Asymmetric dimethylation and citrullination in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, and effects of anti-TCR/anti-TNF-α antibody-based therapy.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D). We determined by GC-MS the extent of asymmetric dimethylation (prADMA) and citrullination (prCit) of L-arginine residues in organ proteins (pr) of normoglycaemic control (ngCo, n = 6), acutely diabetic (acT1D, n = 6), chronically diabetic (chT1D, n = 4), and cured (cuT1D, n = 4) rats after anti-TCR/anti-TNF-α therapy. Pancreatic prCit and prADMA did not differ between the groups but were correlated (r = 0.728, P = 0.0003, n = 20). acT1D rats had lower prCit levels in spleen and kidney than ngCo rats. cuT1D rats had higher prADMA levels than chT1D rats only in the spleen. Combination therapy re-established normoglycaemia and increased prADMA in the spleen without altering pancreatic prADMA and prCit. Western blotting demonstrated the presence of different prADMA pattern, especially an ≈ 50-kDa prADMA in spleen and pancreas, and an ≈ 25-kDa prADMA in the pancreas only, with the kidney showing only a very faint and small prADMA. Besides the changes in the pancreas during different metabolic states, the spleen may play a stronger role for the recognition of metabolic changes in T1D than thought thus far.

Anti-TCRß mAb in Combination With Neurogenin3 Gene Therapy Reverses Established Overt Type 1 Diabetes in Female NOD Mice.

Insulin-producing ß cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR ß chain (TCRß) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRß mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRß mAb therapy alone reversed only ∼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRß mAb treatment, ∼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRß mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRß mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRß with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cell‒mediated destruction in mice with established overt diabetes.

The Effect of Inhibitory Signals on the Priming of Drug Hapten-Specific T Cells That Express Distinct Vß Receptors.

Drug hypersensitivity involves the activation of T cells in an HLA allele-restricted manner. Because the majority of individuals who carry HLA risk alleles do not develop hypersensitivity, other parameters must control development of the drug-specific T cell response. Thus, we have used a T cell-priming assay and nitroso sulfamethoxazole (SMX-NO) as a model Ag to investigate the activation of specific TCR Vß subtypes, the impact of programmed death -1 (PD-1), CTL-associated protein 4 (CTLA4), and T cell Ig and mucin domain protein-3 (TIM-3) coinhibitory signaling on activation of naive and memory T cells, and the ability of regulatory T cells (Tregs) to prevent responses. An expansion of the TCR repertoire was observed for nine Vß subtypes, whereas spectratyping revealed that SMX-NO-specific T cell responses are controlled by public TCRs present in all individuals alongside private TCR repertoires specific to each individual. We proceeded to evaluate the extent to which the activation of these TCR Vß-restricted Ag-specific T cell responses is governed by regulatory signals. Blockade of PD-L1/CTLA4 signaling dampened activation of SMX-NO-specific naive and memory T cells, whereas blockade of TIM-3 produced no effect. Programmed death-1, CTLA4, and TIM-3 displayed discrete expression profiles during drug-induced T cell activation, and expression of each receptor was enhanced on dividing T cells. Because these receptors are also expressed on Tregs, Treg-mediated suppression of SMX-NO-induced T cell activation was investigated. Tregs significantly dampened the priming of T cells. In conclusion, our findings demonstrate that distinct TCR Vß subtypes, dysregulation of coinhibitory signaling pathways, and dysfunctional Tregs may influence predisposition to hypersensitivity.

Inhibition of RORγT Skews TCRα Gene Rearrangement and Limits T Cell Repertoire Diversity.

