Imaging early steps of human T cell activation by antigen-presenting cells.

In this work the Ca2+ response and the morphological changes elicited by Ag in human CD4+ T cells are described at the single cell level. The APC used to present the diphtheria toxoid Ag to a human diphtheria toxoid-specific T cell clone were murine L cell fibroblast transfectants expressing MHC class II molecules. The increase of the intracellular Ca2+ concentration, [Ca2+]i, which is one of the earliest steps of the response to TCR stimulation, was followed by fluorimetry with fura-2 on an imaging system. This response was a specific consequence of successful Ag presentation, because it only took place when fibroblasts expressed both class II MHC molecules and Ag. CD4 molecules were also involved in this intercellular interaction, because the Ca2+ response could be inhibited by preincubating the T cells with an anti-CD4 antibody. The response induced by APC started after a delay of at least 6 min, after which large Ca2+ oscillations took place, with a pseudo period of 100 s at 35 degrees C. The frequency of these oscillations decreased with temperature. The oscillations became progressively more damped during the first 30 to 40 min of cell-to-cell interaction, after which they completely stopped; however, [Ca2+]i remained well above its resting level for more than 1 h after the contact. The Ca2+ oscillations were entirely dependent on Ca2+ influx because they immediately disappeared when external calcium was removed. Similar oscillations were observed when the cells were stimulated with an anti-CD3 antibody. After stimulation with APC, many T cells abandoned their spherical shape and tended to flatten and elongate. This aspect of the T cell response was not observed after stimulation with an anti-CD3 antibody. In the presence of cytochalasin B, the morphologic changes elicited by the APC were blocked, whereas the Ca2+ response was slightly enhanced. However, when T cells were loaded with the Ca2+ chelator BAPTA, both Ca2+ and morphologic changes were inhibited, suggesting that the Ca2+ response plays a permissive role for the morphologic changes.     

Induction of peripheral T cell tolerance by antigen-presenting B cells. II. Chronic antigen presentation overrules antigen-presenting B cell activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4(+) T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.     

Incomplete activation of CD4 T cells by antigen-presenting transitional immature B cells: implications for peripheral B and T cell responsiveness

B cells leave the bone marrow as transitional B cells. Transitional B cells represent a target of negative selection and peripheral tolerance, both of which are abrogated in vitro by mediators of T cell help. In vitro, transitional and mature B cells differ in their responses to B cell receptor ligation. Whereas mature B cells up-regulate the T cell costimulatory molecule CD86 (B7.2) and are activated, transitional B cells do not and undergo apoptosis. The ability of transitional B cells to process and present Ag to CD4 T cells and to elicit protective signals in the absence of CD86 up-regulation was investigated. We report that transitional B cells can process and present Ag as peptide:MHC class II complexes. However, their ability to activate T cells and elicit help signals from CD4-expressing Th cells was compromised compared with mature B cells, unless exogenous T cell costimulation was provided. A stringent requirement for CD28 costimulation was not evident in interactions between transitional B cells and preactivated CD4-expressing T cells, indicating that T cells involved in vivo in an ongoing immune response might rescue Ag-specific transitional B cells from negative selection. These data suggest that during an immune response, immature B cells may be able to sustain the responses of preactivated CD4(+) T cells, while being unable to initiate activation of naive T cells. Furthermore, the ability of preactivated, but not naive T cells to provide survival signals to B cell receptor-engaged transitional immature B cells argues that these B cells may be directed toward activation rather than negative selection when encountering Ag in the context of a pre-existing immune response.     

Ex vivo induction and expansion of antigen-specific cytotoxic T cells by HLA-Ig-coated artificial antigen-presenting cells

Adoptive immunotherapy holds promise as a treatment for cancer and infectious diseases, but its development has been impeded by the lack of reproducible methods for generating therapeutic numbers of antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs). As a result, there are only limited reports of expansion of antigen-specific CTLs to the levels required for clinical therapy. To address this issue, artificial antigen-presenting cells (aAPCs) were made by coupling a soluble human leukocyte antigen-immunoglobulin fusion protein (HLA-Ig) and CD28-specific antibody to beads. HLA-Ig-based aAPCs were used to induce and expand CTLs specific for cytomegalovirus (CMV) or melanoma. aAPC-induced cultures showed robust antigen-specific CTL expansion over successive rounds of stimulation, resulting in the generation of clinically relevant antigen-specific CTLs that recognized endogenous antigen-major histocompatibility complex complexes presented on melanoma cells. These studies show the value of HLA-Ig-based aAPCs for reproducible expansion of disease-specific CTLs for clinical approaches to adoptive immunotherapy.     

