anti-CD3 Cross-linking Research Articles

Immune dysregulation caused by homozygous mutations in CBLB.

CBL-B is an E3 ubiquitin ligase that ubiquitinates proteins downstream of immune receptors to downregulate positive signaling cascades. Distinct homozygous mutations in CBLB were identified in 3 unrelated children with early-onset autoimmunity, one of whom also had chronic urticaria. Patient T cells exhibited hyperproliferation in response to anti-CD3 cross-linking. One of the mutations, p.R496X, abolished CBL-B expression, and a second mutation, p.C464W, resulted in preserved CBL-B expression. The third mutation, p.H285L in the SH2 domain of CBL-B, was expressed at half the normal level in the patient’s cells. Mice homozygous for the CBL-B p.H257L mutation, which corresponds to the patient’s p.H285L mutation, had T and B cell hyperproliferation in response to antigen receptor cross-linking. CblbH257L mice had increased percentages of T regulatory cells (Tregs) that had normal in vitro suppressive function. However, T effector cells from the patient with the p.H285L mutation and CblbH257L mice were resistant to suppression by WT Tregs. Bone marrow–derived mast cells from CblbH257L mice were hyperactivated after FcεRI cross-linking, and CblbH257L mice demonstrated exaggerated IgE-mediated passive anaphylaxis. This study establishes CBL-B deficiency as a cause of immune dysregulation.

Anti-CD3 Bi-Fab crosslinking of T cells enables their fratricide by activation induced cell death (AICD) and cytotoxicity

Abstract We showed in the past anti-CD3 Fabs can form stable bivalent non-covalent dimers we called Bi-Fabs, which appear stimulatory to T cells according to the up-regulation of the early activation marker CD69. Now we show that incubation of naïve T cells with anti-CD3 Bi-Fabs in the presence of proper co-stimulation results in their robust stimulation in comparison to mAb and F(ab’)2 species with same specificity. Nonetheless, anti-CD3 Bi-Fab treatment leads to abortive T cell proliferation with impaired accumulation of divided T cells. After detailed studies to dissect the mechanisms of T cell death enabled by Bi-Fab molecules in comparison with anti-CD3 mAb and F(ab′)2 recognizing the same epitope, we observed that anti-CD3 Bi-Fabs promote T cell fratricidal killing via activation induced cell death (AICD) and CD8 cytolytic activity (CTL) more efficiently than the mAb and F(ab′)2 counterparts. While low resolution structural studies indicate that Bi-Fab and F(ab’)2 adopt similar shapes, molecular dynamic simulation shows that F(ab’)2 is a highly flexible molecule, whereas Bi-Fabs are more rigid and of linear conformation, sampling a smaller range of Fab-Fab angles than F(ab’)2. We hypothesize anti-CD3 Bi-Fab is superior as T cell killer stimulatory modality over mAb and F(ab’)2 of equal specificity due to Bi-Fab’s greater rigidity when crosslinking in trans TCR/CD3 molecules located in neighboring T cells. These findings may be applicable into the design of Ig-based immunotherapeutic drugs targeting the TCR/CD3 complex. Supported by MU start-up funds

Dynamic interaction of PSGL-1 with the T cell receptor regulates the extent of T cell activation

Abstract We previously identified PSGL-1 (P-selectin glycoprotein-1) as a T cell-intrinsic, cell surface checkpoint inhibitor during chronic viral infection. In this model, we found that cross-linking PSGL-1 with antibody reduced signaling through the T cell receptor (TCR) in exhausted T cells and further upregulated PD-1 expression. However, the function of PSGL-1 in the early activation of naïve T cells has not been fully investigated. We found that, crosslinking PSGL-1 on naïve T cells augments TCR signaling, including enhanced Erk and Akt phosphorylation and Nur77 expression. In addition, T cells expressed more activation markers, including PD-1, proliferated to a greater extent, and secreted more cytokines when PSGL-1 was crosslinked compared to TCR stimulation alone, indicating a role as a costimulatory molecule. Strikingly, we have discovered that PSGL-1 co-localizes with the TCR upon stimulation. CD3 and PSGL-1 can be found in the same micro vesicles following anti-CD3 crosslinking, indicating a close connection between PSGL-1 and the TCR. However, the effects of PSGL-1 ligation are similar to what is seen in T cells that lack PSGL-1 expression, except with respect to PD-1 expression. We propose that decreased induction of PD-1 with PSGL-1 deficiency supports greater effector T cell responses. Further studies will address the function of PSGL-1 in cytoskeletal rearrangement and the formation of the immune synapse. Altogether, these data indicate that the extent of T cell activation is regulated by an association of PSGL-1 with the TCR.

