Expression of a specific glycosyltransferase enzyme regulates T cell death mediated by galectin-1

Galectin-1 induces apoptosis of immature thymocytes and activated T cells, suggesting that galectin-1 regulates cell death in the thymus during selection and in the periphery following an immune response. Although it is known that galectin-1 recognizes lactosamine (Gal-GlcNAc) as a minimal ligand, this disaccharide is ubiquitously expressed on a variety of cell surface glycoproteins. Thus, susceptibility to galectin-1 may be regulated by the presentation of lactosamine on specific oligosaccharide structures created by specific glycosyltransferase enzymes. The core 2 beta-1, 6-N-acetylglucosaminyltransferase (core 2 GnT) creates a branched structure on O-glycans that can be elongated to present multiple lactosamine sequences. In the thymus, the core 2 GnT is expressed in galectin-1-sensitive thymocyte subsets. In the periphery, an oligosaccharide epitope created by the core 2 GnT is expressed on galectin-1-sensitive activated T-cells. In this report, we demonstrate that expression of the core 2 GnT was necessary and sufficient for galectin-1-induced death of murine T cell lines. In addition, overexpression of the core 2 GnT in mice increased the susceptibility of double positive thymocytes to galectin-1. These data demonstrate that expression of a specific glycosyltransferase can control susceptibility to galectin-1, suggesting that developmentally regulated glycosyltransferase expression may be a mechanism to modulate cell death during T cell development and function.     

Hepatitis C virus core protein leads to immune suppression and liver damage in a transgenic murine model

Hepatitis C virus (HCV) is remarkably efficient in establishing persistent infection, possibly mediated by an impaired immune response to HCV infection. There is compelling evidence that HCV can infect immune cells, such as macrophages, B cells, and T cells. It has been previously reported that HCV core, the first protein expressed during the early phase of viral infection, contains the immunomodulatory function of suppressing host immune responses. This altered function of immune cells caused by HCV infection may explain the ineffective immune response to HCV. To further characterize the immunomodulatory role of HCV core in vivo, we generated transgenic (TG) mice by directing the expression of core protein to T lymphocytes by using the CD2 promoter. T-lymphocyte responses, including the production of gamma interferon and interleukin-2, were significantly diminished in these mice compared to their non-TG littermates. The inhibition of T-lymphocyte responsiveness may be due to the increased susceptibility of peripheral T lymphocytes to Fas-mediated apoptosis. Surprisingly, significant lymphocyte infiltration was observed in the portal tracts of livers isolated from core TG mice, associated with increasing serum alanine aminotransferase levels. Moreover, no intrahepatic lymphocytes or liver damage was found in non-TG littermates and core TG mice bred to Fas-deficient lpr mice. These results suggest that HCV core drives liver injury by increasing Fas-mediated apoptosis and liver infiltration of peripheral T cells.     

In vivo overexpression of Core2 N-Acetylglucosaminyltransferase prevents repopulation of the bone marrow with colony forming cells but fails to affect normal T cell development

UDP-GlcNAc:Galβ1 → 3GalNAc-R β1 → 6N-acetylglucosaminyltransferase (Core2 N-acetyl-glucosaminyltransferase, C2GnT; EC 2.4.1.102) forms β1 → 6N-acetyl-glucosaminyl linkages in O-glycoproteins and creates branches for the addition of N-acetyl-lactosamine antennae. Changes in C2GnT activity have been associated with immune disorders, malignancies, and T-cell ontogeny. In this study, we used SCID (severe combined immune deficiency) mice to determine the effects of C2GnT overexpression on hemopoiesis, and in particular, on thymocyte development. BALB/c bone marrow cells transfected with C2GnT using the retroviral murine stem cell vector were used to repopulate SCID mice. Mice were analysed 3 weeks to 3 months after bone marrow transfer. Elevated levels of C2GnT activity in bone marrow, spleen, and thymus from mice repopulated with C2GnT transfected bone marrow cells indicated that C2GnT was overexpressed in recipient mice. In C2GnT repopulated mice, up to 50% of T cells showed an increase in CD43 130-kDa expression, compared with T cells from control animals, indicative of an elevated C2GnT activity in these cells. Furthermore, T-cell subset numbers appeared to be normal, suggesting that C2GnT overexpression did not alter T-cell ontogeny. Interestingly, C2GnT overexpression negatively affected the repopulation of myeloid cells. Only insignificant numbers of interleukin-3/granulocyte-macrophage colony stimulating factor (IL-3/GM-CSF) responsive bone marrow cells were found to be retrovirally transfected in C2GnT repopulated mice, whereas up to 50% of IL-3/GM-CSF responsive bone marrow cells were found to be retrovirally transfected in corresponding controls. These data indicate that in vivo overexpression of C2GnT negatively interferes with the myeloid differentiation pathway but does not affect T-cell development. J. Cell. Physiol. 176:350–358, 1998. © 1998 Wiley-Liss, Inc.     

