Intracellular CD154 expression reflects antigen-specific CD8+ t cells but shows less sensitivity than intracellular cytokine and MHC tetramer staining

A recent report showed that analysis of CD154 expression in the presence of the secretion inhibitor Brefeldin A (Bref A) could be used to assess the entire repertoire of antigen-specific CD4(+) T helper cells. However, the capacity of intracellular CD154 expression to identify antigen-specific CD8(+) T cells has yet to be investigated. In this study, we compared the ability of intracellular CD154 expression to assess antigen-specific CD8(+) T cells with that of accepted standard assays, namely intracellular cytokine IFN-gamma staining (ICS) and MHC class I tetramer staining. The detection of intracellular CD154 molecules in the presence of Bref A reflected the kinetic trend of antigen-specific CD8+ T cell number, but unfortunately showed less sensitivity than ICS and tetramer staining. However, ICS levels peaked and saturated 8 h after antigenic stimulation in the presence of Bref A and then declined, whereas intracellular CD154 expression peaked by 8 h and maintained the saturated level up to 24 h post-stimulation. Moreover, intracellular CD154 expression in antigen-specific CD8+ T cells developed in the absence of CD4(+) T cells changed little, whereas the number of IFN-gamma-producing CD8(+) T cells decreased abruptly. These results suggest that intracellular CD154 could aid the assessment of antigen-specific CD8(+) T cells, but does not have as much ability to identify heterogeneous CD4(+) T helper cells. Therefore, the combined analytical techniques of ICS and tetramer staining together with intracellular CD154 assays may be able to provide useful information on the accurate phenotype and functionality of antigen-specific CD8(+) T cells.     

A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles

Recently activated, but not resting, CD4(+) T cells express CD154, providing costimulatory signals to B cells and antigen-presenting cells (APCs). Therefore, de novo CD154 expression after stimulation identifies antigen-specific CD4(+) T cells. Previous assays were limited by the transient nature of surface CD154 expression; we overcame this by including fluorescently conjugated CD154-specific antibody during stimulation. Our assay is fully compatible with intracellular cytokine staining, and can be used for stimulations as long as 24 h. Notably, it is nonlethal, providing a means to purify viable antigen-specific CD4(+) T cells for further analysis. Using this assay, we found that stimulated cells expressing tumor necrosis factor (TNF)-alpha, interleukin (IL)-2 or interferon (IFN)-gamma were predominantly CD154(+). Furthermore, some cells expressing none of these cytokines also expressed CD154, suggesting that CD154 marks cells with other effector functions. For vaccine- or pathogen-specific responses, we found substantial heterogeneity in expression of CD154 and cytokines, suggesting previously unrecognized diversity in abilities of responding cells to stimulate APCs through CD40.     

CD154 blockade alters innate immune cell recruitment and programs alloreactive CD8+ T cells into KLRG-1(high) short-lived effector T cells

CD154/CD40 blockade combined with donor specific transfusion remains one of the most effective therapies in prolonging allograft survival. Despite this, the mechanisms by which these pathways synergize to prevent rejection are not completely understood. Utilizing a BALB/c (H2-K(d)) to B6 (H2-K(b)) fully allogeneic skin transplant model system, we performed a detailed longitudinal analysis of the kinetics and magnitude of CD8(+) T cell expansion and differentiation in the presence of CD154/CD40 pathway blockade. Results demonstrated that treatment with anti-CD154 vs. DST had distinct and opposing effects on activated CD44(high) CD62L(low) CD8(+) T cells in skin graft recipients. Specifically, CD154 blockade delayed alloreactive CD8(+) T cell responses, while DST accelerated them. DST inhibited the differentiation of alloreactive CD8(+) T cells into multi-cytokine producing effectors, while CD40/CD154 blockade led to the diminution of the KLRG-1(low) long-lived memory precursor population compared with either untreated or DST treated animals. Moreover, only CD154 blockade effectively inhibited CXCL1 expression and neutrophil recruitment into the graft. When combined, anti-CD154 and DST acted synergistically to profoundly diminish the absolute number of IFN-γ producing alloreactive CD8(+) T cells, and intra-graft expression of inflammatory chemokines. These findings demonstrate that the previously described ability of anti-CD154 and DST to result in alloreactive T cell deletion involves both delayed kinetics of T cell expansion and differentiation and inhibited development of KLRG-1(low) memory precursor cells.     