Testing for HLA/peptide tetramer-binding to the T cell receptor complex on human T lymphocytes

HLA/peptide tetramers are frequently used for ex vivo monitoring of disease- or vaccine-induced T cell immune responses and for T cell epitope identification. However, when low-levels HLA/peptide tetramer-positive T cell populations are encountered, it is difficult to ascertain whether this represents a true T cell receptor (TCR)-mediated interaction or background signal. To address this issue, we have developed a method for both HLA class I and class II tetramer assays to confirm tetramer-binding to the TCR/CD3 complex. Preincubation of T cells with anti-CD3 mAb SPV-T3b and subsequent crosslinking interferes with the binding of HLA/peptide tetramers to the TCR/CD3 complex and thereby indicates to what extent HLA/peptide tetramer binds through interaction with TCR/CD3 complex. SPV-T3b pretreatment results in a 2- to 10-fold decrease in tetramer-binding intensity to antigen-specific CD8+ or CD4+ T cells, whereas background reactivity of HLA/peptide tetramers containing HIV-derived peptide in HIV-negative donors remained unchanged. SPV-T3b pretreatment forms a valuable tool to verify tetramer-based detection of antigen-specific T cells during the monitoring of immune responses in clinical studies.

Exogenous Immunoglobulin Downregulates T Cell Receptor Signaling and Cytokine Production

BACKGROUND: Immune globulin, a polyvalent solution of pooled human serum immunoglobulin, when administered intravenously (IGIV) is used as replacement therapy in patients with hypogammaglobulinemia. In addition, IGIV has immunosuppressive properties and is FDA-approved to treat idiopathic thrombocytopenic purpura; it has also been used to treat several other autoimmune diseases including autoimmune hemolytic anemia and Kawasaki Disease. The mechanisms of action underlying IGIVs immunomodulatory effects are poorly understood, although a few reports have shown IGIV can inhibit T cell activation-induced upregulation of CD38 and CD69 in vitro and variably affect cytokine secretion by Th1 and Th2 cells. We hypothesized that the immunosuppressive effects of IGIV might be lymphocyte subset specific and mediated through T cell antigen receptor signaling interference, thus modulating Th1/Th2 cytokine production.METHODS: Mature (CD45RO) and naïve (CD45RA) lymphocytes from adult and umbilical cord blood were cultured for 8 days in the presence of IGIV (Gamunex™, 0–2.0 mg/mL). T, B, and natural killer (NK) lymphocyte subsets were then quantified by flow cytometry (FACS) over a time course. Additionally, the integrity of the T cell antigen receptor signaling pathway was assessed by FACS, quantifying expression of CD25 and CD69 following anti-CD3 cross-linking. Finally, multiplexed microsphere-based immunoassays were used to quantify cytokine secretion from IGIV-treated cells.RESULTS: IGIV, in a dose-dependent manner, reduced lymphocyte proliferation up to 3-fold after 6 days in culture in both cord and adult blood. IGIV also decreased activation-induced upregulation of CD25 and CD69, with the highest concentrations of IGIV leading to a 70% reduction in cells with an activated phenotype. This inhibitory effect was sustained over a 7-day period in both CD4 and CD8 T cell subsets and was independent of maturation markers (CD45RA/CD45RO). The percentage and absolute number of NK cells varied with IGIV treatment, but a marked increase was observed by day 6. IGIV also selectively reduced the absolute number and percentage of CD45RO+ T cells in cord blood over a time course. Direct cell surface Ig binding was not detected on T cell subsets. IGIV, in a dose-dependent manner, significantly decreased Th2 cytokine secretion, specifically IL-5, IL-10, and IL-13. This effect was more pronounced in cord blood lymphocytes but also was observed in adult blood lymphocytes. IGIV treatment also decreased Th1 cytokine secretion, particularly IL-2 and IFN-γ.CONCLUSIONS: IGIV inhibits T cell proliferation and activation as well as Th1 and Th2 cytokine secretion in a dose- and time-dependent manner. These effects may be responsible for the immunophenotypic changes observed—specifically a reduction in mature T cells and expansion of NK cells. While it is not entirely clear how IGIV is mediating its effects, the inhibition of Th1 and Th2 cytokine production and reduction of T cell activation signals in vitro may explain its role in blocking autoimmune responses in the clinical setting. Further studies are required to identify more precisely the molecular target and mechanism of action of IGIV in vivo.

Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase

A small population of plasmacytoid DCs (pDCs) in mouse tumor-draining LNs can express the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO). We show that these IDO+ pDCs directly activate resting CD4+CD25+Foxp3+ Tregs for potent suppressor activity. In vivo, Tregs isolated from tumor-draining LNs were constitutively activated and suppressed antigen-specific T cells immediately ex vivo. In vitro, IDO+ pDCs from tumor-draining LNs rapidly activated resting Tregs from non-tumor-bearing hosts without the need for mitogen or exogenous anti-CD3 crosslinking. Treg activation by IDO+ pDCs was MHC restricted, required an intact amino acid-responsive GCN2 pathway in the Tregs, and was prevented by CTLA4 blockade. Tregs activated by IDO markedly upregulated programmed cell death 1 ligand 1 (PD-L1) and PD-L2 expression on target DCs, and the ability of Tregs to suppress target T cell proliferation was abrogated by antibodies against the programmed cell death 1/PD-L (PD-1/PD-L) pathway. In contrast, Tregs activated by anti-CD3 crosslinking did not cause upregulation of PD-Ls, and suppression by these cells was unaffected by blocking the PD-1/PD-L pathway. Tregs isolated from tumor-draining LNs in vivo showed potent PD-1/PD-L-mediated suppression, which was selectively lost when tumors were grown in IDO-deficient hosts. We hypothesize that IDO+ pDCs create a profoundly suppressive microenvironment within tumor-draining LNs via constitutive activation of Tregs.

Cytotoxic T lymphocytes express intracellular stores of FasL via mechanisms distinct from cytolytic granule exocytosis (B16)

Abstract Cytotoxic T lymphocytes (CTL) lyse target cells through the release of cytolytic granules and cell surface expression of Fas ligand (FasL). We found that there is prestored FasL pool in CTL, which underwent rapid TCR-regulated cell surface expression upon target cell engagement. Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same control as degranulation. The aim of the current study is to investigate if degranulation of cytolytic granules and FasL expression pathways of CTL are controlled by differential mechanisms. FasL cell surface expression and degranulation, as measured by CD107a cell surface expression in CTLs were compared by flow cytometry under different conditions. FasL underwent an immediate and transient cell surface delivery following either TCR stimulation by anti-CD3 antibody crosslinking, or phorbol ester treatment, conditions that do not permit degranulation. Furthermore, treatment with pharmacological reagents indicated that the preformed FasL cell surface transport is PI3-kinase (PI3K)-, calcineurin- and microtubule-independent, which are all indispensable for degranulation. Finally, stored FasL resides in vesicles distinct from cytolytic granules as assessed by confocal microscopy and subcellular fractionation. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to regulation and localization. This work was funded by the Canadian Institutes of Health Research (CIHR).

Lenalidomide (CC5013, Revlimid®) Promotes T Helper-1 Function Bias and Increases Responsiveness to Autologous Bone Marrow Antigens in Myelodysplastic Syndrome (MDS).

