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.     

Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors

Summary: Antigen recognition by the T cell receptor (TCR) complex induces the formation of a TCR signalosome by recruiting various signaling molecules, generating the recognition signals for T cell activation. The activation status and functional outcome are positively and negatively regulated by dynamic organization of the signalosome and by costimulation signals. We have studied the negative regulation of T cell activation, particularly through inhibitory adapters and costimulation receptors that are little expressed in resting cells but are induced upon T cell activation. We described Grb‐associated binder 2 (Gab2) and cytotoxic T lymphocyte antigen‐4 (CTLA‐4) as a representative inhibitory adapter and a negative costimulation receptor, respectively, both of which exhibit negative feedback. Gab2 functions as a signal branch for activation vs. inhibition, as phosphorylation of either Src homology 2 (SH2) domain‐containing leukocyte phosphoprotein of 76 kDa (SLP‐76) or Gab2 by zeta‐associated protein of 70 kDa (ZAP‐70) determines the fate of the response. As a professional inhibitory receptor, CTLA‐4 inhibits T cell response by competition of ligand binding with positive costimulator receptor CD28, and also induces inhibitory signaling. The trafficking and the cell surface expression of CTLA‐4 are dynamically regulated and induced. CTLA‐4 is accumulated in lysosomes and secreted to the T cell–APC contact site upon TCR stimulation. As T cell activation proceeds, these inhibitory adapters and costimulation receptors are induced and suppress/regulate the responses as negative feedback.     

Cell interaction in immune response]

The recognition of antigens by specific T- and B-lymphocytic receptors underlies an immune response. However, the formation of a potential signal for the activation of lymphocytes requires an additional their stimulation (costimulation). The main source of costimulation signals is the interaction of the surface molecules of lymphocytes and accessory cells. The interaction between the T-cell surface molecules CD28 and costimulatory molecules of antigen-presenting cells (CD80 or CD86) is the most important point of the T-helper cell activation. The interaction between B-cell molecule CD40 and T-helper surface molecule CD154 is the key event of B-cell (and other antigen-presenting cell) activation. When costimulation is absent, antigen recognition induces specific lymphocytic anergy or apoptosis. Defects of costimulatory molecular expression or function can cause immunodeficiency. For example, hereditary defect of CD154 expression causes the hyper-IgM syndrome. The soluble forms of some costimulatory molecules are considered to be potential immunomodulators.     

Genetic analysis of CD28 signaling

T cell activation is central to initiating an immune response. Two signals are required: an antigen-specific signal through the T cell receptor (TCR) and an antigen-independent costimulatory signal, primarily through CD28 in naïve T cells. Although many of the molecules involved in TCR signal transduction have been identified, the signaling pathways downstream of CD28 involved in costimulation are not well-defined. Through mutagenesis, we have generated a panel of Jurkat T cell lines in which CD28 costimulation fails to upregulate the RE/AP composite element of the IL-2 promoter. Biochemical analysis and genetic rescue of the defects in these cell lines will lead to a better understanding of CD28 signal transduction.     

Costimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25

Rearrangements in the actin cytoskeleton play a pivotal role for costimulation-induced formation of the immunological synapse and T cell activation. Yet, little is known about the actin-binding proteins that link costimulation to rearrangements in the actin cytoskeleton. Here we demonstrate that phosphorylation of the actin bundling protein L-plastin in response to costimulation through TCR/CD3 plus CD2 or CD28, respectively, is important for the activation of human peripheral blood T lymphocytes (PBT). Mass spectrometry and site-directed mutagenesis revealed that Ser5 represents the only phospho-acceptor site of L-plastin in PBT. Wild-type L-plastin (wt-LPL) and a non-phosphorylatable 5A-L-plastin (5A-LPL) equally relocalized to the immunological synapse between PBT and APC. Yet importantly, cells expressing 5A-LPL showed a significantly lower expression of the T cell activation molecules CD25 and CD69 on the cell surface than cells expressing wt-LPL. This effect is due to a failure in the transport of CD25 and CD69 to the cell surface since the total amount of these proteins within the cells remained unchanged. In conclusion, phosphorylation of the actin bundling protein L-plastin represents a so-far-unknown mechanism by which costimulation controls the transport of activation receptors to the T cell surface.     

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.     

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.     

