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.     

The male minor transplantation antigen preferentially activates recipient CD4+ T cells through the indirect presentation pathway in vivo

To evaluate the priming and trafficking of male Ag-reactive CD4(+) T cells in vivo, we developed an adoptive transfer model, using Marilyn (Mar) TCR transgenic T cells that are specific for the H-Y minor transplantation Ag plus I-A(b). By manipulating donor and recipient strain combinations, we permitted the Mar CD4(+) T cells to respond to the H-Y Ag after processing and presentation by recipient APCs (indirect pathway), or to the male Ag as expressed on donor APCs (direct pathway). Mar CD4(+) T cells responding through the indirect pathway specifically proliferated and expressed activation markers between days 2 and 4 posttransplant, migrated to the graft 2-3 days before cessation of graft heartbeat, and were detected in close proximity to transplant-infiltrating recipient APCs. Intriguingly, adoptively transferred Mar T cells did not respond to male heart or skin grafts placed onto syngeneic MHC class II-deficient female recipients, demonstrating that activation of Mar T cell preferentially occurs through cognate interactions with processed male Ag expressed on recipient APCs. The data highlight the potency of indirect processing and presentation pathways in vivo and underscore the importance of indirectly primed CD4(+) T cells as relevant participants in both the priming and effector phases of acute graft rejection.     

Differential requirements for Th1 and Th17 responses to a systemic self-antigen

T cell-APC interactions are essential for the initiation of effector responses against foreign and self-antigens, but the role of these interactions in generating different populations of effector T cells in vivo remains unclear. Using a model of CD4(+) T cell responses to a systemic self-antigen without adjuvants or infection, we demonstrate that activation of APCs augments Th17 responses much more than Th1 responses. Recognition of systemic Ag induces tolerance in self-reactive CD4(+) T cells, but induction of CD40 signaling, even under tolerogenic conditions, results in a strong, Ag-specific IL-17 response without large numbers of IFN-γ-producing cells. Transfer of the same CD4(+) T cells into lymphopenic recipients expressing the self-antigen results in uncontrolled production of IL-17, IFN-γ, and systemic inflammation. If the Ag-specific T cells lack CD40L, production of IL-17 but not IFN-γ is decreased, and the survival time of recipient mice is significantly increased. In addition, transient blockade of the initial MHC class II-dependent T cell-APC interaction results in a greater reduction of IL-17 than of IFN-γ production. These data suggest that Th17 differentiation is more sensitive to T cell interactions with APCs than is the Th1 response, and interrupting this interaction, specifically the CD40 pathway, may be key to controlling Th17-mediated autoimmunity.     

On the role of APC-activation for in vitro versus in vivo T cell priming

Professional antigen-presenting cells take up antigens for processing and presentation in association with MHC class I and II molecules. When APCs receive the right stimuli, they undergo a maturation process and migrate to secondary lymphoid organs to trigger T cell activation. In this study, we compared side-by-side in vivo and in vitro activation of T cells. Transgenic CD8(+) T cells specific for the p33 epitope, derived from the lymphocytic choriomeningitis virus glycoprotein, were labeled with CFSE and injected into syngeneic mice or alternatively, co-cultured in vitro with APCs. The p33 epitope was delivered as free peptide or genetically fused to virus-like particles. Whereas proliferation of specific T cells was comparable in both systems, the production of IFN-gamma and the expression of CD25 showed important differences. Induction of effector function and expression of activation markers were strongly enhanced in vitro by both the free peptide and VLPs. Surprisingly, addition of CpG-containing immune-stimulating DNA for activation of APCs dramatically increased effector T cell differentiation in vitro, whereas no enhancement could be observed in vitro. Thus, activation of professional APCs was mandatory for induction of effector CD8(+) T cell responses in vivo, while this step was largely dispensable in vitro.     

