mutations in H2-D(d) and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-D(d) alpha(1) helix or the alpha(2) helix abrogated Ly-49D-mediated cytotoxicity, but these changes had no significant effect on Ly-49A-dependent inhibition. Each of three alpha(2) domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-D(d) interactions require distinct determinants compared with Ly-49A/H2-D(d) interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells.

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-D(d). To compare Ly-49D and Ly-49A interactions with H2-D(d), we created mutations in H2-D(d) and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-D(d) alpha(1) helix or the alpha(2) helix abrogated Ly-49D-mediated cytotoxicity, but these changes had no significant effect on Ly-49A-dependent inhibition. Each of three alpha(2) domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-D(d) interactions require distinct determinants compared with Ly-49A/H2-D(d) interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D-ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti- Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor- bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab')2 anti-Ly-49D (12A8) and F(ab')2 anti-H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2(d) mice (BALB/c, B10.D2, and DBA/2) but not from H-2(b) or H-2(k) mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells.

The Ly49 natural killer (NK) cell receptors are class I MHC-specific inhibitory receptors that are distributed to overlapping NK cell subsets. The formation of the Ly49 receptor repertoire was examined with transgenic mice that express Ly49A in all NK cells. In MHC class I-deficient mice, the Ly49A transgene did not prevent expression of endogenous Ly49 genes. However, in H-2(d) mice that express a Ly49A ligand, the transgene caused clear alterations in the endogenous Ly49 repertoire. The frequency of NK cells expressing another H-2(d)-specific receptor, Ly49G2(+), was substantially reduced. Reduced numbers of cells expressing endogenous Ly49A was suggested by reduced endogenous Ly49A mRNA levels. These results support the existence of an MHC-dependent education process that limits the number of NK cells that coexpress multiple self-specific Ly49 receptors. Ligand-dependent downregulation of Ly49 cell surface levels was also examined. Cell-surface downregulation occurred even when the transgene was expressed at low levels. The results demonstrate that downregulation of Ly49A cell surface levels is a posttranscriptional event, and argue against a model in which Ly49 receptors are calibrated to specific cell surface levels depending on the available class I ligands.

The Ly49 natural killer (NK) cell receptors are class I MHC-specific inhibitory receptors that are distributed to overlapping NK cell subsets. The formation of the Ly49 receptor repertoire was examined with transgenic mice that express Ly49A in all NK cells. In MHC class I-deficient mice, the Ly49A transgene did not prevent expression of endogenous Ly49 genes. However, in H-2(d) mice that express a Ly49A ligand, the transgene caused clear alterations in the endogenous Ly49 repertoire. The frequency of NK cells expressing another H-2(d)-specific receptor, Ly49G2(+), was substantially reduced. Reduced numbers of cells expressing endogenous Ly49A was suggested by reduced endogenous Ly49A mRNA levels. These results support the existence of an MHC-dependent education process that limits the number of NK cells that coexpress multiple self-specific Ly49 receptors. Ligand-dependent downregulation of Ly49 cell surface levels was also examined. Cell-surface downregulation occurred even when the transgene was expressed at low levels. The results demonstrate that downregulation of Ly49A cell surface levels is a posttranscriptional event, and argue against a model in which Ly49 receptors are calibrated to specific cell surface levels depending on the available class I ligands.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice.

Natural killer (NK) cells and some T cells are endowed with receptors specific for class I major histocompatibility complex (MHC) molecules that can inhibit cellular effector functions. The function of the Ly49 receptor family has been studied in vitro, but no gene transfer experiments have directly established the role of these receptors in NK cell functions. We show here that transgenic expression of the H-2Dd-specific Ly49A receptor in all NK cells and T cells conferred class I-specific inhibition of NK cell-mediated target cell lysis as well as of T cell proliferation. Furthermore, transgene expression prevented NK cell-mediated rejection of allogeneic H-2d bone marrow grafts by irradiated mice. These results demonstrate the function and specificity of Ly49 receptors in vivo, and establish that their subset-specific expression is necessary for the discrimination of MHC-different cells by NK cells in unmanipulated mice.

The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.

