Role of janus tyrosine kinase -3 (JAK3) and signal transducer and activator of transcription (STAT5) pathway in T lymphocyte activation

T cell activation and expansion is essential for immune response against foreign antigens. However, uncontrolled T cell activity can be manifested as a number of lymphoid derived diseases such as autoimmunity, graft versus host disease, and lymphoma. The purpose of this research was to test the central hypothesis that the Jak3/Stat5 pathway is critical for T cell function. To accomplish this objective, two novel Jak3 inhibitors, AG490 and PNU156804, were identified and their effects characterized on Jak3/Stat5 activation and T cell growth. Inhibition of Jak3 selectively disrupted primary human T lymphocyte growth in response to Interleukin-2 (IL-2), as well as other γ c cytokine family members including IL-4, IL-7, IL-9, and IL-15. Inhibition of Jak3 ablated IL-2 induced Stat5 but not TNF-α mediated NF-κβ DNA binding. Loss of Jak3 activity did not affect T cell receptor mediated signals including activation of p56Lck and Zap70, or IL-2 receptor a chain expression. To examine the effects of Jak3/Stat5 inhibition within a mature immune system, we employed a rat heart allograft model of Lewis (RT1 1) to ACI (RT1a). Heart allograft survival was significantly prolonged following Jak3/Stat5 inhibition when rats were treated with AG490 (20mg/kg) or PNU156804 (80mg/kg) compared to non-treated control animals. This effect was synergistically potentiated when Jak3 inhibitors were used in combination with a signal 1/2 disrupter, cyclosporine, but only additively potentiated with another signal 3 inhibitor, rapamycin. This suggested that sequential inhibition of T cell function is more effective. To specifically address the role of Stat5 in maintaining T cell activity, novel Stat5 antisense oligonucleotides were synthesized and characterized in vitro. Primary human T cells and T-cell tumor lines treated with Stat5 antisense oligonucleotide (7.5 μM) rapidly underwent apoptosis, while no changes in cell cycle were observed as measured by FACS analysis utilizing Annexin-V-Fluorescein and Propidium iodide staining. Evidence is provided to suggest that caspase 8 and 9 pathways mediate this event. Thus, Stat5 may act rather as a negative regulator of apoptotic signals and not as a positive regulator of cell cycle as previously proposed. We conclude that the Jak3/Stat5 pathway is critical for γc cytokine mediated gene expression necessary for T cell expansion and normal immune function and represents an therapeutically relevant effector pathway to combat T cell derived disease. ^

Microarray analysis of autoimmune diseases by machine learning procedures

—Microarray-based global gene expression profiling, with the use of sophisticated statistical algorithms is providing new insights into the pathogenesis of autoimmune diseases. We have applied a novel statistical technique for gene selection based on machine learning approaches to analyze microarray expression data gathered from patients with systemic lupus erythematosus (SLE) and primary antiphospholipid syndrome (PAPS), two autoimmune diseases of unknown genetic origin that share many common features. The methodology included a combination of three data discretization policies, a consensus gene selection method, and a multivariate correlation measurement. A set of 150 genes was found to discriminate SLE and PAPS patients from healthy individuals. Statistical validations demonstrate the relevance of this gene set from an univariate and multivariate perspective. Moreover, functional characterization of these genes identified an interferon-regulated gene signature, consistent with previous reports. It also revealed the existence of other regulatory pathways, including those regulated by PTEN, TNF, and BCL-2, which are altered in SLE and PAPS. Remarkably, a significant number of these genes carry E2F binding motifs in their promoters, projecting a role for E2F in the regulation of autoimmunity.

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.