Mucin-type O-glycans and leukosialin

Mucin-type O-glycans on leukocytes acquire functions once they contain core 2 branches, which can be synthesized by core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT). Recently, understanding the roles of mucin-type O-glycans has been significantly advanced by generating transgenic mice overexpressing C2GnT or knockout mice defective in C2GnT. This review article summarizes previous results implicating the roles of mucin-type O-glycans and the most recent studies to test such a hypothesis. These results, taken together, demonstrate that mucin-type O-glycans either facilitate or attenuate cell adhesion depending on the structures of non-reducing termini.     

T-cell-mediated immunity to lymphocytic choriomeningitis virus in beta2-integrin (CD18)- and ICAM-1 (CD54)-deficient mice.

The T-cell response to lymphocytic choriomeningitis virus was studied in mice with deficient expression of beta2-integrins or ICAM-1. In such mice, the generation of virus-specific cytotoxic T lymphocytes was only slightly impaired and bystander activation was as extensive as that observed in wild-type mice. T-cell-mediated inflammation, assessed as primary footpad swelling and susceptibility to intracerebral infection, was slightly compromised only in beta2-integrin-deficient mice. However, adoptive immunization of mutant mice soon after local infection did reveal a reduced capacity to support the inflammatory reaction, indicating that under conditions of more limited immune activation both molecules do play a role in formation of the inflammatory exudate. Finally, virus control was found to be somewhat impaired in both mutant strains. In conclusion, our results indicate that although LFA-1-ICAM-1 interaction is important for certain aspects of the T-cell-mediated response to viruses, T-cell activation is surprisingly intact in these mutant mice, indicating extensive functional redundancy within cell interaction molecules.     

IL-2, -4, and -15 differentially regulate O-glycan branching and P-selectin ligand formation in activated CD8 T cells.

The glycosyltransferase core 2 beta1-6 N-acetylglucosaminyl transferase (C2GnT1 or C2GlcNAcT1) is responsible for formation of branched structures on O-glycans present on cell surface glycoproteins. The O-glycan branch created by C2GnT1 is physiologically important insofar as only this structure can be extended and modified to yield P-selectin ligands that promote initial interactions between extravasating lymphocytes and endothelia. In mature T cells, C2GnT1 activity is thought to be induced as an intrinsic consequence of T cell activation. Through analysis of C2GnT1-dependent epitopes on CD43 and CD45RB we have found that in activated CD8(+) T cells expression of C2GnT1 was dependent upon exposure to specific cytokines rather than being induced as a direct consequence of activation. Activated CD8(+) cells became receptive to strong induction of C2GnT1 expression and P-selectin ligand expression in response to IL-2, moderate induction by IL-15, and minimal induction in response to IL-4. Our observations clarify the relationship between T cell activation and C2GnT1 expression, demonstrate the differential impact of distinct cytokines on expression of C2GnT1 activity and P-selectin ligand, and reinforce the concept that the cytokine milieu subsequent to activation can influence adhesion systems that dictate lymphocyte homing properties.     

Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.     