The present study examined self-tolerance for T cell receptor (TCR) alpha beta intestinal intraepithelial lymphocytes (iIELs) using the 2C transgenic (Tg) mouse model specific for a peptide antigen (Ag) presented by the class I major histocompatibility complex H-2Ld. Although Tg+ T cells were largely deleted from the periphery of Ag+ mice, equivalent numbers of Tg iIELs were present in Ag+ compared to Ag- mice. Tg iIELs in Ag- mice contained CD8 alpha beta, CD8 alpha alpha, and CD4-CD8- subsets, whereas only CD8 alpha alpha and CD4-CD8- Tg iIEL subsets were detected in Ag+ mice. Analysis of surface markers revealed that Tg iIELs in Ag+ mice expressed decreased levels of Thy-1 and increased CD45R/B220 as compared to Ag- Tg iIELs. In response to activation with exogenous peptide or immobilized anti-TCR mAB, iIELs from Ag- mice proliferated at high levels and produced interleukin (IL)-2 and interferon (IFN)-gamma, while Tg+ iIELs from Ag+ mice proliferated at low levels and failed to produce detectable IL-2 or IFN-gamma. Activation of sorted iIEL subsets from Ag- mice revealed that CD8 alpha alpha and CD4-CD8- subsets produced low levels of IL-2 and IFN-gamma in response to activation with antigen-presenting cells and added peptide or immobilized anti-TCR mAb, while CD8 alpha beta + iIELs responded to endogenous levels of peptide. In response to APC and exogenous peptide, sorted iIEL subsets from Ag+ mice produced IL-2 and IFN-gamma, and proliferated at greatly reduced levels compared to corresponding subsets from Ag- mice. Analysis of cytokine mRNA levels revealed that activation in vitro induced IL-2 mRNA only in Ag-, but not Ag+ iIELs, whereas a high level of IL-4 mRNA induction was detected in Tg+ iIELs from Ag+ mice, and to a lesser degree, from Ag- mice. These data suggest that tolerance for Tg+ iIELs resulted in the deletion of CD8 alpha beta + subsets and the persistence of Tg+ iIEL subsets with decreased sensitivity to endogenous levels of self-peptide. A comparison of the cytokine profiles expressed by Tg+ iIEL subsets in Ag- and Ag+ mice suggested that tolerance induction had involved the functional deviation of cells from TC1 (T helper-1-like) to a less inflammatory TC2 (T helper-2-like) phenotype capable of mediating humoral immune responses in the mucosa.

The identity of allogeneic peptide/major histocompatibility complex (MHC) complexes that elicit vigorous T cell responses has remained an interesting problem for both practical and theoretical reasons. Although a few abundant MHC class I-bound peptides have been purified and sequenced, identifying the unique T cell-stimulating peptides from among the thousands of existing peptides is still a very difficult undertaking. In this report, we identified the antigenic peptide that is recognized by an alloreactive bm1 anti-B6 T cell clone using a novel genetic strategy that is based upon measurement of T cell receptor occupancy in single T cells. Using lacZ-inducible T cells as a probe, we screened a splenic cDNA library in transiently transfected antigen-presenting cells (APCs) and isolated a cDNA clone that allowed expression of the appropriate peptide/Kb MHC complex in APC. The antigenic octapeptide (SVVEFSSL) exactly matched the consensus Kb MHC motif, but was surprisingly encoded by a non-ATG defined translation reading frame. Furthermore, the abundance of the naturally processed analog in untransfected cells was estimated to be

The identity of allogeneic peptide/major histocompatibility complex (MHC) complexes that elicit vigorous T cell responses has remained an interesting problem for both practical and theoretical reasons. Although a few abundant MHC class I-bound peptides have been purified and sequenced, identifying the unique T cell-stimulating peptides from among the thousands of existing peptides is still a very difficult undertaking. In this report, we identified the antigenic peptide that is recognized by an alloreactive bm1 anti-B6 T cell clone using a novel genetic strategy that is based upon measurement of T cell receptor occupancy in single T cells. Using lacZ-inducible T cells as a probe, we screened a splenic cDNA library in transiently transfected antigen-presenting cells (APCs) and isolated a cDNA clone that allowed expression of the appropriate peptide/Kb MHC complex in APC. The antigenic octapeptide (SVVEFSSL) exactly matched the consensus Kb MHC motif, but was surprisingly encoded by a non-ATG defined translation reading frame. Furthermore, the abundance of the naturally processed analog in untransfected cells was estimated to be

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.