Background: The myelodysplastic syndromes (MDS) comprise a spectrum of stem cell malignancies with natural histories that vary from indolent mild cytopenias to rapid transformation to acute leukemia. MDS patients have impaired T cell antigen-induced proliferation and reduced T helper-1 (Th-1) cytokine production. Lenalidomide, an immuno-modulatory drug structurally-related to thalidomide, is FDA-approved for the treatment of MDS with chromosome 5q deletion; however, its mechanism of action is not fully characterized. We hypothesize that immune modulation by lenalidomide will be an effective adjunct to vaccine therapy for patients with MDS.Methods: The immunoregulatory effects of lenalidomide were investigated both in vitro and in vivo. Peripheral blood mononuclear cells (PBMCs) from MDS patients and normal controls were stimulated with anti-CD3 cross-linking, allogeneic dendritic cells (allo-DCs), autologous dendritic cells (auto-DCs), and patient-derived autologous bone marrow mononuclear cells (BM-MNC) as antigen sources in the presence of DMSO (vehicle control) and lenalidomide [0.625 μM to 40 μM]. Proliferation of specific CD4+ and CD8+ T cell populations was assessed by Brdu incorporation and intracellular cytokine production by flow cytometry. Preliminary studies were performed to examine the combined effects of the GMCSF/K562 “bystander” vaccine (gift of Dr. I. Borrello, Johns Hopkins University) and lenalidomide on antigen-induced T cell proliferation in PBMC from both normal donors and MDS patients.Results: Lenalidomide augmented a Th-1-biased cytokine (IFN-γ, TNF-α and IL-2) response from normal donors (n=5) and MDS patients (n=5). The Th-1-biased increase in cytokine production accompanied erythroid response in MDS patients treated with 10 mg of lenalidomide for 16 weeks (n=4 responders and 3 non-responders) (List et al, NEJM 2005; 351:549). Augmentation of antigen-dependent proliferation accompanied cytokine responses both in vitro and in vivo. Next, we examined the effects of lenalidomide on in vitro response to autologous and allogeneic antigens. We found that pre-treatment T cell proliferation in response to auto-DC priming was not distinguishable from background. However, proliferation in response to auto-BM-MNCs used as a source of autologous tumor antigens was significantly increased by lenalidomide in CD3+, CD4+, and CD8+ T cell populations (P=0.002, 0.04, and 0.04, respectively). Proliferation after allo-DC exposure was also significantly enhanced by lenalidomide treatment (P<0.05). GMCSF/K562 “bystander” vaccine-increased proliferation to allo-DC antigens in CD4+ and CD8+ T cells without exposure to lenalidomide (n=4) (167% increase vs. 245% increase, respectively). When allo-DC-stimulated PBMCs were treated with lenalidomide alone, CD4+ and CD8+ proliferation was increased by 47% and 39% respectively. The combination of lenalidomide and the GMCSF/K562 vaccine further enhanced T cell proliferation to allo-DC stimulation (325% and 397% for CD4+ and CD8+ populations, respectively).Conclusion: Lenalidomide significantly augments T cell immune function in MDS, and potentiates immune response to the GMCSF/K562 “bystander” vaccine. We conclude that lenalidomide represents an attractive adjunct to vaccines for clinical investigation in MDS.

Activin A Functions as a Th2 Cytokine in the Promotion of the Alternative Activation of Macrophages

Activin A, a member of the TGF-beta superfamily, is a pluripotent growth and differentiation factor. In this study, we report that murine Th cells produce activin A upon activation. Activin activity in the cultured CD4+ T cells was induced by anti-CD3 cross-linking. Activin betaA mRNA level was increased in response to activation, indicating that activin production in CD4+ T cells is regulated at the mRNA level. Activin production was detected exclusively in CD4+CD25- T cells, but not in CD4+CD25+ regulatory T cells. When CD4+ T cells were differentiated into Th cell subsets, higher activin secretion was detected when cultured under Th2-skewing conditions. The mRNA level of activin betaA was abundant in Th2, but not in Th1 cells. Furthermore, secretion of activin was significantly higher in activated Th2 clones than in Th1 clones. The activin betaA-proximal promoter contains a binding site for c-Maf, a Th2-specific transcriptional factor, at close proximity with an NF-AT binding site. c-Maf was able to synergize with NF-AT to transactivate activin betaA gene, and both factors are implicated in activin betaA transcription in Th2 cells. Activin A induced macrophages to express arginase-1 (M-2 phenotype), whereas it inhibited inducible NO synthase expression (M-1 phenotype) induced by IFN-gamma. Taken together, these observations suggest that activin A is a novel Th2 cytokine that promotes differentiation of macrophages toward the M-2 phenotype.

