Ontogeny of T cell tolerance to peripherally expressed antigens

Transgenic expression of the influenza virus hemagglutinin (HA) in the pancreatic islet β cells of InsHA mice leads to peripheral tolerance of HA-specific T cells. To examine the onset of tolerance, InsHA mice were immunized with influenza virus A/PR/8 at different ages, and the presence of nontolerant T cells was determined by the induction of autoimmune diabetes. The data revealed a neonatal period wherein T cells were not tolerant and influenza virus infection led to HA-specific β cell destruction and autoimmune diabetes. The ability to induce autoimmunity gradually waned, such that adult mice were profoundly tolerant to viral HA and were protected from diabetes. Because cross-presentation of islet antigens by professional antigen-presenting cells had been reported to induce peripheral tolerance, the temporal relationship between tolerance induction and activation of HA-specific T cells in the lymph nodes draining the pancreas was examined. In tolerant adult mice, but not in 1-week-old neonates, activation and proliferation of HA-specific CD8+ T cells occurred in the pancreatic lymph nodes. Thus, lack of tolerance in the perinatal period correlated with lack of activation of antigen-specific CD8+ T cells. This work provides evidence for the developmental regulation of peripheral tolerance induction.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+ T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8+ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+ T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

Genetic basis of tolerance to O2 deprivation in Drosophila melanogaster

The ability to tolerate a low-O2 environment varies widely among species in the animal kingdom. Some animals, such as Drosophila melanogaster, can tolerate anoxia for prolonged periods without apparent tissue injury. To determine the genetic basis of the cellular responses to low O2, we performed a genetic screen in Drosophila to identify loci that are responsible for anoxia resistance. Four X-linked, anoxia-sensitive mutants belonging to three complementation groups were isolated after screening more than 10,000 mutagenized flies. The identified recessive and dominant mutations showed marked delay in recovery from O2 deprivation. In addition, electrophysiologic studies demonstrated that polysynaptic transmission in the central nervous system of the mutant flies was abnormally long during recovery from anoxia. These studies show that anoxic tolerance can be genetically dissected.

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor β1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor β1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.

Allelic variation of a dehydrin gene cosegregates with chilling tolerance during seedling emergence

Dehydrins (DHNs, LEA D-11) are plant proteins present during environmental stresses associated with dehydration or low temperatures and during seed maturation. Functions of DHNs have not yet been defined. Earlier, we hypothesized that a ≈35-kDa DHN and membrane properties that reduce electrolyte leakage from seeds confer chilling tolerance during seedling emergence of cowpea (Vigna unguiculata L. Walp.) in an additive and independent manner. Evidence for this hypothesis was not rigorous because it was based on correlations of presence/absence of the DHN and slow electrolyte leakage with chilling tolerance in closely related cowpea lines that have some other genetic differences. Here, we provide more compelling genetic evidence for involvement of the DHN in chilling tolerance of cowpea. We developed near-isogenic lines by backcrossing. We isolated and determined the sequence of a cDNA corresponding to the ≈35-kDa DHN and used gene-specific oligonucleotides derived from it to test the genetic linkage between the DHN presence/absence trait and the DHN structural gene. We tested for association between the DHN presence/absence trait and both low-temperature seed emergence and electrolyte leakage. We show that allelic differences in the Dhn structural gene map to the same position as the DHN protein presence/absence trait and that the presence of the ≈35-kDa DHN is indeed associated with chilling tolerance during seedling emergence, independent of electrolyte leakage effects. Two types of allelic variation in the Dhn gene were identified in the protein-coding region, deletion of one Φ-segment from the DHN-negative lines and two single amino acid substitutions.

Constitutive expression of the cold-regulated Arabidopsis thaliana COR15a gene affects both chloroplast and protoplast freezing tolerance

Cold acclimation in plants is associated with the expression of COR (cold-regulated) genes that encode polypeptides of unknown function. It has been widely speculated that products of these genes might have roles in freezing tolerance. Here we provide direct evidence in support of this hypothesis. We show that constitutive expression of COR15a, a cold-regulated gene of Arabidopsis thaliana that encodes a chloroplast-targeted polypeptide, enhances the in vivo freezing tolerance of chloroplasts in nonacclimated plants by almost 2°C, nearly one-third of the increase that occurs upon cold acclimation of wild-type plants. Significantly, constitutive expression of COR15a also affects the in vitro freezing tolerance of protoplasts. At temperatures between −5 and −8°C, the survival of protoplasts isolated from leaves of nonacclimated transgenic plants expressing COR15a was greater than that of protoplasts isolated from leaves of nonacclimated wild-type plants. At temperatures between −2 and −4°C, constitutive expression of COR15a had a slight negative effect on survival. The implications of these data regarding possible modes of COR15a action are discussed.