Target cell expression of major histocompatibility complex (MHC) class I molecules correlates with resistance to lysis by natural killer (NK) cells. Prior functional studies of the murine NK cell surface molecule, Ly-49, suggested its role in downregulating NK cell cytotoxicity by specifically interacting with target cell H-2Dd molecules. In support of this hypothesis, we now demonstrate a physical interaction between H-2Dd and Ly-49 in both qualitative and quantitative cell-cell binding assays employing a stable transfected Chinese hamster ovary (CHO) cell line expressing Ly-49 and MHC class I transfected target cells. Binding occurred only when CHO cells expressed Ly-49 at high levels and targets expressed H-2Dd by transfection. Monoclonal antibody blocking experiments confirmed this interaction. These studies indicate that the specificity of natural killing is influenced by NK cell receptors that engage target cell MHC class I molecules.

Target cell expression of major histocompatibility complex (MHC) class I molecules correlates with resistance to lysis by natural killer (NK) cells. Prior functional studies of the murine NK cell surface molecule, Ly-49, suggested its role in downregulating NK cell cytotoxicity by specifically interacting with target cell H-2Dd molecules. In support of this hypothesis, we now demonstrate a physical interaction between H-2Dd and Ly-49 in both qualitative and quantitative cell-cell binding assays employing a stable transfected Chinese hamster ovary (CHO) cell line expressing Ly-49 and MHC class I transfected target cells. Binding occurred only when CHO cells expressed Ly-49 at high levels and targets expressed H-2Dd by transfection. Monoclonal antibody blocking experiments confirmed this interaction. These studies indicate that the specificity of natural killing is influenced by NK cell receptors that engage target cell MHC class I molecules.

In studies of the resistance of inbred mice to infection with Trypanosoma cruzi Peru, mouse strain B10.S was the only strain which survived the infection resulting from the inoculation of 10(3) trypomastigotes. This is the only inbred mouse strain studied to survive infection. To investigate the effect of the H-2 haplotype on survival, C57BL/10 congenic mouse strains bearing H-2S recombinant haplotypes and mouse strains A.SWSn/J and SJL/J were tested for their ability to overcome the T. cruzi infection. None of the recombinant strains tested, including B10.S(7R), B10.S(8R), B10.S(9R), and B10.HTT, survived the infection, indicating that at least two or more regions of the H-2 locus must be H-2S to ensure survival. Strains A.SWSn/J and SJL/J with the H-2S haplotype did not survive, indicating that the genetic background outside the H-2 complex also influences survival. The congenic F1 hybrid (C57BL/10 X B10.S) F1 exhibited intermediate survival levels when compared with the parental strains, indicating that H-2S survival is affected by gene dosage. The F1 hybrid strain [B10.S(7R) X B10.S(8R)]F1, which possesses the complete H-2S haplotype in the trans configuration, did not survive T. cruzi infection, suggesting that H-2S-mediated survival does not operate by trans complementation.

A novel approach for the analysis of membrane proteins involved in ligand-induced surface receptor patching and capping is described. The technique is based on the use of immunolactoperoxidase (immuno-LPO) conjugates which catalyze the iodination of those surface proteins with available tyrosine groups that are located in the immediate vicinity of the patch or cap of a particular antigen. We have used the patching and capping of the H-2 (histocompatibility) antigen on mouse thymocytes to illustrate this method. However, this technique should be generally applicable to any cell surface proteins which can be induced to form patches or caps by a specific ligand. Cytochemical analysis indicates that the immuno-LPO conjugates induce the same patching and capping of the H-2 antigen as does the unconjugated antibody. Biochemical analysis of the 125I-labeled proteins by SDS polyacrylamide gel electrophoresis indicates that a large membrane protein (mol wt of approximately 200,000 daltons) is closely associated with H-2 patches and caps. Since a number of other prominent membrane proteins are not labeled by this procedure, selective redistribution of certain surface proteins must be occurring during H-2 antibody-induced patching and capping.