A T cell receptor antagonist peptide induces T cells that mediate bystander suppression and prevent autoimmune encephalomyelitis induced with multiple myelin antigens

Experimental autoimmune encephalomyelitis (EAE) induced with myelin proteolipid protein (PLP) residues 139–151 (HSLGKWLGHPDKF) can be prevented by treatment with a T cell receptor (TCR) antagonist peptide (L144/R147) generated by substituting at the two principal TCR contact residues in the encephalitogenic peptide. The TCR antagonist peptide blocks activation of encephalitogenic Th1 helper cells in vitro, but the mechanisms by which the antagonist peptide blocks EAE in vivo are not clear. Immunization with L144/R147 did not inhibit generation of PLP-(139–151)-specific T cells in vivo. Furthermore, preimmunization with L144/R147 protected mice from EAE induced with the encephalitogenic peptides PLP-(178–191) and myelin oligodendrocyte protein (MOG) residues 92–106 and with mouse myelin basic protein (MBP). These data suggest that the L144/R147 peptide does not act as an antagonist in vivo but mediates bystander suppression, probably by the generation of regulatory T cells. To confirm this we generated T cell lines and clones from animals immunized with PLP-(139–151) plus L144/R147. T cells specific for L144/R147 peptide were crossreactive with the native PLP-(139–151) peptide, produced Th2/Th0 cytokines, and suppressed EAE upon adoptive transfer. These studies demonstrate that TCR antagonist peptides may have multiple biological effects in vivo. One of the principal mechanisms by which these peptides inhibit autoimmunity is by the induction of regulatory T cells, leading to bystander suppression of EAE. These results have important implications for the treatment of autoimmune diseases where there are autopathogenic responses to multiple antigens in the target organ.

Generation of CD8+ T cells specific for transporter associated with antigen processing deficient cells

Cells with impaired transporter associated with antigen processing (TAP) function express low levels of cell surface major histocompatibility complex (MHC) class I molecules, and are generally resistant to lysis by MHC class I restricted cytotoxic T lymphocytes (CTLs). Here we report the generation of MHC class I restricted CD8+ CTLs that surprisingly require target cell TAP deficiency for efficient recognition. C57BL/6 (B6) mice immunized with syngenic B7–1 (CD80) expressing TAP-deficient cells generated a potent CTL response against both TAP-deficient RMA-S tumor cells and TAP-deficient Con A blasts, whereas the corresponding TAP-expressing target cells were considerably less susceptible or resistant to lysis. The CTL epitopes recognized were expressed also by the human TAP-deficient cell line T2, transfected with appropriate MHC class I molecules. B6 mice immunized with B7–1-transfected TAP-deficient RMA-S cells were protected from outgrowth of a subsequent RMA-S tumor challenge. These findings are discussed in relation to the biochemical nature of MHC class I dependent CTL epitopes associated with impaired TAP function, as well as implications for immunotherapy and autoimmunity.

In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen

T cells recognizing poorly displayed self determinants escape tolerance mechanisms and persist in the adult repertoire. The process by which these T cells are primed is not clear, but once activated, they can cause autoimmunity. Here, we show that dendritic cells treated with interleukin 6 (IL-6) process and present determinants from a model native antigen in a qualitatively altered hierarchy, activating T cells in vitro and in vivo against determinants that were previously cryptic because of poor display. IL-6 does not induce conventional maturation of dendritic cells but alters the pH of peripheral, early endosomal compartments and renders the cells more susceptible to killing by chloroquine. Acidification of endosomes by ouabain mimics the effect of IL-6 and allows processing of the same cryptic determinant. These results suggest that cytokines such as IL-6 could initiate and help to propagate an autoimmune disease process by differentiating dendritic cells into a state distinct from that induced by normal maturation.

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.

The KMDB/MutationView: a mutation database for human disease genes

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med.keio.ac.jp.

Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse

Quantitative and qualitative defects in CD1-restricted natural killer T cells have been reported in several autoimmune-prone strains of mice, including the nonobese diabetic (NOD) mouse. These defects are believed to be associated with the emergence of spontaneous autoimmunity. Here we demonstrate that both CD1d-null NOD and CD1d-null NOD/BDC2.5 T cell receptor transgenic mice have an accelerated onset and increased incidence of diabetes when compared with CD1d+/− and CD1d+/+ littermates. The acceleration of disease did not seem to result from changes in the T helper (Th)1/Th2 balance because lymphocytes purified from lymphoid organs and pancreatic islets of wild-type and CD1d-null mice secreted equivalent amounts of IFN-γ and IL-4 after stimulation. In contrast, the pancreata of CD1d-null mice harbored significantly higher numbers of activated memory T cells expressing the chemokine receptor CCR4. Notably, the presence of these T cells was associated with immunohistochemical evidence of increased destructive insulitis. Thus, CD1d-restricted T cells are critically important for regulation of the spontaneous disease process in NOD mice.