Dependency of direct pathway CD4+ T cells on CD40-CD154 costimulation is determined by nature and microenvironment of primary contact with alloantigen

Blockade of the CD40-CD154 costimulatory pathway can inhibit CD4(+) T cell-mediated alloimmune responses. The aim of this study was to define the in vivo requirement for CD40-CD154 costimulation by CD4(+) T cells that respond to alloantigen following direct recognition. We used TCR-transgenic CD4(+) T cells that are reactive to the MHC class II alloantigen, H2A(s). An experimental in vivo model was established that allowed direct comparison of the fate of a trace population of H2A(s)-reactive CD4(+) T cells when challenged with different forms of H2A(s+) alloantigen under conditions of CD40-CD154 costimulation blockade. In this study, we demonstrate that an i.v. infusion of H2A(s+) leukocytes in combination with anti-CD154 therapy rapidly deletes H2A(s)-reactive CD4(+) T cells. In contrast, following transplantation of an H2A(s+) cardiac allograft, H2A(s)-reactive CD4(+) T cell responses were unaffected by blocking CD40-CD154 interactions. Consistent with these findings, combined treatment with donor leukocytes and anti-CD154 therapy was found to be more effective in prolonging the survival of cardiac allografts compared with CD154 mAb treatment alone. The dominant mechanism by which donor leukocyte infusion and anti-CD154 therapy facilitate allograft acceptance is deletion of donor-reactive direct pathway T cells. No evidence for the generation of regulatory cells by this combined therapy was found. Taken together, these results clearly demonstrate that naive alloreactive CD4(+) T cells have distinct requirements for CD40-CD154 costimulation depending on the form and microenvironment of primary alloantigen contact.     

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.     

IL-2 and IL-15 regulate CD154 expression on activated CD4 T cells

The cellular and humoral immune system is critically dependent upon CD40-CD154 (CD40 ligand) interactions between CD40 expressed on B cells, macrophages, and dendritic cells, and CD154 expressed primarily on CD4 T cells. Previous studies have shown that CD154 is transiently expressed on CD4 T cells after T cell receptor engagement in vitro. However, we found that stimulation of PBLs with maximal CD28 costimulation, using beads coupled to Abs against CD3 and CD28, led to a very prolonged expression of CD154 on CD4 cells (>4 days) that was dependent upon autocrine IL-2 production. Previously activated CD4 T cells could respond to IL-2, or the related cytokine IL-15, by de novo CD154 production and expression without requiring an additional signal from CD3 and CD28. These results provide evidence that CD28 costimulation of CD4 T cells, through autocrine IL-2 production, maintains high levels of CD154 expression. This has significant impact on our understanding of the acquired immune response and may provide insight concerning the mechanisms underlying several immunological diseases.     

Role of CD40-dependent down-regulation of CD154 in impaired induction of CD154 in CD4(+) T cells from HIV-1-infected patients

CD40-CD154 interaction is pivotal for cell-mediated immunity. There are contradictory reports on whether HIV-1 infection impairs CD154 induction. The interaction between CD40 and CD154 is important not only because it results in activation of APCs but also because it controls CD154 by diminishing expression of this molecule. Compared with healthy controls, CD4(+) T cells from HIV-1(+) patients had impaired induction of CD154 when T cell activation was mediated by CD40(+) APCs. In contrast, T cell activation in the absence of these cells resulted in normal CD154 expression. CD154 induction in HIV-1(+) patients and controls were similar upon blockade of CD40-CD154 binding. Defective regulation of CD154 appeared to occur downstream of the control of mRNA levels because up-regulation of CD154 mRNA was not impaired by HIV-1 infection. This work identifies CD40 as a mediator of impaired CD154 induction in HIV-1 infection and explains why this defect was not detected by studies where T cell activation was triggered independently of CD40(+) APCs. In addition, dysregulation of CD154 in HIV-1 infection likely contributes to immunodeficiency because diminished expression of CD154 induced by CD40 is of functional relevance, resulting in decreased dendritic cell maturation.     

GRAIL is up-regulated in CD4+ CD25+ T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype

GRAIL (gene related to anergy in lymphocytes) is an ubiquitin-protein isopeptide ligase (E3) ubiquitin ligase necessary for the induction of CD4(+) T cell anergy in vivo. We have extended our previous studies to characterize the expression pattern of GRAIL in other murine CD4(+) T cell types with a described anergic phenotype. These studies revealed that GRAIL expression is increased in naturally occurring (thymically derived) CD4(+) CD25(+) T regulatory cells (mRNA levels 10-fold higher than naive CD25(-) T cells). Further investigation demonstrated that CD25(+) Foxp3(+) antigen-specific T cells were induced after a "tolerizing-administration" of antigen and that GRAIL expression correlated with the CD25(+) Foxp3(+) antigen-specific subset. Lastly, using retroviral transduction, we demonstrated that forced expression of GRAIL in a T cell line was sufficient for conversion of these cells to a regulatory phenotype in the absence of detectable Foxp3. These data demonstrate that GRAIL is differentially expressed in naturally occurring and peripherally induced CD25(+) T regulatory cells and that the expression of GRAIL is linked to their functional regulatory activity.     

Expression of the interleukin-7 receptor alpha chain (CD127) on virus-specific CD8+ T cells identifies functionally and phenotypically defined memory T cells during acute resolving hepatitis B virus infection

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.     