Recent studies have elucidated the molecular mechanism of RORγT transcriptional regulation of Th17 differentiation and function. RORγT was initially identified as a transcription factor required for thymopoiesis by maintaining survival of CD4+CD8+ (DP) thymocytes. While RORγ antagonists are currently being developed to treat autoimmunity, it remains unclear how RORγT inhibition may impact thymocyte development. In this study, we show that in addition to regulating DP thymocytes survival, RORγT also controls genes that regulate thymocyte migration, proliferation, and T cell receptor (TCR)α selection. Strikingly, pharmacological inhibition of RORγ skews TCRα gene rearrangement, limits T cell repertoire diversity, and inhibits development of autoimmune encephalomyelitis. Thus, targeting RORγT not only inhibits Th17 cell development and function but also fundamentally alters thymic-emigrant recognition of self and foreign antigens. The analysis of RORγ inhibitors has allowed us to gain a broader perspective of the diverse function of RORγT and its impact on T cell biology.

Position-dependent silencing of germline Vß segments on TCRß alleles containing preassembled VßDJßCß1 genes.

The genomic organization of TCRbeta loci enables Vbeta-to-DJbeta2 rearrangements on alleles with assembled VbetaDJbetaCbeta1 genes, which could have deleterious physiologic consequences. To determine whether such Vbeta rearrangements occur and, if so, how they might be regulated, we analyzed mice with TCRbeta alleles containing preassembled functional VbetaDJbetaCbeta1 genes. Vbeta10 segments were transcribed, rearranged, and expressed in thymocytes when located immediately upstream of a Vbeta1DJbetaCbeta1 gene, but not on alleles with a Vbeta14DJbetaCbeta1 gene. Germline Vbeta10 transcription was silenced in mature alphabeta T cells. This allele-dependent and developmental stage-specific silencing of Vbeta10 correlated with increased CpG methylation and decreased histone acetylation over the Vbeta10 promoter and coding region. Transcription, rearrangement, and expression of the Vbeta4 and Vbeta16 segments located upstream of Vbeta10 were silenced on alleles containing either VbetaDJbetaCbeta1 gene; sequences within Vbeta4, Vbeta16, and the Vbeta4/Vbeta16-Vbeta10 intergenic region exhibited constitutive high CpG methylation and low histone acetylation. Collectively, our data indicate that the position of Vbeta segments relative to assembled VbetaDJbetaCbeta1 genes influences their rearrangement and suggest that DNA sequences between Vbeta segments may form boundaries between active and inactive Vbeta chromatin domains upstream of VbetaDJbetaCbeta genes.

TCR beta feedback signals inhibit the coupling of recombinationally accessible V beta 14 segments with DJ beta complexes.

Ag receptor allelic exclusion is thought to occur through monoallelic initiation and subsequent feedback inhibition of recombinational accessibility. However, our previous analysis of mice containing a V(D)J recombination reporter inserted into Vbeta14 (Vbeta14(Rep)) indicated that Vbeta14 chromatin accessibility is biallelic. To determine whether Vbeta14 recombinational accessibility is subject to feedback inhibition, we analyzed TCRbeta rearrangements in Vbeta14(Rep) mice containing a preassembled in-frame transgenic Vbeta8.2Dbeta1Jbeta1.1 or an endogenous Vbeta14Dbeta1Jbeta1.4 rearrangement on the homologous chromosome. Expression of either preassembled VbetaDJbetaC beta-chain accelerated thymocyte development because of enhanced cellular selection, demonstrating that the rate-limiting step in early alphabeta T cell development is the assembly of an in-frame VbetaDJbeta rearrangement. Expression of these preassembled VbetaDJbeta rearrangements inhibited endogenous Vbeta14-to-DJbeta rearrangements as expected. However, in contrast to results predicted by the accepted model of TCRbeta feedback inhibition, we found that expression of these preassembled TCR beta-chains did not downregulate recombinational accessibility of Vbeta14 chromatin. Our findings suggest that TCRbeta-mediated feedback inhibition of Vbeta14 rearrangements depends on inherent properties of Vbeta14, Dbeta, and Jbeta recombination signal sequences.

Self-peptides prolong survival in murine autoimmunity via reduced IL-2/IL-7-mediated STAT5 signaling, CD8 coreceptor, and V alpha 2 down-regulation.