Wiskott-Aldrich Syndrome at the nexus of autoimmune and primary immunodeficiency diseases

Wiskott-Aldrich Syndrome (WAS) is a X-linked primary immunodeficiency disorder also marked by a very high (up to 70%) incidence of autoimmunity. Wiskott-Aldrich Syndrome arises from mutations in the Wiskott-Aldrich Syndrome protein (WASp), a cytoplasmic protein that links signaling by cell surface receptors such as the T-cell receptor and integrins to actin polymerization. WASp promotes the functions of multiple cell types that support immune responses, but also is important for the function of regulatory T cells and in TCR-induced apoptosis, two negative mechanisms of immune regulation that maintain peripheral immune tolerance. Here we review the nature of immune defects and autoimmunity in WAS and WASp deficient mice and discuss how this single gene defect can simultaneously impair immune responses to pathogens and promote autoimmunity. The myriad cellular immune defects found in WAS make this Mendelian syndrome an interesting model for the study of more complex immune diseases that arise from the interplay of environmental and multiple genetic risk factors.     

Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.     

Effects of overexpression of growth hormone on T cell activity in transgenic mice

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overexpressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.     

Abnormal priming of CD4(+) T cells by dendritic cells expressing hepatitis C virus core and E1 proteins

Patients infected with hepatitis C virus (HCV) have an impaired response against HCV antigens while keeping immune competence for other antigens. We hypothesized that expression of HCV proteins in infected dendritic cells (DC) might impair their antigen-presenting function, leading to a defective anti-HCV T-cell immunity. To test this hypothesis, DC from normal donors were transduced with an adenovirus coding for HCV core and E1 proteins and these cells (DC-CE1) were used to stimulate T lymphocytes. DC-CE1 were poor stimulators of allogeneic reactions and of autologous primary and secondary proliferative responses. Autologous T cells stimulated with DC-CE1 exhibited a pattern of incomplete activation characterized by enhanced CD25 expression but reduced interleukin 2 production. The same pattern of incomplete lymphocyte activation was observed in CD4(+) T cells responding to HCV core in patients with chronic HCV infection. However, CD4(+) response to HCV core was normal in patients who cleared HCV after alpha interferon therapy. Moreover, a normal CD4(+) response to tetanus toxoid was found in both chronic HCV carriers and patients who had eliminated the infection. Our results suggest that expression of HCV structural antigens in infected DC disturbs their antigen-presenting function, leading to incomplete activation of anti-HCV-specific T cells and chronicity of infection. However, presentation of unrelated antigens by noninfected DC would allow normal T-cell immunity to other pathogens.     

IL-12, STAT4-dependent up-regulation of CD4(+) T cell core 2 beta-1,6-n-acetylglucosaminyltransferase, an enzyme essential for biosynthesis of P-selectin ligands

TCR activation of naive T cells in the presence of IL-12 drives polarization toward a Th1 phenotype and synthesis of P- and E-selectin ligands. Fucosyltransferase VII (Fuc-T VII) and core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT) are critical for biosynthesis of selectin ligands. P-selectin glycoprotein ligand-1 is the best characterized ligand for P-selectin and also binds E-selectin. The contributions of TCR and cytokine signaling pathways to up-regulate Fuc-T VII and C2GnT during biosynthesis of E- and P-selectin ligands, such as P-selectin glycoprotein ligand 1, are unknown. IL-12 signals via the STAT4 pathway. Here, naive DO11.10 TCR transgenic and STAT4(-/-) TCR transgenic CD4(+) T cells were stimulated with Ag and IL-12 (Th1 condition), IL-4 (Th2), or neutralizing anti-IL-4 mAb only (Th0). The levels of Fuc-T VII and C2GnT mRNA in these cells were compared with their adhesive interactions with P- and E-selectin in vitro under flow. The data show IL-12/STAT4 signaling is necessary for induction of C2GnT, but not Fuc-TVII mRNA, and that STAT4(-/-) Th1 cells do not traffic normally to sites of inflammation in vivo, do not interact with P-selectin, and exhibit a partial reduction of E-selectin interactions under shear stress in vitro. Ag-specific TCR activation in CD4(+) T cells was sufficient to trigger induction of Fuc-TVII, but not C2GnT, mRNA and expression of E-selectin, but not P-selectin, ligands. Thus, Fuc-T VII and C2GnT are regulated by different signals during Th cell differentiation, and both cytokine and TCR signals are necessary for the expression of E- and P-selectin ligands.     

