CD40-CD40L interaction in immunity against protozoan infections

Activation of the immune system against protozoan infections relies particularly on two specific signals provided by cognate interaction of T cells with antigen presenting cells (APCs). The first signal is attributed to binding of the T-cell receptor (TCR) to peptide/MHC complexes on the surface of APCs, whereas the second signal is triggered through binding of several costimulatory molecules on the surface of APCs with their corresponding receptors on T cells. Among these costimulatory signallings, CD40/CD40L interactions have been particularly investigated in protozoan infection models with regard to their potential to amplify cell-mediated immunity against intracellular parasites. This article reviews current studies of the potential role of CD40/CD40L interaction in the modulation of immune responses against some protozoan parasites and highlights recent developments regarding manipulation of this interaction for promoting control of parasite infections.     

Accessory molecule regulation of naive CD4 T cell activation

Naive CD4 T cell activation is a complex process involving many steps. T cell receptor (TCR) signals, provided by interaction with peptide/MHC on antigen-presenting cells (APC), control many events associated with activation. The extent of TCR signaling and the magnitude of the T cell response is in turn controlled by accessory molecules on APC, which stabilize T-APC interactions. Full T cell activation additionally requires multiple costimulatory signals, generated upon ligation of T cell coreceptors by accessory molecules, and these lead to IL-2 production, proliferation and differentiation of the naive cell into an effector state. This review summarizes the role played by accessory molecules in naive CD4 activation and discusses how integration of signals from these molecules, with signals from the TCR, may determine the outcome of T-APC interaction. The available data provide explanations for why only APC which express high levels of multiple costimulatory/adhesion molecules, such as dendritic cells and activated B cells, induce efficient naive T cell responses, and suggest that ICAM-1/LFA-1 and B7/CD28 interactions are major pathways used to initiate naive T cell activation.     

CD4+ T‐cell activation is differentially modulated by bacteria‐primed dendritic cells,but is generally down‐regulated by n‐3 polyunsaturated fatty acids

Appropriate activation of CD4⁺ T cells is fundamental for efficient initiation and progression of acquired immune responses. Here, we showed that CD4⁺ T‐cell activation is dependent on changes in membrane n‐3 polyunsaturated fatty acids (PUFAs) and is dynamically regulated by the type of signals provided by dendritic cells (DCs). Upon interaction with DCs primed by different concentrations and species of gut bacteria, CD4⁺ T cells were activated according to the type of DC stimulus. The levels of CD80 were found to correlate to the levels of expression of CD28 and to the proliferation of CD4⁺ T cells, while the presence of CD40 and CD86 on DCs inversely affected inducible costimulator (ICOS) and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) levels in CD4⁺ T cells. For all DC stimuli, cells high in n‐3 PUFAs showed reduced ability to respond to CD28 stimulation, to proliferate, and to express ICOS and CTLA‐4. Diminished T‐cell receptor (TCR) and CD28 signalling was found to be responsible for n‐3 PUFA effects. Thus, the dietary fatty acid composition influences the overall level of CD4⁺ T‐cell activation induced by DCs, while the priming effect of the DC stimuli modulates CD80, CD86 and CD40 levels, thereby affecting and shaping activation of acquired immunity by differential regulation of proliferation and costimulatory molecule expression in CD4⁺ T cells.     

T cells compete by cleaving cell surface CD27 and blocking access to CD70‐bearing APCs

T cells compete against each other for access to molecules on APCs in addition to peptide/MHC complexes. However, the identity of cell surface molecules that influence T‐cell competition, other than peptide/MHC, have yet to be defined. Here, we identify CD70, a TNF ligand expressed on activated APCs, as an important mediator of T‐cell competition for APCs. Upon engagement of CD27 by CD70, CD27 is proteolytically cleaved from the surface of the interacting CD8⁺ T cell and captured by CD70 expressing dendritic cells. The capture of CD27 effectively masks CD70 on APCs, disallowing the interaction with CD27 on other competing T cells. Collectively, our data indicate that T cells compete against each other for access to the TNF‐ligand CD70, an interaction that affects the duration and potency of T cell/DC interactions, thus influencing the repertoire of responding CD8⁺ T cells to self or foreign antigens.     