Presentation of acquired peptide-MHC class II ligands by CD4+ regulatory T cells or helper cells differentially regulates antigen-specific CD4+ T cell response

Activated T cells can acquire membrane molecules from APCs through a process termed trogocytosis. The functional consequence of this event has been a subject of debate. Focusing on transfer of peptide-MHC class II (MHC-II) complexes from APCs to CD4(+) T cells after activation, in this study we investigated the molecule acquisition potential of naturally occurring regulatory T cells (Tregs) and CD4(+) Th cells. We show that acquisition of membrane molecules from APCs is an inherent feature of CD4(+) T cell activation. Triggering of the TCR enables CD4(+) T cells to acquire their agonist ligands as well as other irrelevant membrane molecules from the interacting APCs or bystander cells in a contact-dependent manner. Notably, trogocytosis is a continuous process during cell cycle progression, and Th cells and Tregs have comparable capacity for trogocytosis both in vitro and in vivo. The captured peptide-MHC-II molecules, residing in sequestered foci on the host cell surface, endow the host cells with Ag-presenting capability. Presentation of acquired peptide-MHC-II ligands by Th cells or Tregs has either stimulatory or regulatory effect on naive CD4(+) T cells, respectively. Furthermore, Th cells with captured peptide-MHC-II molecules become effector cells that manifest better recall responses, and Tregs with captured ligands exhibit enhanced suppression activity. These findings implicate trogocytosis in different subsets of CD4(+) T cells as an intrinsic mechanism for the fine tuning of Ag-specific CD4(+) T cell response.     

Ocular surface APCs are necessary for autoreactive T cell-mediated experimental autoimmune lacrimal keratoconjunctivitis

As specialized sentinels between the innate and adaptive immune response, APCs are essential for activation of Ag-specific lymphocytes, pathogen clearance, and generation of immunological memory. The process is tightly regulated; however, excessive or atypical stimuli may ignite activation of APCs in a way that allows self-Ag presentation to autoreactive T cells in the context of the necessary costimulatory signals, ultimately resulting in autoimmunity. Studies in both animal models and patients suggest that dry eye is a chronic CD4(+) T cell-mediated ocular surface autoimmune-based inflammatory disease. Using a desiccating stress-induced mouse model of dry eye, we establish the fundamental role of APCs for both the generation and maintenance of ocular-specific autoreactive CD4(+) T cells. Subconjunctival administration of liposome-encapsulated clodronate efficiently diminished resident ocular surface APCs, inhibited the generation of autoreactive CD4(+) T cells, and blocked their ability to cause disease. APC-dependent CD4(+) T cell activation required intact draining cervical lymph nodes, as cervical lymphadenectomy also inhibited CD4(+) T cell-mediated dry eye disease. In addition, local depletion of peripheral conjunctival APCs blocked the ability of dry eye-specific CD4(+) T cells to accumulate within the ocular surface tissues, suggesting that fully primed and targeted dry eye-specific CD4(+) T cells require secondary activation by resident ocular surface APCs for maintenance and effector function. These data demonstrate that APCs are necessary for the initiation and development of experimental dry eye and support the standing hypothesis that dry eye is a self-Ag-driven autoimmune disease.     

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.     

CD40 on APCs is needed for optimal programming, maintenance, and recall of CD8+ T cell memory even in the absence of CD4+ T cell help

CD40 stimulation is one of the many signals that can activate APCs and we have recently shown it to have a unique function in generating maximum primary CD8(+) T cell responses. However, whether CD40 signaling plays a role in memory CD8(+) T cell responses is still not completely understood. In this study, we show that in the absence of CD40 on all APCs or specifically on dendritic cells, memory CD8(+) T cells are generated but at significantly reduced levels. This reduction is due to a contribution of CD40 at several different steps in the generation of CD8(+) memory. In the initial T cell response, CD40 contributes to maximizing not only the number of effector cells that are generated but also the programming of ones that will differentiate into memory. Subsequently, CD40 is needed to maintain maximal numbers of the committed memory cells in a manner that is independent of the immunizing Ag. Finally, when memory CD8(+) T cells are reactivated there is a variable requirement for CD40 depending on whether CD40 or CD4(+) Th cells were present during the primary response. Therefore, CD40 signaling on APCs plays an important role in all phases of a memory CD8(+) T cell response.     

