T cell receptor-induced lipid raft recruitment of the I kappa B kinase complex is necessary and sufficient for NF-kappa B activation occurring in the cytosol

TCR-induced NF-kappa B activation is necessary for the innate immune response and involves induced lipid raft recruitment of the I kappa B kinase (IKK) complex. In this study, we systematically investigated lipid raft recruitment of members of the NF-kappa B activation pathway in human T cells. All upstream components leading to IKK activation were found constitutively or inducibly in lipid rafts, while the NF-kappa B/I kappa B complex and phosphorylated forms of IKK alpha/beta, I kappa B alpha and p65 are exclusively found in the cytosolic fraction. Disruption of raft organization precluded NF-kappaB activation induced by T cell costimulation, but IL-1-triggered NF-kappa B activation remained unaffected. Targeting of the IKK complex to lipid rafts caused constitutive IKK activation and NF-kappa B DNA binding, which was further triggered upon T cell costimulation. Various experimental approaches revealed that costimulation-induced IKK alpha/beta activation loop phosphorylation is independent from IKK beta-mediated transautophosphorylation, but rather involves phosphorylation by the IKK-interacting protein NIK and its upstream activator COT.     

The level of B7 homologue 1 expression on brain DC is decisive for CD8 Treg cell recruitment into the CNS during EAE

DC in the CNS have emerged as the major rate‐limiting factor for immune invasion and subsequent neuroinflammation during EAE. The mechanism of how this is regulated by brain‐localized DC remains unknown. Here, we describe the ability of brain‐localized DC expressing B7‐H1 molecules to recruit CD8⁺ T cells to the site of inflammation. Using intracerebral microinjections of B7‐homologue 1‐deficient DC, we demonstrate a substantial brain infiltration of CD8⁺ T cells displaying a regulatory phenotype (CD122⁺) and function, resulting in a decrease of EAE peak clinical values. The recruitment of regulatory‐type CD8⁺ T cells into the CNS and the role of brain DC expressing B7‐homologue 1 molecules in this process open up the possibility of DC‐targeted therapeutic manipulation of neuroinflammatory diseases.     

ERMAP is a B7 family-related molecule that negatively regulates T cell and macrophage responses

T cell activation and tolerance are tightly regulated by costimulatory and coinhibitory molecules. B7 family members play a crucial role in regulating immune responses. In this study, we identified erythroid membrane-associated protein (ERMAP) as a novel T cell inhibitory molecule. ERMAP shares significant sequence and structural homology with existing B7 family members in its extracellular domain. The ERMAP protein is expressed on the cell surface of resting and activated antigen-presenting cells (APCs) and in some tumor tissues. The putative ERMAP receptor is expressed on activated CD4 and CD8 T cells and macrophages. Both mouse and human ERMAP-IgG2a Fc (ERMAP-Ig) fusion proteins inhibit T cell functions in vitro. Administration of ERMAP-Ig protein ameliorates autoimmune diseases, including experimental autoimmune encephalomyelitis and type 1 diabetes, in mice. Anti-ERMAP antibody enhances macrophage phagocytosis of cancer cells in vitro. Furthermore, administration of an anti-ERMAP antibody inhibits tumor growth in mice likely by blocking the inhibitory effects of ERMAP on T cells and macrophages. Our results suggest that therapeutic interaction with the ERMAP inhibitory pathway may represent a novel strategy for treating patients with autoimmune disease or cancer.     

CCR6 regulates EAE pathogenesis by controlling regulatory CD4+ T‐cell recruitment to target tissues

The T‐cell subsets, characterized by their cytokine production profiles and immune regulatory functions, depend on correct in vivo location to interact with accessory or target cells for effective immune responses. Differentiation of naive CD4⁺ T cells into effectors is accompanied by sequentially regulated expression of the chemokine receptors responsible for cell recruitment to specific tissues. We studied CCR6 function in EAE, a CD4⁺ T‐cell‐mediated CNS disease characterized by mononuclear infiltration and demyelination. CCR6^?/? mice showed an altered time course of EAE development, with delayed onset, a higher clinical score, and more persistent symptoms than in controls. An imbalanced cytokine profile and reduced Foxp3⁺ cell frequency characterized CNS tissues from CCR6^?/? compared with CCR6^+/+ mice during the disease effector phase. Transfer of CCR6^+/+ Treg to CCR6^?/? mice the day before EAE induction reduced the clinical score associated with an increased in infiltrating Foxp3⁺ cells and recovery of the cytokine balance in CCR6^?/? mouse CNS. Competitive assays between CCR6^+/+ and CCR6^?/? Treg adoptively transferred to CCR6^?/? mice showed impaired ability of CCR6^?/? Treg to infiltrate CNS tissues in EAE‐affected mice. Our data indicate a CCR6 requirement by CD4⁺ Treg to downregulate the CNS inflammatory process and neurological signs associated with EAE.     