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.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

CD40 is necessary for activation of naïve T cells by a dendritic cell line in vivo but not in vitro

The importance of CD40–CD40L interactions during CD4⁺ T‐cell activation has been extensively investigated over the years; however, it still remains questionable whether the interaction is a prerequisite for dendritic cell (DC)‐mediated antigen‐specific priming in vivo. Naïve CD4⁺ T cells require two signals for proper activation and induction of differentiation: signal 1 is provided by peptide antigens in the context of the major histocompatibility complex (MHC) class II, while signal 2 is delivered by costimulatory molecules such as CD80 or CD86 present on the antigen‐presenting cell (APC). It is well known that the expression of CD80/CD86 is upregulated after interaction between CD40 on APCs and CD40L expressed by at least partly activated T cells. We used a DC line, JawsII, to compare the importance of CD40 expression and downstream signalling in vitro and in vivo. JawsII cells represent pre‐immature bone marrow‐derived DCs expressing low levels of MHC molecules, low levels of B7 molecules and no CD40. We have previously shown that JawsII cells, despite the lack of CD40 expression, are capable of priming naïve allogeneic T cells in vitro. In correlation with the current literature, we present data showing that constitutive expression of CD40 significantly increases the priming capacity of JawsII cells in vitro. In addition, we show that CD40 expression is required for JawsII cell‐dependent T‐cell priming in vivo.     

The role of CD28 and cytotoxic T‐lymphocyte antigen‐4 (CTLA-4) in regulatory T‐cell biology

Summary: The profound influence of CD28 and cytotoxic T‐lymphocyte antigen‐4 (CTLA‐4) on T‐cell immunity has been known for over a decade, yet the precise roles played by these molecules still continue to emerge. Initially viewed as molecules that provide cell‐intrinsic costimulatory and inhibitory signals, recent evidence suggests that both CD28 and CTLA‐4 are also important in the homeostasis and function of a population of suppressive cells, termed regulatory T cells (Tregs). Here we review the main features of the CD28 and CTLA‐4 system and examine how these impact upon Treg biology.     

Effect of CD80 and CD86 on T Cell Cytokine Production

Conjugation of the T cell receptor (TCR) with antigen/MHC proteins must be accompanied by conjugation of T cell counterreceptors (CD28 or CTLA-4) with costimulatory molecules CD80 or CD86 (B7-1 or B7-2) on antigen presenting cells (APC) to avert T cell anergy, and to provide essential signals for T cell activation and cytokine production. However, T cells and APC express changing patterns of counterreceptors and costimulatory molecules during the immune response. To determine the involvement of CD80 and CD86 in costimulation of T cell cytokine production, T cells were incubated with peritoneal exudate macrophages, which express CD80 and CD86, and stimulated in vitro for 48 or 72 hrs with anti-CD3 in the presence or absence of blocking antibody to CD80 or CD86. Alternatively, enriched anti-CD3 stimulated T cells were costimulated with antibody to CD28 and CTLA-4. Production of T cell IL-2, IL-4, and IL-5 was depressed in the presence of anti-CD86 but not anti-CD80. Production of IFN-γ was significantly blocked by either anti-CD80 and anti-CD86. Anti-CD28 was a potent costimulator of IFN-γ and IL-2 production, but a less potent costimulator of IL-4 and IL-5 production. The data suggest that T cell counterreceptors and APC costimulatory molecules act with varying efficacies at stimulating production of T cell cytokines.     

Integrated T-cell receptor and costimulatory signals determine TGF-β-dependent differentiation and maintenance of Foxp3+ regulatory T cells

Foxp3‐expressing Tregs play a non‐redundant role in protecting against immune pathologies. Foxp3⁺ Tregs can arise intra‐ and extra‐thymically, however, the signals directing their differentiation and maintenance in the periphery are not well understood. We show that stimulation of mouse naïve CD4⁺ T cells in vitro with optimal doses of anti‐CD3/anti‐CD28 resulted in high frequencies of Foxp3⁺ T cells via a TGF‐β‐dependent mechanism. Addition of TGF‐β and retinoic acid overcame the inhibition of Foxp3 expression observed during high‐strength anti‐CD3/anti‐CD28 stimulation. Reducing the strength of TCR or costimulatory signals with inhibitors of mammalian target of rapamycin (mTOR) or MEK/ERK signalling also enhanced expression of Foxp3 in a TGF‐β‐dependent manner. Addition of TGF‐β was further required to maintain Foxp3 expression in ex vivo derived Foxp3⁺ Tregs upon prolonged anti‐CD3/anti‐CD28 signalling. Thus, induction/maintenance of Foxp3 expression by TGF‐β is modulated by the integrated strength of TCR/costimulatory signals.     