Resting B lymphocytes are activated, proliferate, and differentiate into antibody-secreting cells when cultured with long-term lines of major histocompatibility complex (MHC)-restricted, antigen-specific T cell in the presence of the antigen for which the T cells are specific. Under optimal conditions, essentially all B cells are activated and approximately 35% enter S phase in the absence of antigens for which the B cells are specific. Activation and proliferation are observed in cells from both normal mice and mice with the xid-determined immune defect. Highly purified B cells bearing Ia molecules for which the T cells are "cospecific" can present antigen to T cells with the resulting T cell stimulation leading to the activation and proliferation of the antigen-presenting B cells. However, B cells that do not bear Ia molecules for which the T cells are cospecific are also activated and proliferate if antigen and a source of antigen-presenting B cells or macrophage-rich cells of proper histocompatibility type are present. Thus, resting B cells, both normal and "xid", can be activated by non-MHC restricted factors without receptor cross-linkage. Experiments are presented that support the concept that local production and action of such unrestricted activating factors may be responsible for the MHC-restriction of T cell-B cell interaction seen in many circumstances.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

Treatment of BALB/c mice with purified pig antiidiotype to 11-4.1 (anti-H-2Kk) monoclonal antibody has been found previously to induce the appearance of idiotype-bearing molecules (Id') in the serum of these mice, in the absence of detectable antigen-binding activity. In the present study we examined the effect of subsequent immunization of such antiidiotype-primed mice with the original H-2Kk antigen. Skin grafting of virgin BALB/c mice with BALB.K skin did not generate any detectable Id' antibodies when tested by enzyme-linked immunosorbent assay (ELISA). In contrast, grafting of antiidiotype-primed mice with BALB.K skin specifically boosted ther serum level of Id' molecules. Challenge of antiidiotype-primed mice with either B10.D2 or rat skin had no effect on the production of such Id' molecules. Absorption studies demonstrated that the majority of Id' molecules induced by H-2Kk antigenic stimulus and detected in ELISA are antigen-nonbinding molecules, thus indicating specific restimulation by the original H-2Kk antigen of nonbinding idiotype-positive B cell clones. The relevance of these findings to the existence of network interactions in the immune response to H-2 antigens is discussed.

Cloned CTLs QM3 and QM7 isolated from a bulk CTL line B10.QBR anti-B10.MBR recognized a combination of the H-2Kb molecule and an I-Ak subregion gene product. Such a combinatorial specificity was revealed by complementation of the target antigen in F1 animals between two negative parental strains carrying H-2Kb and I-Ak, respectively. We confirmed the involvement of the H-2Kb molecule by blocking killing with anti-Kb mAb and failure of certain mutant H-2Kb genes to complement with I-Ak to generate the determinant in F1 animals. Although the nature of the I-Ak subregion gene product is not definitive, there was a correlation between the expression of Ia antigens on the cell surface and susceptibility of the cells to lysis by these CTLs, suggesting that it is the classical I-Ak class II antigen.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Class II-restricted T cell clones specific for myelin basic protein (MBP) have been generated from PL/J and (PL/J X SJL/J)F1 [((PLSJ)F1] mice following sensitization to rat MBP. Of 17 T cell clones generated from (PLSJ)F1 mice, 5 are I-Au(A alpha uA beta u) restricted, one is restricted to I-As(A alpha sA beta s), 10 are restricted to hybrid I-A(u X s)F1 (A alpha sA beta u) determinants, and one clone is restricted to hybrid I-E(u X s) (E alpha uE beta s) molecules. Thus, of 16 I-A-restricted T cell clones generated from (PLSJ)F1 mice, only one is I-As-restricted, reflecting a lack of priming to MBP in association with I-As. T cell clones restricted to I-Au and to I-E (E alpha u E beta s) molecules recognize mouse (self) MBP. Furthermore, only the five T cell clones restricted to I-Au molecules recognize a determinant in common with mouse (self) MBP within the encephalitogenic N-terminal peptide. Three such I-Au restricted T cell clones, derived independently, cause paralysis in 100% of (PL/J X SJL/J)F1 mice tested. Acute, chronic unremitting, and chronic relapsing paralysis are all induced following injection of these clones. Administration of greater numbers of cloned T cells causes acute and fatal experimental allergic encephalomyelitis, while administration of lower numbers of cloned T cells is associated with chronic unremitting and relapsing paralysis. Paralysis induced with T cell clones shares many clinical, immunologic, and histologic aspects with human demyelinating diseases such as multiple sclerosis. Histopathology reveals perivascular lymphocytic infiltration, demyelination, and remyelination. These studies demonstrate the utility of T cell clones for analyzing the association between class II major histocompatibility complex molecules and disease susceptibility.