Failure of correlation between B7 expression and activation of interleukin-2-secreting T cells

It is well established that triggering interleukin-2 (IL-2) secretion by helper T cells requires the T cell to receive at least two discrete signals. One signal is transduced by the CD3 complex, usually as the result of T cell receptor (TcR) occupancy, the second, or co-stimulatory, signal involves a non-cognate interaction between cell surface accessory molecules on the antigen-presenting cell (APC) and the T cell. A molecular interaction that has been implicated in the provision of co-stimulatory signals is that between B7/BB1 on the APC and its ligands, CD28 and CTL-A4 on the T cell. We have studied the ability of HLA-class II antigen-positive human T cells and a population of DR1-expressing transfected human fibroblasts to stimulate a proliferative response by human T cell clones, and by freshly isolated peripheral blood T cells. Despite their high levels of B7 expression, the T cell clones, were unable to induce proliferation or IL-2 secretion by DR-restricted, antigen-specific T cells. In contrast, the DR1-expressing transfectants, that were B7 negative, induced a strong proliferative response. When these two populations of DR-expressing cells were used to stimulate a primary alloresponse the results were reversed, in that the T cell clones induced a strong alloresponse but the transfected fibroblasts induced no proliferation. These results suggest that the expression of B7 may be necessary for co-stimulation of unprimed T cells, but not of established T cell clones. Furthermore the data show that the expression of B7 by an APC does not necessarily lead to IL-2 production or protection from the induction of tolerance. The mechanisms responsible for the inability of these T cells to provide full activation signals when used as APC is currently under investigation.     

Prolonged costimulation is required for naive T cell activation

Costimulation by members of the B7 family of molecules is critical for the activation of naive CD4+ T cells. While prolonged TCR signaling is necessary for T cell activation, the duration of costimulatory signals required has not been established. In this study, murine bone marrow-derived dendritic cells (DC) and na?ve CD4+ T cells were used to determine the temporal costimulatory requirements for naive T cell activation. By blocking CD80/CD86 costimulation at various time points during DC-T cell interaction and using the CFSE technique to assess the dynamics of T cell proliferation, we found that prolonged costimulation was required for naive T cells to enter and progress through the cell cycle over a wide range of peptide concentrations. Prolonged costimulation was also important for IL-2 production and CD25/CD69 expression by naive T cells. Video microscopy demonstrated that DC and naive T cells formed stable conjugates that persisted for more than 6 h. Thus, persistent CD80/CD86 signaling during prolonged interactions with DC allows naive T cells to enter the cell cycle and programs the daughter cells to undergo subsequent divisions.     

T Helper-Independent Activation of Human CD8+ Cells: the Role of CD28 Costimulation

The concept that activation of MHC class I-restricted CD8⁺ cells entirely depends on help from MHC class II-restricted CD4⁺ T cells has recently been supplemented with an alternative model in which CD8⁺ cells can directly be activated by MHC class I-expressing professional antigen-presenting cells (APC), which are able to deliver an accessory signal. The authors analysed the role of CD28-mediated costimulation for T helper cell-independent activation of purified human CD8⁺ T cells in two different in vitro models. Freshly isolated CD8⁺ cells could be activated (proliferation, IL-2 production and cytotoxic activity) by anti-CD3-presenting FcγR⁺ mouse cells transfected with the human CD28 ligand, CD80, as the only accessory signal. On the other hand, activation of CD8⁺ cells by allogeneic MHC class I on EBV-transformed B cells, which express two different CD28 ligands, CD80 and CD86, also proceeded very efficiently (proliferation, cytotoxic activity and CD25 expression), but was either not, or only partially, blocked by anti-CD80 and anti-CD86 MoAb or CTLA-4Ig. This indicates that other costimulatory signals are also effective, and that CD28 triggering is not absolutely required for initial T-cell activation. CsA and CD80/CD86-blocking agents were synergistic in completely inhibiting activation of CD8⁺ cells in the MLR with allogeneic B-cell lines. This combination also induced non-responsiveness of CD8⁺ cells upon restimulation in the absence of blocking agents. Therefore, although professional APC can apparently provide multiple costimulatory signals for direct activation of CD8⁺ T cells, the signal derived from CD80/CD86 is unique in providing CsA-resistance.     

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.     

Locally produced complement fragments C5a and C3a provide both costimulatory and survival signals to naive CD4+ T cells

Costimulatory signals are critical to T cell activation, but how their effects are mediated remains incompletely characterized. Here, we demonstrate that locally produced C5a and C3a anaphylatoxins interacting with their G protein-coupled receptors (GPCRs), C5aR and C3aR, on APCs and T cells both upstream and downstream of CD28 and CD40L signaling are integrally involved in T cell proliferation and differentiation. Disabling these interactions reduced MHC class II and costimulatory-molecule expression and dramatically diminished T cell responses. Importantly, impaired T cell activation by Cd80-/-Cd86-/- and Cd40-/- APCs was reconstituted by added C5a or C3a. C5aR and C3aR mediated their effects via PI-3 kinase-gamma-dependent AKT phosphorylation, providing a link between GPCR signaling, CD28 costimulation, and T cell survival. These local paracrine and autocrine interactions thus operate constitutively in naive T cells to maintain viability, and their amplification by cognate APC partners thus is critical to T cell costimulation.     