A novel role for CD28 in thymic selection: elimination of CD28/B7 interactions increases positive selection

While the importance of the CD28/B7 costimulation pathway is well established for mature T cells, the role of CD28 in thymocyte selection is less well defined. The role of CD28 in both negative and positive selection was assessed using H-Y-specific TCR-transgenic (Tg) RAG-2-deficient (H-Yrag) mice. Negative selection in male H-Yrag mice was not affected by deficiency in CD28 or B7. Surprisingly, absence of CD28 or B7 in H-Yrag females resulted in increased numbers of CD8 single-positive (SP) thymocytes. The CD8 SP thymocytes found in these females were mature and functionally competent. Furthermore, double-positive (DP) thymocytes from CD28-knockout (CD28KO) or B7.1/B7.2 double-KO (B7DKO) females had higher levels of both CD5 and TCR than those from WT females, consistent with a stronger selecting signal. CD28KO H-Yrag fetal thymic organ cultures also had elevated numbers of thymic CD8 SP cells, reflecting increased thymic differentiation and not recirculation of peripheral T cells. Finally, increased selection of mature CD4 and CD8 SP T cells was observed in non-TCR-Tg CD28KO and B7DKO mice, indicating that this function of CD28-B7 interaction is not unique to a TCR-Tg model. Together these findings demonstrate a novel negative regulatory role for CD28 in inhibiting differentiation of SP thymocytes, probably through inhibition of thymic selection.     

The Peyer's patch is a critical immunoregulatory site for mucosal tolerance in experimental autoimmune encephalomylelitis (EAE)

Expression of MCP-1 in the central nervous system (CNS) is associated with various neuroinflammatory diseases, including multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In this study, we found that MCP-1 was decreased in the CNS but increased in the gut following oral administration of myelin basic protein (MBP) correlating with protection from EAE. To study the trafficking and the fate of T cells during oral tolerance, MBP-specific TCR transgenic (Tg) CD4(+) T cells were labeled using 5,6-carboxy-succinimidyl-fluorescein-ester (CFSE) and transferred intravenously to syngeneic B10.PL recipients before feeding with either MBP or PBS. We observed that the CFSE-labeled T cells traffic to the peripheral lymphoid tissue and the Peyer's patches (PP). The labeled T cells proliferate in vivo in both the lymph node and the PP 48h after MBP feeding, but the cells are maintained in the PP longer than in the LN. CFSE-labeled cells in the PP have high levels of CD69 and Fas expression which is accompanied by increased apoptosis after MBP feeding. Our observations suggest that oral administration of autoantigen induces an elevation of MCP-1 in the gut, early T cell trafficking and activation in the periphery and the PP, followed by deletion of autoreactive T cells in the PP.     

关节炎中B7:CD28/CTLA-4共刺激途径的实验研究

目的：探讨Ｂ＆：ＣＤ２８／ＣＴＬＡ－４共刺激信号大关节炎发病机制中的作用。方法：经牛Ⅱ型胶原诱导ＳＤ大鼠建立关节炎动物模型;采用ＣＴＬＡ－４Ｉｇ作为负性调节因子,分析其对关节炎大鼠中特异性体液反应和组织病理学表现的影响。结果：ＣＴＬＡ＿４Ｉｇ可阻止ＳＤ大鼠发生胶原诱发 关节炎,降低大导ＢＩＩＣ的体液免疫反应。结论：Ｂ７：ＣＤ２８／ＣＴＬＡ－４共刺激信号在诱发Ｔ细胞介导的自身免疫病中起作用,ＣＴＬＡ     

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.     