The role of antigen surface density, and its relationship to the function of the Lyt-2/3 complex, in recognition and triggering of allospecific cytotoxic T lymphocyte (CTL) precursors has been studied by using a novel type of Class I protein-bearing artificial membrane. The cell-size membranes, termed pseudocytes (artificial cells), can be handled like cells but have a well-defined and easily quantitated surface composition. Class I antigen on these membranes stimulated generation of secondary in vitro allogeneic CTL responses as effectively as allogeneic spleen cells, provided that lymphokines were added to the cultures. Antigen density on the pseudocyte surfaces could be varied over a wide range and quantitated by papain cleavage and fluorescence-activated cell sorter analysis. Recognition and triggering of precursor CTL was found to be dramatically dependent on the surface density of antigen and displayed a marked threshold density requirement, below which little or no response occurred. Examination of the effects of anti-Lyt-2 antibody on responses to pseudocytes provided direct evidence for a reciprocal relationship between antigen density and susceptibility to antibody blockade. The results strongly suggest that antigen density is likely to have important biological consequences in control of immune responses. They also show that if Lyt-2/3 functions by interaction with a ligand, then that ligand is the Class I protein.

Novel cell-sized, supported artificial membranes bearing class I antigens have been prepared by a simple dialysis procedure and then used to study the requirements for antigen recognition by precursor cytotoxic T lymphocytes (CTL). The membranes were made by mixing lipid, H-2 antigen, and C18 alkylated 5 microns silica beads in deoxycholate, and dialyzing to remove the detergent. The H-2 antigen-bearing, cell-sized beads, termed pseudocytes (artificial cells), were able to simulate generation of secondary CTL responses with the same specificity as alloantigen-bearing spleen cells. Comparative analyses demonstrated that the size of an antigen-bearing structure, and thus its potential for multivalent interaction, was a critical determinant of effectiveness of antigen recognition, and showed that H-2 antigen was recognized as effectively on cell-sized beads as on allogeneic spleen cells. Generation of a response to antigen on the cell-sized beads was completely dependent on addition of lymphokines to the cultures. Thus, unlike liposomes, H-2 antigen on beads was not available to accessory cells for stimulation of Ia-dependent production of lymphokines by T helper cells. These results, as well as direct observations by microscopy, strongly indicate that antigen is recognized on the surface of the beads. Despite effective stimulation of secondary CTL responses, antigen on beads was completely inactive in stimulating a primary CTL response by naive spleen cells. The results of mixing experiments by using beads and alloantigen-bearing cells or plasma membrane vesicles indicate that the lack of a primary response may result from a requirement for a soluble factor(s) that is not needed for generation of secondary responses. The unique advantages of cell-size supported membranes for studying antigen recognition by T cells are discussed. The beads can be handled and used like antigen-bearing cells in functional assays, while possessing well-defined, readily varied, and easily quantitated composition.

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants.

A novel approach for the analysis of membrane proteins involved in ligand-induced surface receptor patching and capping is described. The technique is based on the use of immunolactoperoxidase (immuno-LPO) conjugates which catalyze the iodination of those surface proteins with available tyrosine groups that are located in the immediate vicinity of the patch or cap of a particular antigen. We have used the patching and capping of the H-2 (histocompatibility) antigen on mouse thymocytes to illustrate this method. However, this technique should be generally applicable to any cell surface proteins which can be induced to form patches or caps by a specific ligand. Cytochemical analysis indicates that the immuno-LPO conjugates induce the same patching and capping of the H-2 antigen as does the unconjugated antibody. Biochemical analysis of the 125I-labeled proteins by SDS polyacrylamide gel electrophoresis indicates that a large membrane protein (mol wt of approximately 200,000 daltons) is closely associated with H-2 patches and caps. Since a number of other prominent membrane proteins are not labeled by this procedure, selective redistribution of certain surface proteins must be occurring during H-2 antibody-induced patching and capping.

Treatment of BALB/c mice with purified pig antiidiotype to 11-4.1 (anti-H-2Kk) monoclonal antibody has been found previously to induce the appearance of idiotype-bearing molecules (Id') in the serum of these mice, in the absence of detectable antigen-binding activity. In the present study we examined the effect of subsequent immunization of such antiidiotype-primed mice with the original H-2Kk antigen. Skin grafting of virgin BALB/c mice with BALB.K skin did not generate any detectable Id' antibodies when tested by enzyme-linked immunosorbent assay (ELISA). In contrast, grafting of antiidiotype-primed mice with BALB.K skin specifically boosted ther serum level of Id' molecules. Challenge of antiidiotype-primed mice with either B10.D2 or rat skin had no effect on the production of such Id' molecules. Absorption studies demonstrated that the majority of Id' molecules induced by H-2Kk antigenic stimulus and detected in ELISA are antigen-nonbinding molecules, thus indicating specific restimulation by the original H-2Kk antigen of nonbinding idiotype-positive B cell clones. The relevance of these findings to the existence of network interactions in the immune response to H-2 antigens is discussed.