Although the pathogenic role of B cells and CD4 T cells has been studied extensively, less is known about the role of CD8 T cells in autoimmunity and self-tolerance. To evaluate the role of CD8 T cells in autoimmunity and its modulation using self-peptides, we used mice expressing soluble OVA (sOVA) under control of the keratin-14 promoter. Spontaneous autoimmunity occurred when sOVA mice were crossed with OT-I mice, whose CD8 T cells carry a Valpha2/Vbeta5-transgenic TCR with specificity for the OVA(257-264) peptide. Eighty-three percent of OVA/OT-I mice died during the first 2 wk of life due to multiple organ inflammation. In contrast, preventive or therapeutic OVA(257-264) peptide injections induced a dose-dependent increase in survival. Healthy survivors exhibited reductions in peripheral CD8 T cells, CD8 coreceptor, and Valpha2 expression. Furthermore, CD8 T cells from healthy mice were anergic and could not be activated by exogenous IL-2. A block in IL-2/IL-7 signaling via the STAT5 pathway provided the basis for low surface expression of the CD8 coreceptor and failure of IL-2 to break CD8 T cell anergy. Thus, the soluble TCR ligand triggered multiple tolerance mechanisms in these sOVA/OT-I mice, making this treatment approach a potential paradigm for modulating human autoimmune diseases.

Inhibition of collagen-induced arthritis by DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2.

BACKGROUND: Arthritogenic T lymphocytes with common T cell receptor (TCR) Vbeta clonotypes, infiltrating in the articulars of rheumatoid arthritis (RA) patients, play a central role in the pathogenesis of RA. TCR Vbeta5.2 and TCR Vbeta8.2 are the main pathogenic T cell clonotypes in the course of collagen-induced arthritis (CIA) progression in Lewis rats. To investigate a TCR-based immunotherapy for RA, we constructed recombinant DNA vaccines encoding TCR Vbeta5.2 and TCR Vbeta8.2, and evaluated the inhibitive effects of the two vaccines on CIA rats. METHODS: Genes encoding TCR Vbeta5.2 and TCR Vbeta8.2 were amplified by RT-PCR from spleen lymphocytes of Lewis rats and cloned into the eukaryotic expression vector pTargeT. The expression of vaccines was confirmed by RT-PCR and immunohistochemistry. The inhibitive effects of the vaccines on articulars of CIA rats were assessed with arthritis index evaluation and histology. Interferon gamma (IFN-gamma) and interleukin (IL)-4 production by spleen lymphocytes were tested with enzyme-linked immunospot assay (ELISPOT) technique, the changes in peripheral CD4(+) and CD8(+) lymphocyte populations were tested by flow cytometry, and the level of anti-CII antibody in serum was assayed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Recombinant DNA vaccines pTargeT-TCR Vbeta5.2 and pTargeT-pTCR Vbeta8.2 were successfully constructed. Both vaccines inhibited CIA, which alleviated the arthritis index score (P < 0.05), decreased the level of IFN-gamma (P < 0.05), and reduced the ratio of CD4(+)/CD8(+) lymphocytes (P < 0.05) and the anti-CII antibody in serum (P < 0.05). In addition, the histological change in DNA-vaccinated rats was less serious than CIA rats. Compared to pTCR Vbeta 8.2 and pTCR Vbeta 5.2 groups, the group that was injected with a combination of the two vaccines showed stronger inhibitive effects on CIA than either individual vaccine. CONCLUSION: The recombinant plasmids pTargeT-TCR Vbeta5.2 and pTargeT-TCR Vbeta8.2 have obvious inhibatory effects on CIA rats and better effects could be achieved when the vaccines were used in combination.

Contrasting roles for Valpha14+ natural killer T cells in a viral model for multiple sclerosis.