Cognate memory CD4+ T cells generated with dendritic cell priming influence the expansion, trafficking, and differentiation of secondary CD8+ T cells and enhance tumor control

CD4(+) T cells are known to provide support for the activation and expansion of primary CD8(+) T cells, their subsequent differentiation, and ultimately their survival as memory cells. However, the importance of cognate memory CD4(+) T cells in the expansion of memory CD8(+) T cells after re-exposure to Ag has been not been examined in detail. Using bone marrow-derived dendritic cells pulsed with cognate or noncognate MHC class I- and class II-restricted peptides, we examined whether the presence of memory CD4(+) T cells with the same Ag specificity as memory CD8(+) T cells influenced the quantity and quality of the secondary CD8(+) T cell response. After recombinant vaccinia virus-mediated challenge, we demonstrate that, although cognate memory CD4(+) T cells are not required for activation of secondary CD8(+) T cells, their presence enhances the expansion of cognate memory CD8(+) T cells. Cognate CD4(+) T cell help results in an approximate 2-fold increase in the frequency of secondary CD8(+) T cells in secondary lymphoid tissues, and can be accounted for by enhanced proliferation in the secondary CD8(+) T cell population. In addition, cognate memory CD4(+) T cells further selectively enhance secondary CD8(+) T cell infiltration of tumor-associated peripheral tissue, and this is accompanied by increased differentiation into effector phenotype within the secondary CD8(+) T cell population. The consequence of these improvements to the magnitude and phenotype of the secondary CD8(+) T cell response is substantial increase in control of tumor outgrowth.     

Airway eosinophils: allergic inflammation recruited professional antigen-presenting cells

The capacity of airway eosinophils, potentially pertinent to allergic diseases of the upper and lower airways, to function as professional APCs, those specifically able to elicit responses from unprimed, Ag-naive CD4(+) T cells has been uncertain. We investigated whether airway eosinophils are capable of initiating naive T cell responses in vivo. Eosinophils, isolated free of other APCs from the spleens of IL-5 transgenic mice, following culture with GM-CSF expressed MHC class II and the costimulatory proteins, CD40, CD80, and CD86. Eosinophils, incubated with OVA Ag in vitro, were instilled intratracheally into wild-type recipient mice that adoptively received i.v. infusions of OVA Ag-specific CD4(+) T cells from OVA TCR transgenic mice. OVA-exposed eosinophils elicited activation (CD69 expression), proliferation (BrdU incorporation), and IL-4, but not IFN-gamma, cytokine production by OVA-specific CD4(+) T cells in paratracheal lymph nodes (LN). Exposure of eosinophils to lysosomotropic NH(4)Cl, which inhibits Ag processing, blocked each of these eosinophil-mediated activation responses of CD4(+) T cells. By three-color fluorescence microscopy, OVA Ag-loaded eosinophil APCs were physically interacting with naive OVA-specific CD4(+) T cells in paratracheal LN after eosinophil airway instillation. Thus, recruited luminal airway eosinophils are distinct allergic "inflammatory" professional APCs able to activate primary CD4(+) T cell responses in regional LNs.     

Membrane-bound IL-22 after de novo production in tuberculosis and anti-Mycobacterium tuberculosis effector function of IL-22+ CD4+ T cells

The role of IL-22-producing CD4(+) T cells in intracellular pathogen infections is poorly characterized. IL-22-producing CD4(+) T cells may express some effector molecules on the membrane, and therefore synergize or contribute to antimicrobial effector function. This hypothesis cannot be tested by conventional approaches manipulating a single IL-22 cytokine at genetic and protein levels, and IL-22(+) T cells cannot be purified for evaluation due to secretion nature of cytokines. In this study, we surprisingly found that upon activation, CD4(+) T cells in Mycobacterium tuberculosis-infected macaques or humans could evolve into T effector cells bearing membrane-bound IL-22 after de novo IL-22 production. Membrane-bound IL-22(+) CD4(+) T effector cells appeared to mature in vivo and sustain membrane distribution in highly inflammatory environments during active M. tuberculosis infection. Near-field scanning optical microscopy/quantum dot-based nanoscale molecular imaging revealed that membrane-bound IL-22, like CD3, distributed in membrane and engaged as ～100-200 nm nanoclusters or ～300-600 nm nanodomains for potential interaction with IL-22R. Importantly, purified membrane-bound IL-22(+) CD4(+) T cells inhibited intracellular M. tuberculosis replication in macrophages. Our findings suggest that IL-22-producing T cells can evolve to retain IL-22 on membrane for prolonged IL-22 t(1/2) and to exert efficient cell-cell interaction for anti-M. tuberculosis effector function.     