Critical role for activation of antigen-presenting cells in priming of cytotoxic T cell responses after vaccination with virus-like particles

Virus-like particles (VLPs) are known to induce strong Ab responses in the absence of adjuvants. In addition, VLPs are able to prime CTL responses in vivo. To study the efficiency of this latter process, we fused peptide p33 derived from lymphocytic choriomeningitis virus to the hepatitis B core Ag, which spontaneously assembles into VLPs (p33-VLPs). These p33-VLPs were efficiently processed in vitro and in vivo for MHC class I presentation. Nevertheless, p33-VLPs induced weak CTL responses that failed to mediate effective protection from viral challenge. However, if APCs were activated concomitantly in vivo using either anti-CD40 Abs or CpG oligonucleotides, the CTL responses induced were fully protective against infection with lymphocytic choriomeningitis virus or recombinant vaccinia virus. Moreover, these CTL responses were comparable to responses generally induced by live vaccines, because they could be measured in primary ex vivo (51)Cr release assays. Thus, while VLPs alone are inefficient at inducing CTL responses, they become very powerful vaccines if applied together with substances that activate APCs.     

Human antigen-presenting cells respond differently to gut-derived probiotic bacteria but mediate similar strain-dependent NK and T cell activation

The intestinal microbiota is essential for homeostasis of the local and systemic immune system, and particularly strains of lactic acid bacteria and Escherichia coli have been shown to have balancing effects on inflammatory conditions such as allergy and inflammatory bowel disease. However, in vitro assessment of the immunomodulatory effects of distinct strains may depend strongly on the cell type used as a model. To select the most appropriate model for screening of beneficial bacteria in human cells, the response to strains of intestinal bacteria of three types of antigen-presenting cells (APC) was compared; blood myeloid dendritic cells (DC), monocyte-derived DC and monocytes, and the effector response of natural killer cells and na?ve T cells was characterized. Maturation induced by gut-derived bacteria differed between APC, with blood DC and monocytes responding with the production of IL-6 and tumour necrosis factor-alpha to bacteria, which elicited mainly IL-10 in monocyte-derived DC. In contrast, comparable IFN-gamma production patterns were found in both natural killer cells and T cells induced by all bacteria-matured APC. An inhibitory effect of certain strains on this IFN-gamma production was also mediated by all types of APC. The most potent responses were induced by monocyte-derived DC, which thus constitute a sensitive screening model.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Anergic T cells inhibit the antigen-presenting function of dendritic cells

The phenomena of infectious tolerance and linked-suppression are well established, but the mechanisms involved are incompletely defined. Anergic T cells can inhibit responsive T cells in vitro and prolong skin allograft survival in vivo. In this study the mechanisms underlying these events were explored. Allospecific mouse T cell clones rendered unresponsive in vitro inhibited proliferation by responsive T cells specific for the same alloantigens. The inhibition required the presence of APC, in that the response to coimmobilized anti-CD3 and anti-CD28 Abs was not inhibited. Coculture of anergic T cells with bone marrow-derived dendritic cells (DC) led to profound inhibition of the ability of the DC to stimulate T cells with the same or a different specificity. After coculture with anergic T cells expression of MHC class II, CD80 and CD86 by DC were down-regulated. These effects did not appear to be due to a soluble factor in that inhibition was not seen in Transwell experiments, and was not reversed by addition of neutralizing anti-IL-4, anti-IL-10, and anti-TGF-beta Abs. Taken together, these data suggest that anergic T cells function as suppressor cells by inhibiting Ag presentation by DC via a cell contact-dependent mechanism.     

Semaphorins in interactions between T cells and antigen-presenting cells

Although semaphorins were identified originally as guidance cues for developing neuronal axons, accumulating evidence indicates that several semaphorins are expressed also in the immune system. SEMA4D (CD100), which is expressed constitutively by T cells, enhances the activation of B cells and dendritic cells (DCs) through its cell-surface receptor, CD72. SEMA4A, which is expressed by DCs, is involved in the activation of T cells through interactions with TIM2. So, these semaphorins seem to function in the reciprocal stimulation of T cells and antigen-presenting cells (APCs). Emerging evidence indicates that additional semaphorins and related molecules are involved in T-cell-APC interactions also.     