Mode of action of the COR15a gene on the freezing tolerance of Arabidopsis thaliana

Constitutive expression of the cold-regulated COR15a gene of Arabidopsis thaliana results in a significant increase in the survival of isolated protoplasts frozen over the range of −4.5 to −7°C. The increased freezing tolerance is the result of a decreased incidence of freeze-induced lamellar-to-hexagonal II phase transitions that occur in regions where the plasma membrane is brought into close apposition with the chloroplast envelope as a result of freeze-induced dehydration. Moreover, the mature polypeptide encoded by this gene, COR15am, increases the lamellar-to-hexagonal II phase transition temperature of dioleoylphosphatidylethanolamine and promotes formation of the lamellar phase in a lipid mixture composed of the major lipid species that comprise the chloroplast envelope. We propose that COR15am, which is located in the chloroplast stroma, defers freeze-induced formation of the hexagonal II phase to lower temperatures (lower hydrations) by altering the intrinsic curvature of the inner membrane of the chloroplast envelope.

An Arabidopsis mutant that requires increased calcium for potassium nutrition and salt tolerance

Potassium (K+) nutrition and salt tolerance are key factors controlling plant productivity. However, the mechanisms by which plants regulate K+ nutrition and salt tolerance are poorly understood. We report here the identification of an Arabidopsis thaliana mutant, sos3 (salt-overly-sensitive 3), which is hypersensitive to Na+ and Li+ stresses. The mutation is recessive and is in a nuclear gene that maps to chromosome V. The sos3 mutation also renders the plant unable to grow on low K+. Surprisingly, increased extracellular Ca2+ suppresses the growth defect of sos3 plants on low K+ or 50 mM NaCl. In contrast, high concentrations of external Ca2+ do not rescue the growth of the salt-hypersensitive sos1 mutant on low K+ or 50 mM NaCl. Under NaCl stress, sos3 seedlings accumulated more Na+ and less K+ than the wild type. Increased external Ca2+ improved K+/Na+ selectivity of both sos3 and wild-type plants. However, this Ca2+ effect in sos3 is more than twice as much as that in the wild type. In addition to defining the first plant mutant with an altered calcium response, these results demonstrate that the SOS3 locus is essential for K+ nutrition, K+/Na+ selectivity, and salt tolerance in higher plants.

Tolerance of Arc repressor to multiple-alanine substitutions

Arc repressor mutants containing from three to 15 multiple-alanine substitutions have spectral properties expected for native Arc proteins, form heterodimers with wild-type Arc, denature cooperatively with Tms equal to or greater than wild type, and, in some cases, fold as much as 30-fold faster and unfold as much as 50-fold slower than wild type. Two of the mutants, containing a total of 14 different substitutions, also footprint operator DNA in vitro. The stability of some of the proteins with multiple-alanine mutations is significantly greater than that predicted from the sum of the single substitutions, suggesting that a subset of the wild-type residues in Arc may interact in an unfavorable fashion. Overall, these results show that almost half of the residues in Arc can be replaced by alanine en masse without compromising the ability of this small, homodimeric protein to fold into a stable, native-like structure.

Suppressor T cells regulate the nonanergic cell population that remains after peripheral tolerance is induced to the Mls-1 antigen in T cell receptor Vβ8.1 transgenic mice

We have found suppressor T cells that inhibit the proliferative response of naive CD4+ T cells in T cell receptor (TCR) Vβ8.1 transgenic mice rendered tolerant in vivo by inoculation of Mls-1a-positive cells. This suppression was mediated by CD4+ T cells but not by CD8+ T cells or double-negative (DN) cells, and splenic CD4+ T cells from tolerant mice displayed a greater suppression than lymph node CD4+ T cells. Cell contact was required for efficient suppression, and known inhibitory cytokines such as IL-4, IL-10, and transforming growth factor β were not involved. Suppressor T cells inhibited IL-2 production by naive CD4+ T cells, and the addition of exogenous IL-2 diminished the suppressed activity while having little activity on tolerant T cells. Suppression was abolished by the elimination of CD25+ T cells in the tolerant CD4+ T cell subset. CD25+CD4+ T cells suppressed the proliferative response of the residual fraction of the nonanergic population, namely, 6C10+CD4+ T cells still present in the tolerant mice. However, 6C10−CD4+ T cells still had reduced reactivity to Mls-1a even after CD25+CD4+ T cells were removed and exogenous IL-2 was added. Suppressor cells appear to affect only residual nonanergic cells in situ, thereby facilitating the maintenance of the unresponsive state in vivo. These data provide a framework for understanding suppressor T cells and explain the difficulties and variables in defining their activity in other systems, because suppressor T cells apparently control only a small population of nonanergic cells in the periphery and may be viewed as a homeostatic mechanism.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells

Immunological unresponsiveness established by the elimination or anergy of self-reactive lymphocyte clones is of importance to immunization against tumor-associated antigens. In this study, we have investigated induction of immunity against the human MUC1 carcinoma-associated antigen in MUC1 transgenic mice unresponsive to MUC1 antigen. Immunization of adult MUC1 transgenic mice with irradiated MUC1-positive tumor cells was unsuccessful in reversing unresponsiveness to MUC1. By contrast, fusions of dendritic cells with MUC1-positive tumor cells induced cellular and humoral immunity against MUC1. Immunization with the dendritic cell fusions that express MUC1 resulted in the rejection of established metastases and no apparent autoimmunity against normal tissues. These findings demonstrate that unresponsiveness to the MUC1 tumor-associated antigen is reversible by immunization with heterokaryons of dendritic cells and MUC1-positive carcinoma cells.

Active site mutant transgene confers tolerance to human β-glucuronidase without affecting the phenotype of MPS VII mice

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of β-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gusmps/mps) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human β-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gusmps/mps) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gusmps/mps) mouse. However, they are now tolerant to immune challenge with human β-glucuronidase. This “tolerant MPS VII mouse model” should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

Requirement for natural killer T (NKT) cells in the induction of allograft tolerance

In this study, we investigated the role of Vα14 natural killer T (NKT) cells in transplant immunity. The ability to reject allografts was not significantly different between wild-type (WT) and Vα14 NKT cell-deficient mice. However, in models in which tolerance was induced against cardiac allografts by blockade of lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions, long-term acceptance of the grafts was observed only in WT but not Vα14 NKT cell-deficient mice. Adoptive transfer with Vα14 NKT cells restored long-term acceptance of allografts in Vα14 NKT cell-deficient mice. The critical role of Vα14 NKT cells to mediate immunosuppression was also observed in vitro in mixed lymphocyte cultures in which lymphocyte function-associated antigen-1/intercellular adhesion molecule-1 or CD28/B7 interactions were blocked. Experiments using IL-4- or IFN-γ-deficient mice suggested a critical contribution of IFN-γ to the Vα14 NKT cell-mediated allograft acceptance in vivo. These results indicate a critical contribution of Vα14 NKT cells to the induction of allograft tolerance and provide a useful model to investigate the regulatory role of Vα14 NKT cells in various immune responses.

Peyer's patches are required for oral tolerance to proteins

To clarify the role of Peyer's patches in oral tolerance induction, BALB/c mice were treated in utero with lymphotoxin β-receptor Ig fusion protein to generate mice lacking Peyer's patches. When these Peyer's patch-null mice were fed 25 mg of ovalbumin (OVA) before systemic immunization, OVA-specific IgG Ab responses in serum and spleen were seen, in marked contrast to low responses in OVA-fed normal mice. Further, high T-cell-proliferative- and delayed-type hypersensitivity responses were seen in Peyer's patch-null mice given oral OVA before systemic challenge. Higher levels of CD4+ T-cell-derived IFN-γ, IL-4, IL-5, and IL-10 syntheses were noted in Peyer's patch-null mice fed OVA, whereas OVA-fed normal mice had suppressed cytokine levels. In contrast, oral administration of trinitrobenzene sulfonic acid (TNBS) to Peyer's patch-null mice resulted in reduced TNBS-specific serum Abs and splenic B cell antitrinitrophenyl Ab-forming cell responses after skin painting with picryl chloride. Further, when delayed-type hypersensitivity and splenic T cell proliferative responses were examined, Peyer's patch-null mice fed TNBS were unresponsive to hapten. Peyer's patch-null mice fed trinitrophenyl-OVA failed to induce systemic unresponsiveness to hapten or protein. These findings show that organized Peyer's patches are required for oral tolerance to proteins, whereas haptens elicit systemic unresponsiveness via the intestinal epithelial cell barrier.