Costimulation by CD28 sFv expressed on the tumor cell surface or as a soluble bispecific molecule targeted to the L6 carcinoma antigen

Interaction of the CD80 (B7-1) and CD86 (B7-2) molecules on antigen presenting cells with the receptors CD28 and CTLA-4 on T cells generates signals important in the regulation of immune responses. Because this receptor system involves multiple receptor-ligand interactions, determining the function for individual receptors has been difficult. One approach is the use of antibodies and their derivatives with singular specificity as substitute ligands to explore the activities of these molecules. We have constructed recombinant mono-and bi-specific sFv molecules specific for the CD28 receptor that are capable of binding and generating costimulatory signals to activate T cells. We demonstrate that these soluble molecules are capable of higher levels of costimulation than soluble CD80Ig at equivalent concentrations. We also constructed artificial adhesion receptors on the cell surface using two different CD28-specific sFvIgs fused to the CD80 cytoplasmic and transmembrane domains. In this report, we compared costimulation by a soluble bispecific (αCD28-α6) single chain sFvIg fusion protein to that generated by L6 antigen positive (L6+) H3347 tumor cells transduced with cell surface expressed forms of aCD28 sFv's. We show that the bispecific protein can target potent CD28 costimulatory activity to L6+ tumor cells in vitro . We also show that transfection of the cell surface forms of the two different CD28 sFvIgs into H3347 tumor cells allows them to generate significant costimulatory signals to activated T cells. Finally, we demonstrate that tumor cell presentation of either the soluble bispecific or transduced cell surface sFv generate similar costimulatory effects resulting in T cell activation.     

特发性血小板减少性紫癜共刺激分子表达的研究进展

特发性血小板减少性紫癜（ITP）是一种自身免疫性疾病,其发病机制尚未完全明确,目前已证实,ITP患者体内存在T、B淋巴细胞的异常活化和B淋巴细胞依赖Th细胞辅助而产生自身抗血小板抗体。在T、B淋巴细胞相互作用和产生自身抗体的病理过程中,CD80、CD86与其配体CD28、CTLA4结合及CD40与其配体CD40L相互作用提供的共刺激信号起了重要的作用。研究表明共刺激分子过度表达有可能激活自身反应性T淋巴细胞,导致自身免疫病的发生。     

Molecular Basis for the Autoreactivity Against Thyroid Stimulating Hormone Receptor

The two signal model of T cell activation predicts that a second costimulatory signal provided by antigen presenting cells (APC) is required in conjunction with the antigenic signal to trigger T cell activation. Considerable evidence indicates that indeed, T cell activation requires such a costimulatory signal which results, at least in part, from the interaction of CD28 with its ligands B7 expressed on all antigen-presenting cells (APC). The second prediction of the two signal model is that T cell receptor engagement in the absence of such a costimulatory signal would lead to specific inactivation of antigen reactive cells. Thus, tissue cells that do not express costimulatory signals would not trigger T cell activation but rather lead to specific inactivation of auto-reactive T cells. By such a model, tolerance to peripheral antigens would be permanently re-established.

We review here the evidence suggesting that the CD28-B7 costimulatory pathway might play an important role in T cell tolerance and in the development of autoimmune responses.     

Evidence for the involvement of T cell costimulation through the B-7/CD28 pathway in atherosclerotic plaques from apolipoprotein E knockout mice