Programmed cell death of T lymphocytes during acute viral infection: a mechanism for virus-induced immune deficiency.

Acute viral infections induce immune deficiencies, as shown by unresponsiveness to mitogens and unrelated antigens. T lymphocytes isolated from mice acutely infected with lymphocytic choriomeningitis virus (LCMV) were found in this study to undergo activation-induced apoptosis upon signalling through the T-cell receptor (TcR)-CD3 complex. Kinetic studies demonstrated that this sensitivity to apoptosis directly correlated with the induction of immune deficiency, as measured by impaired proliferation in response to anti-CD3 antibody or to concanavalin A. Cell cycling in interleukin-2 (IL-2) alone stimulated proliferation of LCMV-induced T cells without inducing apoptosis, but preculturing of T cells from acutely infected mice in IL-2 accelerated apoptosis upon subsequent TcR-CD3 cross-linking. T lymphocytes isolated from mice after the acute infection were less responsive to IL-2, but those T cells, presumably memory T cells, responding to IL-2 were primed in each case to die a rapid apoptotic death upon TcR-CD3 cross-linking. These results indicate that virus infection-induced unresponsiveness to T-cell mitogens is due to apoptosis of the activated lymphocytes and suggest that the sensitization of memory cells by IL-2 induced during infection will cause them to die upon antigen recognition, thereby impairing specific responses to nonviral antigens.     

The relative activities of the C2GnT1 and ST3Gal-I glycosyltransferases determine O-glycan structure and expression of a tumor-associated epitope on MUC1

In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.     

Requirement for efficient interactions between CD4 and MHC class II molecules for survival of resting CD4+ T lymphocytes in vivo and for activation-induced cell death

Regulation of homeostasis in the immune system includes mechanisms that promote survival of resting T lymphocytes, and others that control activation-induced cell death (AICD). In this study, we report on the use of a transgenic mouse model to test the role of CD4-MHC class II interactions for the susceptibility of CD4+ T lymphocytes to AICD, and for the survival of resting CD4+ T cells in peripheral lymphoid organs. The only I-Abeta gene expressed in these mice is an Abetak transgene with a mutation that prevents MHC class II molecules from interacting with CD4. We show increased apoptosis in CD4+ T lymphocytes derived from wild-type, but not from mutant Abetak transgenic mice following stimulation with staphylococcal enterotoxin A. Therefore, AICD may be impaired in CD4+ T cells derived from mutant Abetak transgenic mice. Importantly, we observed much higher apoptosis in resting CD4+ T cells from mutant Abetak transgenic mice than from wild-type mice. Furthermore, resting CD4+ T cells from mutant Abetak transgenic mice expressed higher levels of cell surface CD95 (Fas, APO-1). Ab-mediated cross-linking of CD95 further increased apoptosis in CD4+ T cells from mutant Abetak transgenic mice, but not from wild-type mice, suggesting apoptosis involved CD95 signaling. When cocultured with APC-expressing wild-type MHC class II molecules, apoptosis in resting CD4+ T lymphocytes from mutant Abetak transgenic mice was reduced. Our results show for the first time that interactions between CD4 and MHC class II molecules are required for the survival of resting CD4+ T cells in peripheral lymphoid organs.     

Waltzing with WASP

Wiskott-Aldrich syndrome (WAS) is an inherited immune deficiency that is marked by eczema, bleeding and recurrent infections. The lymphocytes and platelets of WAS patients display cytoskeletal abnormalities, and their T lymphocytes show a diminished proliferative response to stimulation through the T-cell receptor-CD3 complex (TCR-CD3). The product of the WAS gene, WAS protein (WASP), binds to the small GTPase Cdc42. Small GTPases of the Rho family are crucial for the regulation of the actin-based cytoskeleton. WASP and its relative NWASP might play an important role in regulating the actin cytoskeleton. Since both WASP and NWASP have the potential to bind to multiple proteins, they might serve as a hub to coordinate the redistribution of many cellular signals to the actin cytoskeleton. In this review, the authors discuss the possible role of WASP/NWASP and of the newly described protein WIP, which interacts with WASP and NWASP, in coupling signals from the T-cell receptor to the actin-based cytoskeleton.     