Expression of a second receptor rescues self-specific T cells from thymic deletion and allows activation of autoreactive effector function.

Allelic exclusion at the T-cell receptor alpha chain locus is incomplete resulting in the generation of T cells that express two T-cell receptors. The potential involvement of such T cells in autoimmunity has been suggested [Padovan, E., Casorati, G., Dellabona, P., Meyer, S., Brockhaus, M. & Lanzavecchia, A. (1993) Science 262, 422-424; Heath, W. R. & Miller, J. F. A. P. (1993) J. Exp. Med. 178, 1807-1811]. Here we show that expression of a second T-cell receptor can rescue T cells with autospecific receptors from thymic deletion and allow their exit into the periphery. Dual receptor T cells, created by constitutive expression of two transgenic T-cell receptors on a Rag1-/- background, are tolerant to self by maintaining low levels of autospecific receptor, but selfreactive effector function (killing) can be induced through activation via the second receptor. This opens the possibility that T cells carrying two receptors in the periphery of normal individuals contain putatively autoreactive cells that could engage in autoimmune effector functions after recognition of an unrelated environmental antigen.

A common major histocompatibility complex class II allele HLA-DQB1* 0301 is present in clinical variants of pemphigoid.

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease seen primarily in elderly persons. It is characterized clinically by the development of tense bullae and by the presence of an antibasement membrane antibody. In BP, the antigens involved in the autoimmunity are epidermal basement membrane peptides BPAg1 and BPAg2. We have compared high resolution typing of major histocompatibility complex class II loci (HLA-DRB1, DQB1) in 21 patients with BP, 17 with ocular cicatricial pemphigoid (OCP), and 22 with oral pemphigoid (OP) to a panel of 218 haplotypes of normal individuals. We found that the three diseases (BP, OCP, and OP) have significant association with DQB1*0301 (P = 0.005, P < 0.0001, and P = 0.001, respectively). The frequencies of alleles DQB1*0302, 0303, and 06, which share a specific amino acid sequence from position 71 to 77 (Thr-Arg-Ala-Glu-Leu-Val-Thr) were also increased (P = 0.01). We suggest that an identical major histocompatibility complex class II allele (DQB1*0301) is a common marker for enhanced susceptibility and that the same amino acid residues in positions 71-77 (DQB1*0301, -0302, -0305, -0602, -0603 alleles) are found in patients with BP, OCP and OP. Our findings propose that the autoimmune response in the three different clinical variants of pemphigoid, involves the recognition by T cells of a class II region of DQB1, bound to a peptide from the basement membrane of conjunctiva, oral mucosa, and skin.

Insulin-secreting non-islet cells are resistant to autoimmune destruction.

Transgenic nonobese diabetic mice were created in which insulin expression was targeted to proopiomelanocortin-expressing pituitary cells. Proopiomelanocortin-expressing intermediate lobe pituitary cells efficiently secrete fully processed, mature insulin via a regulated secretory pathway, similar to islet beta cells. However, in contrast to the insulin-producing islet beta cells, the insulin-producing intermediate lobe pituitaries are not targeted or destroyed by cells of the immune system. Transplantation of the transgenic intermediate lobe tissues into diabetic nonobese diabetic mice resulted in the restoration of near-normoglycemia and the reversal of diabetic symptoms. The absence of autoimmunity in intermediate lobe pituitary cells engineered to secrete bona fide insulin raises the potential of these cell types for beta-cell replacement therapy for the treatment of insulin-dependent diabetes mellitus.