Antigen-specific T cell activation is thought to influence the initiation and progression of the atherosclerotic plaque. For the T cell activation program to be functional, it is essential not only to facilitate T cell receptor engagement by antigen-presenting cells (APC), but also to deliver requisite costimulatory signals. The most widely comprehended costimulatory pathway involves the ligation of CD28 expressed on T cells by CD80 and CD86 upregulated on APCs. We hypothesized that signals of costimulation should be localized to plaque areas that correspond to the presence of the provoking autoantigen. Atherosclerotic plaques from apoE knockout (KO) mice at different stages of maturation were studied immunohistochemically by monoclonal antibodies to respective markers of primary and secondary lymphocyte activation. Subsequently, flow cytometry studies were conducted in spleen cells from C57BL/6 and apoE KO mice aiming to determine whether the presence of plaques is associated with increased expression of costimulatory signals. We found that regardless of the maturation stage, CD80 and CD86 were evident within the plaques, and colocalized with the presence of markers of dendritic cells and with expression of the extensively investigated autoantigen-oxidized LDL. FACS analysis studies showed that splenocytes from aged atherosclerotic apoE KO mice exhibited increased expression on B cells (which represent APCs) of CD80 and CD86 as compared to their young apoE or na?ve C57BL/6 litters that have no plaques. These results suggest that primary T cell activation may assume a functional proinflammatory program within the plaque by involvement of CD28/B7 costimulatory pathway.     

Differences in responsiveness to CD3 stimulation between naive and memory CD4+ T cells cannot be overcome by CD28 costimulation

Activation of naive CD4⁺ T cells is essential for the induction of primary immune responses. However, this subset is less responsive to signaling via T cell receptor/CD3 (TcR/CD3) complex than memory CD4⁺ cells. For mitogenic activation of T cells, in addition to triggering of the TcR/CD3 complex, costimulatory signals are required that can be generated by surface structures present on the antigen-presenting cells. We investigated here whether differences in responsiveness to TcR/CD3 stimulation of naive and memory cells can be overcome by the costimulatory pathway B7/CD28. Using a B7-dependent system we show that even in the presence of optimal CD28 costimulation, CD4⁺ naive cells still have more stringent TcR/CD3 activation requirements than memory cells. Furthermore, titration of the B7 signal revealed that for activation of naive CD4⁺ cells a higher level of cross-linking of CD28 molecules is required than for memory cells. Thus, our results show that at least two signals are required for activation of both CD4⁺ memory and naive cells, but that for activation of naive cells higher cross-linking of both CD3 and CD28 molecules is necessary.     

CD252 regulates mast cell mediated,CD1d‐restricted NKT‐cell activation in mice

The interaction between tissue‐resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d‐restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha‐galactosylceramide. In contrast, in BM‐derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE‐mediated degranulation. Activated BM‐derived MCs expressing surface CD1d and loaded with alpha‐galactosylceramide were found to induce iNKT‐cell proliferation and the release of IFN‐γ, IL‐13, and IL‐4 in a CD1d‐restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC‐induced IFN‐γ release and iNKT‐cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT‐cell release of both IFN‐γ and IL‐13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses.     

Activated B cells modified by electroporation of multiple mRNAs encoding immune stimulatory molecules are comparable to mature dendritic cells in inducing in vitro antigen-specific T-cell responses

Ex-vivo-activated B cells are an alternative source of antigen-presenting cells (APCs) and a potential replacement for dendritic cells (DCs) in immunotherapy. However, the ability of ex-vivo-activated B cells to function as potent APCs has been a concern, especially when compared to DCs. Our study investigated whether modification of activated B cells with immune stimulatory molecules could enhance the ability of activated B cells to stimulate T cells. We show that murine splenic B cells, activated with a combination of Toll-like receptor agonist and agonistic anti-CD40, stimulated antigen-specific CD8+ T cells more efficiently than cells activated with Toll-like receptor agonist or anti-CD40 alone, probably by down-regulation of the immune regulatory cytokine interleukin-10 (IL-10). However, the activated B cells were still poor T-cell stimulators compared to mature DCs. Therefore, we modified the activated B cells by simultaneous electroporation of multiple messenger RNAs encoding costimulatory molecules (OX40L and 4-1BBL), cytokines (IL-12p35 and IL-12p40) and antigen. We found that de novo expression or overexpression of OX40L, 4-1BBL and IL-12p70 on activated B cells synergistically enhanced proliferation as well as IL-2 and interferon-gamma production by CD8+ T cells. Furthermore, the RNA-modified activated B cells induced antigen-specific cytotoxic T lymphocyte responses as efficiently as mature DCs in vitro. Unexpectedly, modified activated B cells were inferior to mature DCs at in vivo induction of CD8+ T-cell responses. In summary, activated B cells modified to express immune stimulatory molecules are a potent alternative to DCs in immunotherapy.