Activation of CD4+ T cells by delivery of the B7 costimulatory signal on bystander antigen-presenting cells (trans-costimulation)

Increasing evidence in both murine and human systems suggests that the interaction of the T cell surface antigens CD28/CTLA4 with their ligand B7 on the antigen-presenting cells (APC) is the critical costimulatory pathway involved in the induction of maximal T cell activation and the prevention of induction of anergy. It has also been demonstrated that efficient induction of clonal expansion of normal CD4⁺ T cells requires the delivery of the T cell receptor (TCR) ligand and costimulation by the same APC. We demonstrate here that normal murine CD4⁺ T cells can be efficiently activated by soluble anti-CD3 cross-linked by fixed macrophages and by a costimulatory signal delivered by a bystander APC, B7-transfected L cells. The major factor which determined the ability of an APC to provide costimulation in “trans” was the level of cell surface B7 expression. The requirement for B7 costimulation appears to be at initial stage of TCR engagement since optimal T cell activation was only observed when TCR triggering and B7 costimulatory activity were delivered at same time by different APC. Induction of maximal proliferation of both naive CD45RB^hi and memory CD45RB^lo CD4⁺ T cells was B7 dependent and both populations of cells responded equally well to the B7 costimulation delivered in “trans”. Furthermore, trans-costimulation provided by B7 transfected L cells efficiently prevented the induction of anergy in normal murine CD4⁺ T cells induced by anti-CD3 cross-linked by fixed-resting macrophages. Addition of exogenous interleukin-2 (IL-2) and IL-7 to the primary culture in the absence of B7-transfected L cells or addition of IL-2 to the culture containing the B7 transfectant and CTLA4Ig completely prevented the induction of hyporesponsiveness. These findings raise the possibility that in certain pathological states, CD4⁺ T cells in vivo may be activated by costimulation delivered by bystander APC.     

Costimulatory molecules as immunotherapeutic targets in systemic lupus erythematosus

T cells undergo full and productive activation when they traffic to lymph nodes where they encounter dendritic cells displaying foreign antigen in the context of MHC molecules on their surface. Recognition of these antigen–MHC complexes by the T cell’s receptor for antigen, or T cell receptor, provides the first of two obligate signals needed to drive cell proliferation. The second antigen-independent signal is provided by the costimulatory receptor, CD28, as it engages its ligand on the antigen-presenting cells. Failure of the T cell to receive this second signal after antigen recognition leaves the T cell in a state of anergy. Understanding the role of T cell costimulatory receptors in T cell activation has led to the development of novel approaches for regulating immune responses in subjects with cancer or autoimmune disease by experimentally triggering or blocking costimulatory receptor signaling. In this review, we will discuss, first, several costimulatory pathways known to participate or regulate the progression of autoimmune disease, and, second, how manipulation of T cell costimulation and/or costimulation blockade has been used to treat systemic lupus erythematosus.     

ICAM‐1 and B7‐1 provide similar but distinct costimulation for CD8+ T cells,while CD4+ T cells are poorly costimulated by ICAM‐1

The function of purified ICAM‐1 in costimulating CD4⁺ and CD8⁺ T cell responses has been directly compared to that of B7‐1 in a model system that minimizes contributions of other receptor‐ligand interactions. While B7‐1 costimulates both subsets of T cells, ICAM‐1 is much more effective in the costimulation of CD8⁺ cells. ICAM‐1 also synergizes with B7‐1 for the induction of IL‐2 production in CD8⁺ but not CD4⁺ T cells. These differences are not explained by differences in LFA‐1 receptor expression on the two subsets of T cells. The CD8⁺ T cell response to ICAM‐1 costimulation is associated with increased proliferation and IL‐2 production at levels similar to those seen with B7‐1 costimulation, but clonal expansion in response to ICAM‐1 is not as great due to decreased cell survival. ICAM‐1‐mediated costimulation is effective for both naive and memory CT8⁺ T cells, is independent of CD28 engagement, and does not appear to be due solely to effects on adhesion. These results suggest that ICAM‐1‐dependent, B7‐independent costimulation may be important in initiating a CTL response to class I antigen presented by cells that are not professional APC.     

Modification of accessory molecule signaling

The concept of costimulation, the requirement for an independent accessory cellular activation signal that supplements the signal delivered to a lymphocyte by antigen, has been a focal point of progress in understanding the regulation of the immune system. While considerable attention has been directed to new developments related to the activation of cells of the innate immune system through Toll-like receptors, resulting in the production of soluble mediators, augmented expression of cell surface costimulatory molecules on antigen-presenting cells is arguably the most significant early outcome of immune system activation. It is those cell surface molecules that provide the essential afferent costimulatory signals to T cells of the adaptive immune response. Once fully activated, T cells express their own cell surface accessory molecules that permit those T cells to instruct interacting B cells, macrophages, and dendritic cells to further implement an effective immune response. Significantly for patients with autoimmune diseases, the manipulation of costimulatory signals represents a rational and effective approach to modulating the chronic immune system activation that characterizes those diseases. Further elucidation of the complexities of members of the accessory molecule families and their functions should lead to an ever greater capacity for therapeutic modulation of the immune response in autoimmune and inflammatory diseases.     