CD4 T cell help is required for primary CD8 T cell responses to vesicular antigen delivered to dendritic cells in vivo

Insight into the mechanisms by which dendritic cells (DC) present exogenous antigen to T cells is of major importance in the design of vaccines. We examined the effectiveness of free antigen as well as antigen with lipopolysaccharide, emulsified in complete Freund's adjuvant, and antigen encapsulated in liposomes in activating adoptively transferred antigen-specific CD4 and CD8 T cells. When contained in liposomes, 100- to 1000-fold lower antigen amounts were as efficient in inducing proliferation and effector functions of CD4 and CD8 T cells in draining lymph nodes as other antigen forms. CD11c(+)/CD11b(+)/CD205(mod)/CD8alpha(-) DC that captured liposomes were activated and presented this form of antigen in an MHC class I- and class II-restricted manner. CD4 T cells differentiated into Th1 and Th2 effector cells. Primary expansion and cytotoxic activity of CD8 T cells were CD4 T cell-dependent and required the transporter associated with antigen processing (TAP). Finally, adoptively transferred CD4 and CD8 T cells were not deleted after primary immunization and rapidly responded to a secondary immunization with antigen-containing liposomes. In conclusion, encapsulation of antigen in liposomes is an efficient way of delivering antigen to DC for priming of both CD4 and CD8 T cell responses. Importantly, primary CD8 T cell responses were CD4 T cell-dependent.     

T细胞疫苗抑制自身免疫性甲状腺炎的发生

目的　研究T细胞疫苗 (TCV)在小鼠实验性自身免疫性甲状腺炎 (EAT)发生中的阻断作用及可能机制。方法　磁珠分离CD4 T细胞 ,体外活化 ,戊二醛处理获得T细胞疫苗 ,体内接种EAT小鼠。通过EAT评价、细胞因子测定及细胞增殖试验了解TCV对EAT的阻断作用 ;流式细胞术测定小鼠CD4 CD2 5 T细胞百分率。结果　TCV接种后小鼠体内自身抗体水平明显下降 ,甲状腺无明显病理变化 ,IL 2和IFN γ水平以及Tg刺激的淋巴细胞增殖能力均显著降低 ,同时CD4 CD2 5 T细胞百分率较EAT组有明显增高。结论　TCV接种能明显抑制小鼠EAT的发生 ,可能与机体内CD4 CD2 5 Treg细胞的增加有关     

Recovery from EAE is associated with decreased survival of encephalitogenic T cells in the CNS of B7-1/B7-2-deficient mice

Adoptive transfer experiments using C57BL/6 mice lacking B7-1 and B7-2 as recipients of wt (wt) encephalitogenic T cells demonstrate a key role for B7 costimulation during the effector phase of experimental autoimmune encephalomyelitis (EAE). Following transfer of encephalitogenic T cells, B7-1/B7-2-deficient (-/-) recipients develop a transient and mild disease as compared to wt recipients. To understand the mechanism by which B7-1/B7-2 may influence the effector phase of EAE, we analyzed T cells, pro-inflammatory cytokines and chemokines within the CNS of wt and B7-1/B7-2-/- recipients at different times after adoptive transfer of activated myelin specific T cells. There was a marked decline in T cells and inflammatory mediators in the CNS of B7-1/B7-2-/- recipients by day 30 post transfer. B7-1/B7-2-/- mice developed more TUNEL+ apoptotic cells in the parenchyma and greater ratios of TUNEL+ cells/parenchymal foci than wt mice resulting in virtual disappearance of parenchymal foci. Therefore, without B7-1 and B7-2 costimulation in the target organ, there is increased T cell apoptosis and attenuation of inflammation. These results indicate that B7-1 and B7-2 provide critical costimulatory signals for sustaining survival of pathogenic T cells within the central nervous system parenchyma during the effector phase of EAE and suggest novel treatment approaches in the effector phase of autoimmune diseases.     