Golli Protein Negatively Regulates Store Depletion-Induced Calcium Influx in T Cells

Calcium influx is crucial for T cell activation and differentiation. The detailed regulation of this process remains unclear. We report here that golli protein, an alternatively spliced product of the myelin basic protein gene, plays a critical role in regulating calcium influx in T cells. Golli-deficient T cells were hyperproliferative and showed enhanced calcium entry upon T cell receptor stimulation. We further found that golli regulates calcium influx in T cells through the inhibition of the store depletion-induced calcium influx. Mutation of the myristoylation site on golli disrupted its association with the plasma membrane and reversed its inhibitory action on Ca2+ influx, indicating that membrane association of golli was essential for its inhibitory action. These results indicate that golli functions in a unique way to regulate T cell activation through a mechanism involving the modulation of the calcium homeostasis.

Altered proximal T cell receptor (TCR) signaling in human CD4+CD25+ regulatory T cells

CD4+CD25+ regulatory T cells play an important role in peripheral tolerance. Upon T cell receptor (TCR)-mediated activation, the cells fail to proliferate but are induced to have a suppressor function. The intracellular signaling events that lead to their responses have not been elucidated. In this study, we have examined the proximal TCR signaling events in freshly isolated human CD4+CD25+ regulatory T cells after TCR ligation. In contrast to CD4+CD25- T cells, TCR ligation of CD4+CD25+ regulatory T cells by anti-CD3 cross-linking resulted in a lower calcium influx and extracellular signal-regulated kinase 1/2 phosphorylation. Examination of the CD3zeta chain phosphorylation status indicated that CD4+CD25+ regulatory T cells have poor phosphorylation of the protein and consequently, reduced recruitment of zeta-associated protein-70 to the TCR immunoreceptor tyrosine motif. The adaptor protein, Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa, which relays signals to downstream signaling components, also showed reduced phosphorylation, which correlated with reduced VAV guanine nucleotide exchange factors association. Consistent with other findings, the defect is accompanied with impaired actin cap formation, implicating a failure of actin remodeling of the cells. Together, our results demonstrate that CD4+CD25+ regulatory T cells have altered TCR proximal signaling pathways, which could be critical for inducing the distinct behavior of these cells.

T-cell contact-dependent regulation of CC and CXC chemokine production in monocytes through differential involvement of NFκB: implications for rheumatoid arthritis

We and others have reported that rheumatoid arthritis (RA) synovial T cells can activate human monocytes/macrophages in a contact-dependent manner to induce the expression of inflammatory cytokines, including tumour necrosis factor alpha (TNFα). In the present study we demonstrate that RA synovial T cells without further activation can also induce monocyte CC and CXC chemokine production in a contact-dependent manner. The transcription factor NFκB is differentially involved in this process as CXC chemokines but not CC chemokines are inhibited after overexpression of IκBα, the natural inhibitor of NFκB. This effector function of RA synovial T cells is also shared by T cells activated with a cytokine cocktail containing IL-2, IL-6 and TNFα, but not T cells activated by anti-CD3 cross-linking that mimics TCR engagement. This study demonstrates for the first time that RA synovial T cells as well as cytokine-activated T cells are able to induce monocyte chemokine production in a contact-dependent manner and through NFκB-dependent and NFκB-independent mechanisms, in a process influenced by the phosphatidyl-inositol-3-kinase pathway. Moreover, this study provides further evidence that cytokine-activated T cells share aspects of their effector function with RA synovial T cells and that their targeting in the clinic has therapeutic potential.

The RhoH GTPase Interacts with ZAP-70 Kinase and Is Required for T Cell Receptor Signaling and T Cell Development.