Full development of Th2 immunity requires both innate and adaptive sources of CD154

The CD40-CD154 interaction is critical for Th2 response generation during helminth infection and following immunization with helminth-conditioned dendritic cells, yet the key cellular sources of these molecules have still to be defined in vivo. In this study, we demonstrate that the requirement for CD40 expression during murine Th2 response induction is restricted exclusively to the Ag-bearing dendritic cells. In contrast, development of full Th2 immunity required CD154 expression on multiple populations. In this respect, optimal production of IL-5, IL-10, and IL-13 was dependent upon CD154 expression by both CD4(+) T cells and non-lymphoid cells. IL-4 production had less stringent costimulatory requirements, with expression of CD154 on either non-lymphoid cells or T cells alone being sufficient to enable production of this archetypal Th2 cytokine. Disparities in CD154 requirements for T cell and B cell responses were revealed during experimental schistosomiasis where, even in the face of robust Th2 generation, B cell class-switching was entirely dependent upon expression of CD154 by the lymphoid compartment. These data help define the costimulatory interactions that occur during the generation of Th2 immunity, and challenge the widely held view that CD154 expressing T cells are the sole contributors in this process.     

Expression of CD154 by a simian immunodeficiency virus vector induces only transitory changes in rhesus macaques

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Use of soluble MHC class II/peptide multimers to detect antigen-specific T cells in human disease

Most techniques that identify antigen-specific T cells are dependent on the response of these cells to the relevant antigen in culture. Soluble multimers of MHC molecules, when occupied with the same peptide, will bind selectively to T cells specific for that MHC/peptide complex. Techniques to produce fluorescent MHC class II/peptide multimers have recently been developed. These reagents provide a method to facilitate detection and isolation of antigen-specific CD4⁺ T cells and they represent a new research tool to study these cells in patients with immune-mediated diseases.     

Sustained expression of CD154 (CD40L) and proinflammatory cytokine production by alloantigen-stimulated umbilical cord blood T cells

Recent data suggests that graft-versus-host disease (GVHD) is initiated by host APCs. Blockade of CD40:CD154 interactions between APCs and T cells in vivo induces T cell tolerance to host alloantigen and dramatically reduces GVHD. Because allogeneic cord blood (CB) transplantation results in a lower incidence and severity of acute GVHD compared with bone marrow transplantation, we have investigated whether CB T cells can express CD154 in response to stimulation by allogeneic monocyte-derived dendritic cells (MDDC) and have used 5- (and 6-)carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling in combination with intracellular cytokine analysis to assess the proliferation and cytokine profiles of alloantigen-responsive cells. CB T cells stimulated with allogeneic MDDC showed stronger proliferation than adult blood T cells. Surface CD154 expression was detected in the actively dividing CFSElow populations of both the CD4+ and CD4- subsets and was brightest in cells that had divided the most. Assessment of supernatants from MDDC-stimulated CB and adult blood T cells showed no significant difference in the levels of either IFN-gamma or TNF-alpha, but CB T cell supernatants did show a significant lack of detectable IL-2. Intracellular cytokine analysis revealed that dividing CB T cells had been primed to produce IFN-gamma, TNF-alpha, and IL-2 on restimulation. Further phenotype analysis showed that 75% of CB T cells producing IFN-gamma were CD8+. These data suggest that MDDC-stimulated CB T cells express functional CD154 and provide enough costimulation for dendritic cells to prime naive CD8+ CB T cells and induce type 1 cytokine production.     

IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes

Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity. We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12. Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system. We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization. IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses. In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response. IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed.     

CD154 (CD40 ligand)

CD40 ligand, a type II transmembrane protein recently renamed CD154, was originally considered restricted to activated T lymphocytes, functioning as a mediator of T cell-dependent B cell activation, proliferation, and differentiation. However, the spectrum of CD154 expression and function has broadened considerably during recent years, establishing new roles as a central mediator of immunity and inflammation for this member of the tumor necrosis factor (TNF) gene superfamily. The emerging picture indicates that ligation of the receptor CD40 via CD154, most potently in its trimeric form, functions in two ways. CD154 modulates physiologic processes, such as T cell-mediated effector functions and general immune responses required for appropriate host defense, but also triggers the expression of pro-inflammatory mediators, such as cytokines, adhesion molecules, and matrix degrading activities, all of which are associated with the pathogenesis of chronic inflammatory diseases, e.g., autoimmune disorders, arthritis, atherosclerosis, and cancer. Accordingly, CD40/CD154 interactions have advanced as a potential therapeutic target for these diseases, whereby two opposing strategies, interruption as well as enhancement of CD40 signaling, are explored for beneficial outcomes.     

Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression

This protocol details a method to identify CD4+ T cells that respond to antigens. The method relies on detection of CD154, a costimulatory cell surface protein that is expressed by CD4+ T cells upon activation, and can be used to purify live CD4+ T cells of diverse function. To detect CD154, fluorescently labeled antibodies are cultured with cell samples, peptides (or whole antigens) and monensin during a 6- to 24-h stimulation period. (Note that the assay is not compatible with brefeldin A.) After stimulation, cells are stained with any other antibodies of interest and then are analyzed by flow cytometry or purified by cell sorting. Unlike other assays, this method allows simultaneous assessment of other cell phenotypes or functions, is compatible with downstream RNA-based assays and preserves cell viability. This protocol can be completed in 9 h.