Most natural killer (NK) T cells express an invariant Valpha14 T-cell receptor. To explore the contribution of NKT cells in an animal model for multiple sclerosis, Theiler's murine encephalomyelitis virus (TMEV) infection, TMEV-infected mice were treated with Valpha14 antibody. Treatment during the early stage of infection delayed the onset of demyelinating disease with higher interleukin-4 production, whereas administration during the late stage or weekly resulted in more severe demyelination with enhanced virus persistence. The effect of in vivo depletion of NKT cells differed depending on the stage of infection, suggesting contrasting roles for NKT cells over the disease course.

Anti-TCR antibody treatment activates a novel population of nonintestinal CD8 alpha alpha+ TCR alpha beta+ regulatory T cells and prevents experimental autoimmune encephalomyelitis.

CD8alphaalpha+CD4-TCRalphabeta+ T cells are a special lineage of T cells found predominantly within the intestine as intraepithelial lymphocytes and have been shown to be involved in the maintenance of immune homeostasis. Although these cells are independent of classical MHC class I (class Ia) molecules, their origin and function in peripheral lymphoid tissues are unknown. We have recently identified a novel subset of nonintestinal CD8alphaalpha+CD4-TCRalphabeta+ regulatory T cells (CD8alphaalpha Tregs) that recognize a TCR peptide from the conserved CDR2 region of the TCR Vbeta8.2-chain in the context of a class Ib molecule, Qa-1a, and control- activated Vbeta8.2+ T cells mediating experimental autoimmune encephalomyelitis. Using flow cytometry, spectratyping, and real-time PCR analysis of T cell clones and short-term lines, we have determined the TCR repertoire of the CD8alphaalpha regulatory T cells (Tregs) and found that they predominantly use the TCR Vbeta6 gene segment. In vivo injection of anti-TCR Vbeta6 mAb results in activation of the CD8alphaalpha Tregs, inhibition of the Th1-like pathogenic response to the immunizing Ag, and protection from experimental autoimmune encephalomyelitis. These data suggest that activation of the CD8alphaalpha Tregs present in peripheral lymphoid organs other than the gut can be exploited for the control of T cell-mediated autoimmune diseases.

A role for MAPK in feedback inhibition of Tcrb recombination.

The Tcrb locus is subject to a host of regulatory mechanisms that impart a strict cell and developmental stage-specific order to variable (V), diversity (D), and joining (J) gene segment recombination. The Tcrb locus is also regulated by allelic exclusion mechanisms, which restrict functional rearrangements to a single allele. The production of a functional rearrangement in CD4-CD8- double-negative (DN) thymocytes leads to the assembly of a pre-TCR and initiates signaling cascades that allow for DN to CD4+CD8+ double-positive (DP) differentiation, proliferation, and feedback inhibition of further Vbeta to DJbeta rearrangement. Feedback inhibition is believed to be controlled, in part, by the loss of Vbeta gene segment accessibility during the DN to DP transition. However, the pre-TCR signaling pathways that lead to the inactivation of Vbeta chromatin have not been determined. Because activation of the MAPK pathway is documented to promote DP differentiation in the absence of allelic exclusion, we characterized the properties of Vbeta chromatin within DP thymocytes generated by a constitutively active Raf1 (Raf-CAAX) transgene. Consistent with previous reports, we show that the Raf-CAAX transgene does not inhibit Tcrb recombination in DN thymocytes. Nevertheless, DP thymocytes generated by Raf-CAAX signals display normal down-regulation of Vbeta segment accessibility and normal feedback inhibition of the Vbeta to DJbeta rearrangement. Therefore, our results emphasize the distinct requirements for feedback inhibition in the DN and DP compartments. Although MAPK activation cannot impose feedback in DN thymocytes, it contributes to feedback inhibition through developmental changes that are tightly linked to DN to DP differentiation.

Hypervariable region 1 variant acting as TCR antagonist affects hepatitis C virus-specific CD4+ T cell repertoire by favoring CD95-mediated apoptosis.