Engagement of the T cell receptor (TCR) by antigen/major histocompatibility complex initiates a signaling cascade involving activation and recruitment of multiple key intracellular proteins, including the zeta-chain-associated protein 70 (ZAP-70). ZAP-70-deficient mice show thymocyte developmental arrest and mutations of ZAP-70 are associated with severe combined immunodeficiency in humans. However, regulation of these early biochemical processes is not completely understood. RhoH, first identified as a hypermutable gene in non-Hodgkin's lymphomas, belongs to the family of Rho guanosine triphosphatases (Rho GTPases), which are recognized as critical mediators of signaling pathways regulating cell growth, differentiation and function (Gu et al., Science 302: 445-9). The expression of RhoH is restricted to hematopoietic cells, and using in vitro protein-binding and immunoprecipitation assays, we found that RhoH interacts with ZAP-70 in the TCR complex. Therefore, to understand the role of this highly expressed protein in T cell development and function, we have generated RhoH-deficient mice using homologous recombination.RhoH-/- mice are viable and normal in body size. However, the thymuses of 4–6 week-old RhoH-/- mice are significantly smaller and show a 3-fold decrease in cellularity compared with those from WT littermates. RhoH-/- thymocyte development is severely blocked at the CD4+ CD8+ double positive (DP) stage, resulting in markedly fewer CD4 or CD8 single positive (SP) T cells in RhoH-/- thymus (CD4 SP: 10.5+/− 1.6 vs. 1.1+/−0.2; CD8 SP: 6.8+/−0.7 vs. 0.8+/−0.1, cell number x106, WT vs. RhoH−/−, n≥15 mice, p<0.001). Numbers of peripheral T cells are also significantly reduced in RhoH−/− mice. RhoH DP thymocytes exhibit reduced surface expression of TCRb, but normal levels of TCRb mRNA. Further, the few circulating T cells in RhoH−/− mice exhibit normal expression of TCRb. These data suggest that reduced TCRb surface expression is a result, rather than a cause of the developmental arrest. In addition, expression of the activation marker, CD5, is severely reduced on RhoH−/− DP thymocytes (mean fluorescence intensity: 65.3+/−4.7 vs. 7.2+/−0.9, WT vs. RhoH−/−, n≥9 mice, p<0.01). These phenotypes are similar to ZAP-70-deficient mice and suggest that the thymocyte developmental arrest is due to defective TCR signaling pathway in RhoH−/− mice.To determine the potential role of RhoH in TCR signaling, we studied proliferative responses to anti-CD3 antibody cross-linking of RhoH−/− spleen T cells. RhoH−/− cells show significantly defective proliferation (3H-thymidine incorporation: 10.5+/−0.9 vs. 2.1+/−0.2, cpm x103, WT vs. RhoH−/−, n=6, p<0.01) as well as impaired survival (% viable cells: 27+/−2 vs. 6+/−1, WT vs. RhoH−/−, n=3, p<0.01). Additionally, activation of Rac GTPases and p42/p44 mitogen-activated protein kinases, known downstream effectors of the TCR-ZAP70 complex, is significantly reduced in response to TCR stimulation in RhoH−/− T cells as compared with WT cells. Finally, we show that retrovirus-mediated re-expression of RhoH into RhoH−/− bone marrow is sufficient to restore normal thymic development in Rag2−/− recipient mice, demonstrating the specific role of RhoH in thymic development. Together, our studies demonstrate that RhoH is essential for proper thymocyte development and function, and also implicate RhoH as a novel, critical regulator of T cell signaling possibly through its interaction with ZAP-70 in the TCR complex.

The role of Notch and IL‐7 signaling in early thymocyte proliferation and differentiation

We have analyzed the roles of Notch and IL-7 signaling in the proliferation and differentiation of mouse progenitor thymocyte subpopulations cultured on Notch delta-like-1 ligand-expressing OP9 stromal cells. Using bulk and limiting dilution cultures, we show that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into cytoplasmic TCRbeta and surface TCRalpha/beta and TCRgamma/delta expressing T cells. Selection for cytoplasmic TCRbeta-positive cells is dependent on preTalpha expression. Both gamma/delta and alpha/beta TCR expressing T cells arising in culture can be efficiently stimulated by anti-CD3 cross-linking, suggesting that they might be functional. The differentiation of adult, but not fetal, DN1 and DN2 thymocytes into CD4 and/or CD8 expressing cells is inhibited by IL-7. Finally, efficient proliferation and differentiation of DN3 cells requires Notch signaling and preTCR expression, but is independent of IL-7.