Fas-mediated inhibition of CD4+ T cell priming results in dominance of type 1 CD8+ T cells in the immune response to the contact sensitizer trinitrophenyl

One of the unusual properties of chemically reactive haptens is their capacity to simultaneously generate immunogenic determinants for hapten-specific CD8(+) and CD4(+) T cells. Surprisingly, however, a clear dominance of CD8(+) effector T cells is observed in murine contact hypersensitivity to various haptens and upon T cell priming with hapten-modified APCs in vitro. In this study we show that trinitrophenyl-specific CD8(+) T cells actively prevent CD4(+) T cell priming in vitro. This process requires cell-cell contact and is dependent on the expression of Fas on the CD4(+) T cells. Our results reveal an important Fas-dependent mechanism for the regulation of hapten-specific CD4(+) T cell responses by CD8(+) T cells, which causes the dominance of CD8(+) effector T cells and the active suppression of a CD4(+) T cell response. Moreover, our demonstration of reduced contact hypersensitivity to trinitrophenyl in the absence of Fas, but not of perforin and/or granzymes A and B, underlines the important role of Fas as a pathogenetic factor for contact hypersensitivity.     

TLR-activated dendritic cells enhance the response of aged naive CD4 T cells via an IL-6-dependent mechanism

The most effective immunological adjuvants contain microbial products, such as TLR agonists, which bind to conserved pathogen recognition receptors. These activate dendritic cells (DCs) to become highly effective APCs. We assessed whether TLR ligand-treated DCs can enhance the otherwise defective response of aged naive CD4 T cells. In vivo administration of CpG, polyinosinic-polycytidylic acid, and Pam(3)CSK(4) in combination with Ag resulted in the increased expression of costimulatory molecules and MHC class II by DCs, increased serum levels of the inflammatory cytokines IL-6 and RANTES, and increased cognate CD4 T cell responses in young and aged mice. We show that, in vitro, preactivation of DCs by TLR ligands makes them more efficient APCs for aged naive CD4 T cells. After T-DC interaction, there are enhanced production of inflammatory cytokines, particularly IL-6, and greater expansion of the aged T cells, resulting from increased proliferation and greater effector survival with increased levels of Bcl-2. TLR preactivation of both bone marrow-derived and ex vivo DCs improved responses. IL-6 produced by the activated DCs during cognate T cell interaction was necessary for enhanced aged CD4 T cell expansion and survival. These studies suggest that some age-associated immune defects may be overcome by targeted activation of APCs by TLR ligands.     

Steady state dendritic cells present parenchymal self-antigen and contribute to, but are not essential for, tolerization of naive and Th1 effector CD4 cells

Bone marrow-derived APC are critical for both priming effector/memory T cell responses to pathogens and inducing peripheral tolerance in self-reactive T cells. In particular, dendritic cells (DC) can acquire peripheral self-Ags under steady state conditions and are thought to present them to cognate T cells in a default tolerogenic manner, whereas exposure to pathogen-associated inflammatory mediators during the acquisition of pathogen-derived Ags appears to reprogram DCs to prime effector and memory T cell function. Recent studies have confirmed the critical role of DCs in priming CD8 cell effector responses to certain pathogens, although the necessity of steady state DCs in programming T cell tolerance to peripheral self-Ags has not been directly tested. In the current study, the role of steady state DCs in programming self-reactive CD4 cell peripheral tolerance was assessed by combining the CD11c-diphtheria toxin receptor transgenic system, in which DC can be depleted via treatment with diphtheria toxin, with a TCR-transgenic adoptive transfer system in which either naive or Th1 effector CD4 cells are induced to undergo tolerization after exposure to cognate parenchymally derived self-Ag. Although steady state DCs present parenchymal self-Ag and contribute to the tolerization of cognate naive and Th1 effector CD4 cells, they are not essential, indicating the involvement of a non-DC tolerogenic APC population(s). Tolerogenic APCs, however, do not require the cooperation of CD4(+)CD25(+) regulatory T cells. Similarly, DC were required for maximal priming of naive CD4 cells to vaccinia viral-Ag, but priming could still occur in the absence of DC.     