CD28 costimulation is required not only to induce IL-12 receptor but also to render janus kinases/STAT4 responsive to IL-12 stimulation in TCR-triggered T cells

The activation of resting T cells for the acquisition of various functions depends on whether CD28 costimulatory signals are provided upon T cell receptor stimulation. Here, we investigated how CD28 costimulation functions to allow TCR-triggered resting T cells to acquire IL-12 responsiveness. When T cells are stimulated with low doses of anti-CD3 mAb, CD28 costimulation was required for the optimal levels of IL-12 receptor (IL-12R) expression. However, stimulation of T cells with high doses of anti-CD3 alone induced comparable levels of IL-12R expression to those induced upon CD28 costimulation. Nevertheless, there was a substantial difference in IL-12 responsiveness between these two groups of T cells: compared to anti-CD28-costimulated T cells, T cells that were not costimulated with anti-CD28 exhibited decreased levels of Janus kinases (JAK) JAK2/TYK2 and STAT4 phosphorylation and IFN-y production following IL-12 stimulation. Importantly, STAT6 phosphorylation following IL-4 stimulation was not decreased in anti-CD28-uncostimulated T cells. These resutls indicate that CD28 costimulation not only contributes to up-regulating IL-12R expression but is also required to render JAKs/STAT4 responsive to IL-12 stimulation.     

Expression and functional significance of CTLA-4, a negative regulator of T cell activation

The generation of an effective immune response involves antigen-specific T cell expansion and differentiation of effector function. T cell activation requires at least two distinct signals, including signaling via the antigen-specific T cell receptor (TCR) and a costimulatory pathway. Antigen stimulation of T cells can lead either to a productive immune response, characterized by proliferation, differentiation, clonal expansion and effector function, or, in the absence of an appropriate costimulation, to a state of long-lasting unresponsiveness, termed anergy. Anergic T cells fail to proliferate and secrete cytokines in response to secondary stimulation. The interaction between the costimulatory molecule CD28 on T cells and members of the B7 family on antigen-presenting cell results in upregulation of T cell proliferation and cytokine production and induces the expression of the anti-apoptotic protein Bcl-xl. Based of these findings, the two-signal requirement model for T cell activation is generally accepted today. The negative regulatory mechanisms during T cell activation are not well understood, but they are crucial for the maintainance of lymphocyte homeostasis. For several years the functional role of the enigmatic CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4) in T cell activation has been both obscure and controversial. CTLA-4 was initially supposed to provide a costimulatory signal in conjunction with TCR/CD3 signaling. Today we know that CD28 and CTLA-4 molecules may have diametrically opposed functions: signaling via CD28, in conjunctive with TCR, is required for T cell activation, while signaling via CTLA-4 is a negative signal that inhibits T cell proliferation. How the T cell integrates signals through the TCR/CD3 complex, CD28 and CTLA-4 to initiate, maintain and terminate antigen-specific immune response is in fact not fully clarified. In this review, we will focus on the emerging role of CTLA-4 as a negative regulator of T lymphocyte activation and its role in the dynamic interplay of activatory and inhibitory signals.     

Defect in TCR-CD3zeta signaling mediates T cell hypo-responsiveness in mesenteric lymph node

Mesenteric lymph node (MLN) in gut-associated lymphoid tissue plays obligatory roles in the induction of oral tolerance and ignorance to commensals. However, little is known about its immunological characteristics. In this study, we investigated the hypo-responsiveness of MLN CD4(+) T cells, comparing them with spleen CD4(+) T cells. MLN CD4(+) T cells were hypo-proliferative and expressed low levels of Th1-type cytokines in response to antigen or CD3/T cell receptor (TCR) stimulation. The hypo-responsiveness of MLN CD4(+) T cells is linked neither with changes in the regulatory T cell population (CD4(+)CD25(+), CD4(+)Foxp3(+)) nor the apoptotic population. Rather, MLN CD4(+) T cells showed deformity of T cell:APC conjugation and reduced expression of TCR signaling molecules such as CD3zeta, PLC-gamma1, PKC-theta, Zap70, with reduced phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs). Among the alterations in TCR signaling molecules, defective CD3zeta expression is the most evident, and reversal of the anergic state by CD3/CD28 costimulation restored CD3zeta expression levels. Collectively, we suggest that reduced CD3zeta expression and defects in TCR signaling mediate the anergy state of MLN CD4(+) T cells, which play a critical role in maintenance of mucosal tolerance in gut-associated lymphoid tissue.