Crucial Role for Nuclear Factor of Activated T Cells in T Cell Receptor-mediated Regulation of Human Interleukin-17

The biological activities of the inflammatory cytokine interleukin (IL)-17 have been widely studied. However, comparatively little is known about how IL-17 expression is controlled. Here, we examined the basis for transcriptional regulation of the human IL-17 gene. IL-17 secretion was induced in peripheral blood mononuclear cells following anti-CD3 cross-linking to activate the T cell receptor (TCR), and costimulatory signaling through CD28 strongly enhanced CD3-induced IL-17 production. To define cis-acting elements important for IL-17 gene regulation, we cloned 1.25 kb of genomic sequence upstream of the transcriptional start site. This putative promoter was active in Jurkat T cells following CD3 and CD28 cross-linking, and its activity was inhibited by cyclosporin A and MAPK inhibitors. The promoter was also active in Hut102 T cells, which we have shown to secrete IL-17 constitutively. Overexpression of nuclear factor of activated T cells (NFAT) or Ras enhanced IL-17 promoter activity, and studies in Jurkat lines deficient in specific TCR signaling pathways provided supporting evidence for a role for NFAT. To delineate the IL-17 minimal promoter, we created a series of 5' truncations and identified a region between -232 and -159 that was sufficient for inducible promoter activity. Interestingly, two NFAT sites were located within this region, which bound to NFATc1 and NFATc2 in nuclear extracts from Hut102 and Jurkat cells. Moreover, mutations of these sites dramatically reduced both specific DNA binding and reporter gene activity, and chromatin immunoprecipitation assays showed occupancy of NFAT at this region in vivo. Together, these data show that NFAT is the crucial sensor of TCR signaling in the IL-17 promoter.

Mouse EphrinB3 Augments T-cell Signaling and Responses to T-cell Receptor Ligation

Ephrins (EFN) are cell-surface ligands of Ephs, the largest family of cell-surface receptor tyrosine kinases. The function of EFNs in the immune system has not been well studied, although some EFNs and Ephs are expressed at high levels on certain leukocytes. We report here that EFNB3 and its receptors (collectively called EFNB3Rs, as EFNB3 binds to multiple EphBs) were expressed in peripheral T cells and monocytes/macrophages, with T cells being the dominant EFNB3+ and EFNB3R+ cell type. Solid-phase EFNB3-Fc in the presence of suboptimal anti-CD3 crosslinking enhanced T-cell responses in terms of proliferation, activation marker expression, interferon-gamma but not interleukin-2 production, and cytotoxic T-cell activity. EFNB3R costimulation in the presence of phorbol 12-myristate 13- acetate was insensitive to cyclosporin A, similar to CD28 costimulation, suggesting they might share a part of the signaling pathway. After crosslinking, T-cell receptor and EFNB3R congregated into aggregated rafts, and this provided a morphological basis for signaling pathways of T-cell receptor and EFNB3R to interact. Solid-phase EFNB3-Fc augmented p38 and p44/42 MAPK activation further downstream of the signaling pathway. These data suggest that EFNB3 is important in T-cell/T-cell and T-cell/antigen-presenting cell collaboration to enhance T-cell activation and function.

Resistance to Alopecia Areata in C3H/HeJ Mice Is Associated with Increased Expression of Regulatory Cytokines and a Failure to Recruit CD4+ and CD8+ Cells

Grafting alopecia areata affected C3H/HeJ mouse skin to littermates induces alopecia areata, but high dietary soy oil reduces alopecia areata susceptibility. Alopecia areata affected and resistant mice were characterized to evaluate possible mechanisms involved in alopecia areata resistance. Of 44 mice that received alopecia areata affected skin grafts but failed to develop alopecia areata, only two of 22 receiving further alopecia areata affected skin grafts developed alopecia areata, whereas 39 of 44 controls developed alopecia areata. Alopecia areata affected skin contained increased numbers of CD4+ and CD8+ cells, increases in pro inflammatory T helper 1 and T helper 2 type cytokines, and upregulation of CD28, CD40L, and their ligands. In draining lymph nodes, a relatively high number of antigen-presenting cells was recovered, whereas several CD44v variants were downregulated. In contrast, alopecia areata resistant mouse skin did not display increased numbers of CD4+ and CD8+ cells, whereas counter-regulatory cytokines interleukins 4 and 10 were upregulated. High expression of CD28, CD80, CD86, CD40, CTLA4, CD44v variants, and FasL occurred in alopecia areata resistant mouse spleens. In vitro, lymph node cells of susceptible and resistant mice responded equally to a mitogenic stimulus, but only lymph node cells from alopecia areata affected mice displayed an increased response with T cell receptor stimulation via anti-CD3 cross-linking. These results suggest alopecia areata is a cell-mediated autoimmune disease, but alopecia areata affected skin graft hosts may resist alopecia areata onset through active counter-regulatory mechanisms. Because alopecia areata resistant mice showed unimpaired responsiveness and a transient inflammatory response towards the graft, it is suggested that alopecia areata develops as a consequence of an inappropriate immune response regulation.