Deletion of naive CD8 T cells requires persistent antigen and is not programmed by an initial signal from the tolerogenic APC

Activation of naive CD8 T cells in vivo requires the recognition of cognate peptide-MHC complexes on APCs. Depending upon the activation status of the APC, such recognition will promote either a vigorous immune response or T cell tolerance and deletion. Recent studies suggest that the initial signals provided by APCs are sufficient to program the proliferation of naive CD8 T cells and their differentiation into effector cells. In this study, we sought to determine whether an initial encounter with tolerogenic APCs was sufficient to program deletion of naive CD8 T cells. Surprisingly, we find that regardless of whether naive CD8 T cells were stimulated by activated or quiescent APCs, transfer of the activated T cells into an Ag-free host was sufficient to ensure survival. Thus, although the extent of clonal expansion and development of effector function is determined by the activation status of the stimulatory APC, peripheral clonal deletion requires persistent Ag and is not determined by the initial stimulatory event.     

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476-G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.     

A recombinant vector expressing transgenes for four T-cell costimulatory molecules (OX40L,B7-1, ICAM-1, LFA-3) induces sustained CD4+ and CD8+ T-cell activation,protection from apoptosis,and enhanced cytokine production

The role of OX40L on the activation of T cells was investigated using poxvirus vectors expressing OX40L alone or in combination with three other T-cell costimulatory molecules: B7-1, ICAM-1, and LFA-3. Poxvirus vector-infected cells were used to stimulate nai;ve or activated CD4(+) and CD8(+) T cells. These studies demonstrate that (a) OX40L plays a role in sustaining the long-term proliferation of CD8(+) T cells in addition to the known effect on CD4(+) T cells following activation, (b) OX40L enhances the production of Th1 cytokines (IL-2, IFN-gamma, and TNF-alpha) from both CD4(+) and CD8(+) while no change in IL-4 expression was observed, and (c) the anti-apoptotic effect of OX40L on T cells is likely the result of sustained expression of anti-apoptotic genes while genes involved in apoptosis are inhibited. In addition, these are the first studies to demonstrate that the combined use of a vector driving the expression of OX40L with three other costimulatory molecules (B7-1, ICAM-1, and LFA-3) both enhances initial activation and then further potentiates sustained activation of nai;ve and effector T cells.     

Differential requirements for OX40 signals on generation of effector and central memory CD4+ T cells

Memory T cells can be divided into effector memory (T(EM)) and central memory (T(CM)) subsets based on their effector function and homing characteristics. Although previous studies have demonstrated that TCR and cytokine signals mediate the generation of the two memory subsets of CD8(+) T cells, the mechanisms for generation of the CD4(+) T(EM) and T(CM) cell subsets are unknown. We found that OX40-deficient mice showed a marked reduction in the number of CD4(+) T(EM) cells, whereas the number of CD4(+) T(CM) cells was normal. Adoptive transfer experiments using Ag-specific CD4(+) T cells revealed that OX40 signals during the priming phase were indispensable for the optimal generation of the CD4(+) T(EM), but not the CD4(+) T(CM) population. In a different transfer experiment with in vitro established CD4(+)CD44(high)CD62L(low) (T(EM) precursor) and CD4(+)CD44(high)CD62L(high) (T(CM) precursor) subpopulations, OX40-KO T(EM) precursor cells could not survive in the recipient mice, whereas wild-type T(EM) precursor cells differentiated into both T(EM) and T(CM) cells. In contrast, T(CM) precursor cells mainly produced T(CM) cells regardless of OX40 signals, implying the dispensability of OX40 for generation of T(CM) cells. Nevertheless, survival of OX40-KO T(EM) cells was partially rescued in lymphopenic mice. During in vitro recall responses, the OX40-KO T(EM) cells that were generated in lymphopenic recipient mice showed impaired cytokine production, suggesting an essential role for OX40 not only on generation but also on effector function of CD4(+) T(EM) cells. Collectively, the present results indicate differential requirements for OX40 signals on generation of CD4(+) T(EM) and T(CM) cells.