Resting B lymphocytes are activated, proliferate, and differentiate into antibody-secreting cells when cultured with long-term lines of major histocompatibility complex (MHC)-restricted, antigen-specific T cell in the presence of the antigen for which the T cells are specific. Under optimal conditions, essentially all B cells are activated and approximately 35% enter S phase in the absence of antigens for which the B cells are specific. Activation and proliferation are observed in cells from both normal mice and mice with the xid-determined immune defect. Highly purified B cells bearing Ia molecules for which the T cells are "cospecific" can present antigen to T cells with the resulting T cell stimulation leading to the activation and proliferation of the antigen-presenting B cells. However, B cells that do not bear Ia molecules for which the T cells are cospecific are also activated and proliferate if antigen and a source of antigen-presenting B cells or macrophage-rich cells of proper histocompatibility type are present. Thus, resting B cells, both normal and "xid", can be activated by non-MHC restricted factors without receptor cross-linkage. Experiments are presented that support the concept that local production and action of such unrestricted activating factors may be responsible for the MHC-restriction of T cell-B cell interaction seen in many circumstances.

We previously described a somatic cell expressing a variant H-2Dd molecule that did not serve as a target for alloreactive anti-Dd CTL. The mutant cell line had been isolated by its failure to express a serological epitope present on the H-2Dd alpha 3 domain. In the present study the alpha 3 domain of the Dd molecule of this somatic cell variant was sequenced and a single nucleotide change resulting in a glutamic acid to lysine substitution at residue 227 was identified. This change was reproduced in the cloned H-2Dd gene by oligonucleotide-directed mutagenesis. Cells transfected with this mutant gene were not killed by anti-H-2Dd CTL. Because previous studies using hybrid H-2 class I molecules had established that the alpha 3 domain does not express allele-specific determinants recognized by CTL, our results raise the possibility that residues in the alpha 3 domain of H-2 class I molecules are critical for CTL recognition and constitute a conserved (or monomorphic) determinant recognized by CTL.

Class II-restricted T cell clones specific for myelin basic protein (MBP) have been generated from PL/J and (PL/J X SJL/J)F1 [((PLSJ)F1] mice following sensitization to rat MBP. Of 17 T cell clones generated from (PLSJ)F1 mice, 5 are I-Au(A alpha uA beta u) restricted, one is restricted to I-As(A alpha sA beta s), 10 are restricted to hybrid I-A(u X s)F1 (A alpha sA beta u) determinants, and one clone is restricted to hybrid I-E(u X s) (E alpha uE beta s) molecules. Thus, of 16 I-A-restricted T cell clones generated from (PLSJ)F1 mice, only one is I-As-restricted, reflecting a lack of priming to MBP in association with I-As. T cell clones restricted to I-Au and to I-E (E alpha u E beta s) molecules recognize mouse (self) MBP. Furthermore, only the five T cell clones restricted to I-Au molecules recognize a determinant in common with mouse (self) MBP within the encephalitogenic N-terminal peptide. Three such I-Au restricted T cell clones, derived independently, cause paralysis in 100% of (PL/J X SJL/J)F1 mice tested. Acute, chronic unremitting, and chronic relapsing paralysis are all induced following injection of these clones. Administration of greater numbers of cloned T cells causes acute and fatal experimental allergic encephalomyelitis, while administration of lower numbers of cloned T cells is associated with chronic unremitting and relapsing paralysis. Paralysis induced with T cell clones shares many clinical, immunologic, and histologic aspects with human demyelinating diseases such as multiple sclerosis. Histopathology reveals perivascular lymphocytic infiltration, demyelination, and remyelination. These studies demonstrate the utility of T cell clones for analyzing the association between class II major histocompatibility complex molecules and disease susceptibility.