Characterization of the accessory cells involved in suppressor T cell induction

The ability of UV-treated splenic adherent cells (SAC) to induce T cell-mediated immunity and suppressor T cells was analyzed in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. UV irradiation of 0.88 KJ/m2 decreased the capacity of NP-coupled SAC to induce delayed-type hypersensitivity (DTH) responses by about 50%. The ability of uncoupled UV-treated SAC to induce allogeneic DTH response was also imparied, indicating that UV-treated SAC are inefficient at inducing DTH in these systems. TS1 induction by UV-treated NP-SAC was evaluated TS1 induction by UV-treated NP-SAC was evaluated by using adherent cells that were subjected to the same dose of UV irradiation that impaired DTH induction. Intravenous administration of 10(3) or 10(4) UV-treated NP-coupled SAC induced TS1 cells with the same efficiency as non-UV-irradiated cells. The TS1 cells induced in this fashion were antigen specific. Furthermore, to establish that the antigen was not reprocessed by the host, I-J-mismatched, UV-treated NP-SAC were unable to induce TS1 cells. The population of antigen-presenting cells responsible for TS1 induction appear to express both I-A and I-J determinants. TS2 induction by UV-treated accessory cells was also analyzed. TSF1 inducer suppressor factor was pulsed onto graded numbers of either normal or UV-treated adherent cells. The same levels of antigen-specific suppression were induced with normal and UV-treated cells. Finally, TS3 induction by UV-treated NP-SAC was analyzed. UV-treated and normal NP-SAC (3 X 10(3] induced antigen-specific suppression of NP DTH responses. I-J-mismatched, UV-treated NP-SAC failed to induce suppression, suggesting that the hapten was not reprocessed by the host under these experimental conditions. The accessory cell population responsible for TS3 induction appears to express both I-A and I-J determinants. Thus, there are at least two functional distinctions between the antigen-presenting cells that induce immunity vs those that induce suppressor cells. First, UV treatment selectively impairs the antigen-presenting cells, which activate the positive limb of the immune response. Second, I-J determinants appear to be specifically associated with the SAC, which induce suppressor T cells. Although these criteria can be used to distinguish the accessory cells involved in suppressor cell pathways from those controlling helper T cell induction, there were no discernible phenotypic differences among the accessory cells that induce the TS1, TS2, and TS3 subsets.     

Antigen-specific T cell activation and proliferation during oral tolerance induction

One of several routes of achieving immunologic tolerance is through functional inactivation of Ag-specific T cells. Oral administration of Ag can allow survival of the Ag-specific T cells that are functionally anergic. The aim of this study was to investigate whether functional inactivation of Ag-specific T cells is directed through an activation process and to further define the differentiative pathways and functional characteristics of anergic T cells. Mice were transplanted with OVA-specific TCR-transgenic T cells and either fed OVA or immunized s.c. with the OVA peptide 323-339 in CFA. OVA-specific T cells from OVA-fed mice were unresponsive to restimulation in vitro within 48-72 h after treatment. In vivo, however, T cell proliferation was detected by 5, 6-carboxy-succinimidyl-fluoresceine-ester intensity changes in OVA-specific T cells. The mesenteric lymph nodes (LNs) from OVA-fed mice more frequently contained OVA-specific dividing cells in vivo than those in the peripheral LNs, and the reciprocal was observed following s.c. immunization of the OVA peptide in CFA. The induction of anergy in OVA-fed mice was accompanied by rapid up-regulation of CD69 and CTLA-4, later down-regulation of CD45RB on OVA-specific T cells, and a marked decrease in T cell secretion of IL-2, IL-10, and IFN-gamma after OVA restimulation in vitro. Results from this study indicate that the inductive phase of oral tolerance is preceded by Ag-specific T cell activation in vivo, proliferation in the regional draining LNs, and differentiation into a memory-like state. These results indicate that Ag-directed differentiation occurs as a part of T cell tolerance through anergy.     

B lymphocytes as antigen-presenting cells for CD4+ T cell priming in vivo

The contribution of B lymphocytes as APCs for CD4+ T cell priming remains controversial, based on findings that B cells cannot provide the requisite ligating and costimulatory signals for naive T cells to be activated. In the current study, we have examined Ag-specific T:B cell collaboration under circumstances in which B cells take up Ag through Ig receptors in vivo. This results in their activation and an ability to effectively stimulate naive CD4+ T cells both in vitro and in vivo. The aim of this work was to establish some of the key molecular interactions, as well as kinetics, between Ag-specific T and B cells that enable this priming to take place. Our approach was to amplify the starting pools of both Ag-specific T and B cell populations in vivo to track directly the events during initial T:B cell collaborations. We show that the induction of optimal levels of T cell priming to a protein Ag requires the involvement of Ag-specific B cells. The interaction that results between Ag-specific T and B cells prevents the down-modulation of B7 costimulatory molecules usually observed in the absence of appropriate T cells. Moreover, this prevention in down-modulation is independent of CD40:CD40 ligand contact. Finally, we present data suggesting that once Ag-specific T and B cells interact, there is a rapid (1-2-h) down-regulation of antigenic complexes on the surface of the B lymphocytes, possibly to prevent them from engaging other T cells in the vicinity and therefore focus the initial interaction.     