We have described previously that hypervariable region 1 (HVR1) variants of hepatitis C virus (HCV) frequently act as T cell receptor (TCR) antagonists for HVR1-specific helper T cells. These naturally occurring HVR1-antagonistic sequences interfered with the effects of HVR1-agonistic sequences such as TCR down-regulation and early activatory signals. By taking advantage of these findings, in this paper, we have analyzed the fate of these HVR1-specific antagonized CD4+ T cells. We present the evidence that TCR antagonism renders agonist-activated T cells susceptible to bystander CD95-mediated killing by suppressing the expression of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitor proteins. To verify whether the TCR repertoire of a HVR1-specific T cell population could be modified consequently, we used a HVR1-agonistic sequence to induce in vitro CD4+ T cells and another HVR1 sequence with antagonistic property to mediate suppressive phenomena. HVR1-specific T cells were cultured with the agonist alone or with the agonist plus the antagonist. HVR1 specificity and T cell repertoires were followed over time by analyzing TCR beta-variable gene segment by "spectratyping". The results showed that the specificity for the agonist was rapidly spoiled after culture in the presence of the antagonist, and the TCR repertoire was strongly modified as a result of CD95-mediated apoptosis of agonist-specific clonal expansions. These data support the hypothesis that in HCV infection, the generation of TCR antagonists may reshape the T cell repertoire, representing an efficacious immune evasion strategy of a highly mutant pathogen.

Sustained pre-TCR expression in Notch1IC-transgenic rats impairs T cell maturation and selection.

Notch1 is involved in directing cell fate decisions in a variety of developmental scenarios. Extending previous experiments in mice, we generated transgenic rats expressing the intracellular domain of Notch1 in the thymus. Importantly, this leads to sustained expression of the pre-TCR throughout thymocyte development, accompanied by a reduction of alphabetaTCR complexes. In addition, re-expression of RAG-1 and RAG-2 in TCRbeta(+) cells is impaired, and the Valpha repertoire is altered. Consequently, thymocytes in transgenic rats do not undergo positive selection and largely fail to progress to the single positive stage. According to our model, the previously reported effects of Notch1 on the CD4/CD8 cell fate decision may be explained by a differential sensitivity of the two lineages toward altered TCR signaling.

Impairment of thymocyte development by dominant-negative Kuzbanian (ADAM-10) is rescued by the Notch ligand, delta-1.

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.

Exclusion and inclusion of TCR alpha proteins during T cell development in TCR-transgenic and normal mice.

Allelic exclusion of immune receptor genes (and molecules) is incompletely understood. With regard to TCRalphabeta lineage T cells, exclusion at the tcr-b, but not tcr-a, locus seems to be strictly controlled at the locus rearrangement level. Consequently, while nearly all developing TCRalphabeta thymocytes express a single TCRbeta protein, many thymocytes rearrange and express two different TCRalpha chains and, thus, display two alphabetaTCRs on the cell surface. Of interest, the number of such dual TCR-expressing cells is appreciably lower among the mature T cells. To understand the details of TCR chain regulation at various stages of T cell development, we analyzed TCR expression in mice transgenic for two rearranged alphabetaTCR. We discovered that in such TCR double-transgenic (TCRdTg) mice peripheral T cells were functionally monospecific. Molecularly, this monospecificity was due to TCRalpha exclusion: one transgenic TCRalpha protein was selectively down-regulated from the thymocyte and T cell surface. In searching for the mechanism(s) governing this selective TCRalpha down-regulation, we present evidence for the role of protein tyrosine kinase signaling and coreceptor involvement. This mechanism may be operating in normal thymocytes.

Immunogenetic susceptibility of atherosclerotic stroke: implications on current and future treatment of vascular inflammation.