Impaired interleukin-2 synthesis and T cell proliferation following antibody-mediated CD3 and CD2 or CD28 cross-linking in trans: evidence that T cell activation requires the engagement of costimulatory molecules within the immunological synapse

Although the T cell costimulatory molecules CD2 and CD28 are enriched within the immunological synapse (IS), it has been suggested that costimulatory molecules need not be localized to the contact site between a T cell and an antigen-presenting cell (APC) in order to costimulate T cell activation. To determine whether CD2 or CD28 engagement outside of the IS is sufficient to costimulate T cell activation, we compared mouse T cell responses to anti-CD3 and anti-CD2 monoclonal antibodies (mAbs) or anti-CD3 and anti-CD28 mAbs immobilized on the same, i.e., in cis, or on different, i.e., in trans, 10 micron polystyrene microspheres. In comparison to T cells that were stimulated with co-immobilized anti-CD3 and anti-CD2 or anti-CD28 mAbs, DNA synthesis, interleukin (IL)-2 production, and cellular proliferation were all severely impaired following T cell stimulation with anti-CD3 and anti-CD2 mAbs or anti-CD3 and anti-CD28 mAbs on different microspheres. Deficient cellular proliferation and IL-2 synthesis by T cells that experienced CD3 and CD2 or CD28 cross-linking in trans provides evidence that costimulatory molecules must function in the context of the IS for optimal T cell activation.     

CD28/CTLA-4/B7 and CD40/CD40L costimulation and activation of regulatory T cells

Costimulatory signals are crucial for T cell activation. Attempts to block costimulatory pathways have been effective in preventing unwanted immune reactions. In particular, blocking the CD28/cytotoxic T lymphocyte antigen(CTLA)-4/B7 interaction(using CTLA-4Ig) and the CD40/CD40 L interaction(using anti-CD40 L antibodies) prevents T cell mediated autoimmune diseases, transplant rejection and graft vs host disease in experimental models. Moreover, CTLA-4Ig is in clinical use to treat rheumatoid arthritis(abatacept) and to prevent rejection of renal transplants(belatacept). Under certain experimental conditions, this treatment can even result in tolerance. Surprisingly, the underlying mechanisms of immune modulation are still not completely understood. We here discuss the evidence that costimulation blockade differentially affects effector T cells(Teff) and regulatory T cells(Treg). The latter are required to control inappropriate and unwanted immune responses, and their activity often contributes to tolerance induction and maintenance. Unfortunately, our knowledge on the costimulatory requirements of Treg cells is very limited. We therefore summarize the current understanding ofthe costimulatory requirements of Treg cells, and elaborate on the effect of anti-CD40 L antibody and CTLA-4Ig treatment on Treg cell activity. In this context, we point out that the outcome of a treatment aiming at blocking the CD28/CTLA-4/B7 costimulatory interaction can vary with dosing, timing and underlying immunopathology.     

Human mast cells costimulate T cells through a CD28‐independent interaction

Mast cells are innate immune cells usually residing in peripheral tissues, where they are likely to activate T‐cell responses. Similar to other myeloid immune cells, mast cells can function as antigen‐presenting cells. However, little is known about the capacity of human mast cells to costimulate CD4⁺ T cells. Here, we studied the T‐cell stimulatory potential of human mast cells. Peripheral blood derived mast cells were generated and cocultured with isolated CD4⁺ T cells. In the presence of T‐cell receptor triggering using anti‐CD3, mast cells promoted strong proliferation of T cells, which was two‐ to fivefold stronger than the “T‐cell promoting capacity” of monocytes. The interplay between mast cells and T cells was dependent on cell–cell contact, suggesting that costimulatory molecules on the mast cell surface are responsible for the effect. However, in contrast to monocytes, the T‐cell costimulation by mast cells was independent of the classical costimulatory molecule CD28, or that of OX40L, ICOSL, or LIGHT. Our data show that mast cells can costimulate human CD4⁺ T cells to induce strong T‐cell proliferation, but that therapies aiming at disrupting the interaction of CD28 and B7 molecules do not inhibit mast cell mediated T‐cell activation.     

Immunomodulation of the Anti-Islet CD8 T Cell Response by B7-2

The absence of diabetes in NOD mice devoid of B7-2 signifies a critical role played by B7-2 in promoting autoimmunity. We asked whether the CD8 T cell compartment is impacted by the absence of B7-2. We found significantly lower expansion of anti-islet CD8 T cells in B7-2KO mice, although their survival and activation states remained unchanged in the pancreatic lymph nodes (PLNs). CD8 T cells from B7-2KO mice exhibited significantly diminished effector function compared to NOD mice. Adoptive transfer experiments using in vitro activated anti-islet CD8 T cells showed that B7-2 does not control the effector phase of the autoreactive CD8 T cell response. Our data indicate that B7-2 promotes pancreatic autoimmunity by controlling CD8 T cell expansion and effector function, but is dispensable for CD8 T cell activation, survival, and the effector phase of anti-islet CD8 T cell response.     