Core 2 β1,6-N-acetylglucosaminyltransferases accelerate the escape of choriocarcinoma from natural killer cell immunity

Hyperglycosylated human chorionic gonadotropin (H-hCG) is secreted from choriocarcinoma and contains a core2 O-glycan formed by core2 β1,6-N-acetylglucosaminyl transferase (C2GnT). Choriocarcinoma is considered immunogenic as it is gestational and contains paternal chromosomal components. Here we examined the function of C2GnT in the evasion of choriocarcinoma cells from natural killer (NK) cell-mediating killing. We determined that C2GnT is highly expressed in malignant gestational trophoblastic neoplasms. C2GnT KO downregulates core2 O-glycan expression in choriocarcinoma cells, which are more efficiently killed by NK cells than control cells. C2GnT KO cell containing tumor necrosis factor-related apoptosis inducing ligand have lower viability than control cells. Additionally, poly-N-acetyllactosamine in core2 branched oligosaccharides on MHC class I-related chain A (MICA) and mucin1 (MUC1) is significantly reduced in C2GnT KO cells. Meanwhile, the cumulative survival rate of nude mice inoculated with C2GnT KO tumors was higher than that of the control group. These findings suggest that choriocarcinoma cells may escape NK cell-mediated killing via glycosylation of MICA and MUC1.     

Role of dendritic cells in the immune response induced by mouse mammary tumor virus superantigen.

After mouse mammary tumor virus (MMTV) infection, B lymphocytes present a superantigen (Sag) and receive help from the unlimited number of CD4(+) T cells expressing Sag-specific T-cell receptor Vbeta elements. The infected B cells divide and differentiate, similarly to what occurs in classical B-cell responses. The amplification of Sag-reactive T cells can be considered a primary immune response. Since B cells are usually not efficient in the activation of naive T cells, we addressed the question of whether professional antigen-presenting cells such as dendritic cells (DCs) are responsible for T-cell priming. We show here, using MMTV(SIM), a viral isolate which requires major histocompatibility complex class II I-E expression to induce a strong Sag response in vivo, that transgenic mice expressing I-E exclusively on DCs (I-EalphaDC tg) reveal a strong Sag response. This Sag response was dependent on the presence of B cells, as indicated by the absence of stimulation in I-EalphaDC tg mice lacking B cells (I-EalphaDC tg muMT(-/-)), even if these B cells lack I-E expression. Furthermore, the involvement of either residual transgene expression by B cells or transfer of I-E from DCs to B cells was excluded by the use of mixed bone marrow chimeras. Our results indicate that after priming by DCs in the context of I-E, the MMTV(SIM) Sag can be recognized on the surface of B cells in the context of I-A. The most likely physiological relevance of the lowering of the antigen threshold required for T-cell/B-cell collaboration after DC priming is to allow B cells with a low affinity for antigen to receive T-cell help in a primary immune response.     

Expression of Wiskott-Aldrich syndrome protein in dendritic cells regulates synapse formation and activation of naive CD8+ T cells

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin polimerization in hematopoietic cells. Mutations in WASp cause a severe immunodeficiency characterized by defective initiation of primary immune response and autoimmunity. The contribution of altered dendritic cells (DCs) functions to the disease pathogenesis has not been fully elucidated. In this study, we show that conventional DCs develop normally in WASp-deficient mice. However, Ag targeting to lymphoid organ-resident DCs via anti-DEC205 results in impaired naive CD8(+) T cell activation, especially at low Ag doses. Altered trafficking of Ag-bearing DCs to lymph nodes (LNs) accounts only partially for defective priming because correction of DCs migration does not rescue T cell activation. In vitro and in vivo imaging of DC-T cell interactions in LNs showed that cytoskeletal alterations in WASp null DCs causes a reduction in the ability to form and stabilize conjugates with naive CD8(+) T lymphocytes both in vitro and in vivo. These data indicate that WASp expression in DCs regulates both the ability to traffic to secondary lymphoid organs and to activate naive T cells in LNs.