The understanding of the pathophysiology governing atherosclerosis supports a prominent role for inflammation pathways in plaque initiation and progression that result in stroke and myocardial infarction. Elevated levels of inflammatory markers in the blood, such as C-reactive protein and CD40 ligand/CD40, in concert with increased expression of adhesion molecules, chemokines, cytokines, matrix metalloproteinases (MMP), and inflammatory cells in the plaque, characterize the symptomatic atherothrombotic state. Advances in predictive capabilities of vascular events using a number of these biomarkers are beginning to remodel our clinical practice in the use of medications such as statins and angiotensin receptor blockers for stroke prevention. Although the general inflammatory features of atherosclerosis are becoming widely recognized, factors resulting in individual variability in plaque formation and instability remain poorly defined. Emerging literature points toward several acquired and innate susceptibility factors in the immune pathways that may provide insight into why many plaques rapidly evolve from a "stable" to an "unstable" or symptomatic state. First, exposure of plaque memory T-lymphocytes to infectious or endogenous antigens may result in rapid clonal expansion of T-cell variable beta chain subtypes and stimulate macrophages to release MMPs, causing plaque destabilization. The effects of infectious agents can further be influenced by an individual's major histocompatibility complex class II molecule profiles, which can affect susceptibility to specific organisms. Second, functional polymorphisms of genes that regulate the immune pathway can predispose patients to a more robust inflammatory expression after risk factor exposure. Identification of a susceptibility gene profile and immunologic mediators that promote T-cell activation provides a unique opportunity for early identification of stroke risk and targets for future therapy.

Antigen-specific T cell repertoire modification of CD4+CD25+ regulatory T cells.

T cell immune responses are regulated by the interplay between effector and suppressor T cells. Immunization with Ag leads to the selective expansion and survival of effector CD4(+) T cells with high affinity TCR against the Ag and MHC. However, it is not known if CD4(+)CD25(+) regulatory T cells (T(reg)) recognize the same Ag as effector T cells or whether Ag-specific TCR repertoire modification occurs in T(reg). In this study, we demonstrate that after a primary Ag challenge, T(reg) proliferate and TCR repertoire modification is observed although both of these responses were lower than those in conventional T cells. The repertoire modification of Ag-specific T(reg) after primary Ag challenge augmented the total suppressive function of T(reg) against TCR repertoire modification but not against the proliferation of memory CD4(+) T cells. These results reveal that T cell repertoire modification against a non-self Ag occurs in T(reg), which would be crucial for limiting excess primary and memory CD4(+) T cell responses. In addition, these studies provide evidence that manipulation of Ag-specific T(reg) is an ideal strategy for the clinical use of T(reg).

Epicutaneous sensitization with superantigen induces allergic skin inflammation.

BACKGROUND: Atopic dermatitis (AD) is characterized by skin infiltration with eosinophils and lymphocytes and expression of Th2 cytokines in acute skin lesions. The skin of patients with AD is frequently colonized with enterotoxin-secreting strains of Staphylococcus aureus. Staphylococcal enterotoxins have been implicated in the exacerbations of the inflammatory skin lesions in patients with AD. OBJECTIVE: We sought to determine whether epicutaneous (EC) sensitization of mice with staphylococcal enterotoxin B (SEB) results in allergic skin inflammation. METHODS: BALB/c mice were EC-sensitized with SEB. Their skin was examined for allergic inflammation and cytokine expression, and their splenocytes were examined for cytokine secretion in response to SEB. RESULTS: EC sensitization with SEB elicited a local, cutaneous, inflammatory response characterized by dermal infiltration with eosinophils and mononuclear cells and increased mRNA expression of the Th2 cytokine IL-4 but not of the Th1 cytokine IFN-gamma. EC-sensitized mice mounted a systemic Th2 response to SEB evidenced by elevated total and SEB-specific IgG1 and IgE. Although EC sensitization with SEB resulted in selective depletion of SEB-specific T-cell receptor Vbeta8+ cells from the spleen and sensitized skin, splenocytes from SEB-sensitized mice secreted relatively more IL-4 and less IFN-gamma than did saline-sensitized controls, consistent with Th2 skewing of the systemic immune response to the superantigen. CONCLUSION: These results suggest that EC exposure to superantigens skews the immune response toward Th2 cells, leading to allergic skin inflammation and increased IgE synthesis that are characteristic of AD.

TCR comodulation of nonengaged TCR takes place by a protein kinase C and CD3 gamma di-leucine-based motif-dependent mechanism.

One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.