Selective expansion of memory CD4(+) T cells by mitogenic human CD28 generates inflammatory cytokines and regulatory T cells

Costimulatory signals are important for development of effector and regulatory T cells. In this case, CD28 signaling is usually considered inert in the absence of signaling through the TCR. By contrast, mitogenic rat CD28 mAb reportedly expand regulatory T cells without TCR stimulation. We found that a commercially available human CD28 mAb (ANC28) stimulated PBMC without TCR co-ligation or cross-linking; ANC28 selectively expanded CD4(+)CD25(+)FOXP3(-) (Teff) and CD4(+)CD25(+)FOXP3(+) (Treg) cells. ANC28 stimulated the CD45RO(+) CD4(+) (memory) population, whereas CD45RA(+)CD4(+) (naive) cells did not respond. ANC28 also induced inflammatory cytokines. Treg induced by ANC28 retain the Treg phenotype longer than costimulated Treg. Treg induced by ANC28 suppressed CD25(-) T cells through a contact-dependent mechanism. Purity influenced the response of CD4(+)CD25(+ )cells because bead-purified CD4(+)CD25(+ )cells (85-90% pure) responded strongly to ANC28, whereas 98% pure FACS-sorted CD4(+)CD25(bright) (Treg) did not respond. Purified CD4(+)CD25(int) cells responded similarly to the bead-purified CD4(+)CD25(+) cells. Thus, pre-activated CD4(+) T cells (CD25(int)) respond to ANC28 rather than Treg (CD25(bright)). The ability of ANC28 to expand both effectors producing inflammatory cytokines as well as suppressive regulatory T cells might be useful for ex vivo expansion of therapeutic T cells.     

Generating functional CD8+ T cell memory response under transient CD4+ T cell deficiency: implications for vaccination of immunocompromised individuals

Studies based on either MHC class II-knockout or CD4+ T cell-depleted murine models have demonstrated a critical role for CD4+ T cells in the generation of CD8+ T cell memory. However, it is difficult to extend these findings to immunocompromised humans where a complete loss of CD4+ T cells is rarely observed. Here, we have developed a model setting, which allows studies on the generation of CD8+ T cell memory responses in a transient CD4+ T cell-deficient setting similar to that seen in immunocompromised patients. Immunisation with an adenoviral vaccine under transient helpless or help-deficient conditions showed varying degrees of impact on the priming of CD8+ T cell responses. Antigen-specific T cells generated under normal CD4+ T cell help and transient help-deficient conditions showed similar effector phenotype and were capable of proliferation upon secondary antigen encounter. Most importantly, in spite of CD4+ T cell deficiency, the long-term CD8+ T cell memory response remained functionally stable and showed comparable cytotoxic effector function as seen in CD8+ T cells generated with normal CD4+ T cell numbers. These findings provide evidence that in spite of partially impaired activation of a primary CD8+ T cell response, a fully functional and stable memory CTL response can be induced under conditions of severe transient CD4+ T cell deficiency.     

γδ T cells in EAE: Early trafficking events and cytokine requirements

We have previously shown that γδ T cells traffic to the CNS during EAE with concurrently increased expression of β₂‐integrins and production of IFN‐γ and TNF‐α. To extend these studies, we transferred bioluminescent γδ T cells to WT mice and followed their movement through the acute stages of disease. We found that γδ T cells rapidly migrated to the site of myelin oligodendrocyte glycoprotein peptide injection and underwent massive expansion. Within 6 days after EAE induction, bioluminescent γδ T cells were found in the spinal cord and brain, peaking in number between days 10 and 12 and then rapidly declining by day 15. Reconstitution of γδ T cell^?/? mice with γδ T cells derived from β₂‐integrin‐deficient mice (CD11a, ‐b or ‐c) demonstrated that γδ T‐cell trafficking to the CNS during EAE is independent of this family of adhesion molecules. We also examined the role of γδ T‐cell‐produced IFN‐γ and TNF‐α in EAE and found that production of both cytokines by γδ T cells was required for full development of EAE. These results indicate that γδ T cells are critical for the development of EAE and suggest a therapeutic target in demyelinating disease.