Cloned CTLs QM3 and QM7 isolated from a bulk CTL line B10.QBR anti-B10.MBR recognized a combination of the H-2Kb molecule and an I-Ak subregion gene product. Such a combinatorial specificity was revealed by complementation of the target antigen in F1 animals between two negative parental strains carrying H-2Kb and I-Ak, respectively. We confirmed the involvement of the H-2Kb molecule by blocking killing with anti-Kb mAb and failure of certain mutant H-2Kb genes to complement with I-Ak to generate the determinant in F1 animals. Although the nature of the I-Ak subregion gene product is not definitive, there was a correlation between the expression of Ia antigens on the cell surface and susceptibility of the cells to lysis by these CTLs, suggesting that it is the classical I-Ak class II antigen.

The ability to generate specific cytotoxic responses using purified major histocompatibility complex (MHC) antigen in liposomes has made it possible to directly assess the importance of class I carbohydrate moieties in T cell recognition of alloantigen. Deglycosylation of affinity-purified H-2Kk to yield a single glycan-free product did not alter the specificity, the magnitude, nor the dose range of the cytotoxic T lymphocyte (CTL) response to the class I antigen. It can be concluded that carbohydrate moieties are not required to maintain the necessary conformation of the MHC protein, nor to interact with either the antigen-specific receptor or accessory proteins on precursor CTL.

In studies of the resistance of inbred mice to infection with Trypanosoma cruzi Peru, mouse strain B10.S was the only strain which survived the infection resulting from the inoculation of 10(3) trypomastigotes. This is the only inbred mouse strain studied to survive infection. To investigate the effect of the H-2 haplotype on survival, C57BL/10 congenic mouse strains bearing H-2S recombinant haplotypes and mouse strains A.SWSn/J and SJL/J were tested for their ability to overcome the T. cruzi infection. None of the recombinant strains tested, including B10.S(7R), B10.S(8R), B10.S(9R), and B10.HTT, survived the infection, indicating that at least two or more regions of the H-2 locus must be H-2S to ensure survival. Strains A.SWSn/J and SJL/J with the H-2S haplotype did not survive, indicating that the genetic background outside the H-2 complex also influences survival. The congenic F1 hybrid (C57BL/10 X B10.S) F1 exhibited intermediate survival levels when compared with the parental strains, indicating that H-2S survival is affected by gene dosage. The F1 hybrid strain [B10.S(7R) X B10.S(8R)]F1, which possesses the complete H-2S haplotype in the trans configuration, did not survive T. cruzi infection, suggesting that H-2S-mediated survival does not operate by trans complementation.

The role of antigen surface density, and its relationship to the function of the Lyt-2/3 complex, in recognition and triggering of allospecific cytotoxic T lymphocyte (CTL) precursors has been studied by using a novel type of Class I protein-bearing artificial membrane. The cell-size membranes, termed pseudocytes (artificial cells), can be handled like cells but have a well-defined and easily quantitated surface composition. Class I antigen on these membranes stimulated generation of secondary in vitro allogeneic CTL responses as effectively as allogeneic spleen cells, provided that lymphokines were added to the cultures. Antigen density on the pseudocyte surfaces could be varied over a wide range and quantitated by papain cleavage and fluorescence-activated cell sorter analysis. Recognition and triggering of precursor CTL was found to be dramatically dependent on the surface density of antigen and displayed a marked threshold density requirement, below which little or no response occurred. Examination of the effects of anti-Lyt-2 antibody on responses to pseudocytes provided direct evidence for a reciprocal relationship between antigen density and susceptibility to antibody blockade. The results strongly suggest that antigen density is likely to have important biological consequences in control of immune responses. They also show that if Lyt-2/3 functions by interaction with a ligand, then that ligand is the Class I protein.

Recognition of polymorphic determinants on class I or class II MHC Ag is required for T lymphocyte responses. Using cell-size artificial membranes (pseudocytes) bearing H-2 class I Ag it is demonstrated that T cells can, in addition, recognize nonpolymorphic determinants on class I proteins. Pseudocytes bearing class I alloantigen stimulate in vitro generation of secondary allogeneic CTL responses. At a suboptimal alloantigen surface density, incorporation of class I molecules identical to those of the responder cells (self-H-2) or from third-party cells resulted in dramatically enhanced responses, whereas incorporation of class II proteins had no effect. The receptor that mediates recognition of conserved class I determinants has not been identified, but results of antibody blocking studies are consistent with the Lyt-2/3 complex of CTL having this role. Thus, class I proteins on Ag-bearing cells can have two distinct roles in T cell activation, one involving recognition of polymorphic determinants by the Ag-specific receptor and the other involving recognition of conserved determinants.