T-Cell Antigen Receptor Peptides Inhibit Signal Transduction within the Membrane Bilayer

Previous studies have shown that a synthetic peptide (core peptide, CP) corresponding to a 9-amino-acid region in the transmembrane domain of the α subunit of the T-cell antigen receptor (TCR) can suppress T-cell function in vitro and in vivo. The aim of these experiments was to determine the cellular site and molecular mechanism of CP inhibition in T cells. The cytochrome c-sensitive TCR-expressing hybridoma (2B4) was stimulated with pigeon cytochrome c antigen, anti-CD3 crosslinking, or PMA and ionomycin, in the presence or absence of CP, and the resulting IL-2 produced was measured in a bioassay using an IL-2-dependent cell line (CTLL-2). In the presence of CP, IL-2 production was inhibited following antigen-induced stimulation. By contrast, when stimulated with cross-linking antibodies to the CD3 complex or with PMA and ionomycin, both of which activate T cells downstream of the TCR antigen recognition site, CP had no effect on IL-2 production. These experiments suggest that CP interferes with TCR function by inhibiting T-cell activation at the transmembrane/receptor level. In addition, we show that CP inhibits early TCR signal transduction events such as TCR ζ chain phosphorylation following stimulation with either antigen or anti-CD3-crosslinking antibodies, although this is unlikely to be the mechanism leading to the reduced IL-2 production.

Deficiency of small GTPase Rac2 affects T cell activation.

Rac2 is a hematopoietic-specific GTPase acting as a molecular switch to mediate both transcriptional activation and cell morphological changes. We have examined the effect of Rac2 deficiency during T cell activation. In Rac2(-/-) T cells, proliferation was reduced upon stimulation with either plate-bound anti-CD3 or T cell receptor-specific antigen. This defect is accompanied with decreased activation of mitogen activated protein kinase extracellular signal-regulated kinase (ERK)1/2 and p38, and reduced Ca(2)+ mobilization. TCR stimulation-induced actin polymerization is also reduced. In addition, anti-CD3 cross-linking-induced T cell capping is reduced compared with wild-type T cells. These results indicate that Rac2 is important in mediating both transcriptional and cytoskeletal changes during T cell activation. The phenotypic similarity of Rac2(-/-) to Vav(-/-) cells implicates Rac2 as a downstream mediator of Vav signaling.

Tamoxifen alleviates disease severity and decreases double negative T cells in autoimmune MRL-lpr/lpr mice.

Previous study suggested that MRL-lpr/lpr mice treated with tamoxifen (TAM) had less severe proteinuria, reduced serum titre of anti-dsDNA autoantibodies and an increased survival rate. To investigate further the regulatory mechanisms of TAM on MRL-lpr/lpr female mice, a total dose of 200 microg per mice (5.5 mg/kg) was given every 2 weeks subcutaneously, while the control mice were injected with oil only. After being treated with TAM four times, the mice were killed and cellular functions were evaluated. The TAM-treated groups had smaller sized spleen and lymph nodes. Flow cytometric analysis of splenocytes had a significantly lower percentage of cell number of T cells and double negative T cells (CD4- CD8- T cells). There was no difference in cytokine production (interleukin (IL)-2, IL-4, IL-5, IL-10 and interferon-gamma (IFN-gamma)) from splenocytes stimulated with concanavalin A (Con A) or cytokines (IL-6) secreted by peritoneal exudate cells when stimulated with lipopolysaccharide (LPS). However, IL-2 from lymph node cells was significantly higher on TAM-treated mice. Finally, splenocytes or purified T cells stimulated with anti-CD3 antibody plus cross-linking immunoglobulin G (IgG) of the TAM-treated group had higher 3H-incorporation of proliferation assay compared with that of control groups. In vitro study further demonstrated that IL-2-activated proliferation of lymph node double negative (DN) T cells can be inhibited by TAM treatment in a dose-dependent manner. Our finding demonstrated that TAM may potentially influence T cells and modulate the immune function, which offers a novel approach to explore the feasibility of hormone therapy for autoimmune diseases.