The involvement of antigen-presenting cells and suppressor cells in the ultraviolet radiation-induced inhibition of secondary cytotoxic T cell sensitization

When mice were treated with ultraviolet (UV) radiation before immunization with the skin sensitizers, trinitro-chlorobenzene or dinitrofluorobenzene, they showed greatly depressed levels of priming for a secondary in vitro cytotoxic response against haptenated cells. Because the irradiation and skin painting were done on separate sites, these results suggest that the UV radiation had a systemic effect. The priming response of irradiated mice to allogeneic stimulation was normal, indicating some antigen selectivity to the inhibitory effect of UV radiation. A defect in antigen-presenting cells, previously demonstrated in UV-irradiated mice, was found to be largely responsible for the depressed priming response observed in these animals. In addition, the UV-irradiated, immunized mice possessed suppressor cells that were capable of blocking priming for cytotoxic responses against haptenated cells in normal mice.     

Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma. Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.     

T cell receptor-induced activation and apoptosis in cycling human T cells occur throughout the cell cycle

Previous studies have found conflicting associations between susceptibility to activation-induced cell death and the cell cycle in T cells. However, most of the studies used potentially toxic pharmacological agents for cell cycle synchronization. A panel of human melanoma tumor-reactive T cell lines, a CD8+ HER-2/neu-reactive T cell clone, and the leukemic T cell line Jurkat were separated by centrifugal elutriation. Fractions enriched for the G0-G1, S, and G2-M phases of the cell cycle were assayed for T cell receptor-mediated activation as measured by intracellular Ca(2+) flux, cytolytic recognition of tumor targets, and induction of Fas ligand mRNA. Susceptibility to apoptosis induced by recombinant Fas ligand and activation-induced cell death were also studied. None of the parameters studied was specific to a certain phase of the cell cycle, leading us to conclude that in nontransformed human T cells, both activation and apoptosis through T cell receptor activation can occur in all phases of the cell cycle.     

Signals involved in T cell activation. I. Phorbol esters enhance responsiveness but cannot replace intact accessory cells in the induction of mitogen-stimulated T cell proliferation

The role of accessory cells (AC) in the initiation of mitogen-induced T cell proliferation was examined by comparing the effect of intact macrophages (M phi) with that of 4-beta-phorbol 12-myristate 13-acetate (PMA). In high-density cultures, purified guinea pig T cells failed to proliferate in response to stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), or PMA alone. The addition of M phi to PHA or Con A but not PMA-stimulated cultures restored T cell proliferation. The addition of PMA to high-density T cell cultures stimulated with PHA or Con A also permitted [3H]thymidine incorporation, but was less effective than intact M phi in this regard. This action of PMA was dependent on the small number of AC contaminating the T cell cultures as evidenced by the finding that PMA could not support mitogen responsiveness of T cells that had been depleted of Ia-bearing cells by planning, even when these cells were cultured at high density. When PMA was added to T cell cultures supported by optimal numbers of M phi, catalase-reversible suppression of responses was noted. Even in cultures containing catalase, PMA failed to enhance responsiveness above that supported by optimal numbers of M phi. A low-density culture system was used to examine in greater detail the possibility that PMA could completely substitute for M phi in promoting T cells activation. In low-density cultures, mitogen-induced T cell proliferation required intact M phi. PMA could not support responses even in cultures supplemented with interleukin 1-containing M phi supernatants or purified interleukin 2 alone or in combination. Similar results were found in high-density cultures of T cells depleted of Ia-bearing cells. These results support a model of T cell activation in which AC play at least two distinct roles. The initiation of the response requires a signal conveyed by an intact M phi, which cannot be provided by either a M phi supernatant factor or PMA. The response can be amplified by additional M phi or M phi supernatant factors. PMA can substitute for M phi in this regard and can provide the signal necessary for amplification of T cell proliferation supported by small numbers of intact AC.     

A functional recombinant single-chain T cell receptor fragment capable of selectively targeting antigen-presenting cells

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complexes (MHC) by the T cell receptor (TCR). Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently a potent anti-human p53 CD8(+) cytotoxic T lymphocyte (CTL) response has been developed in HLA-A2 transgenic mice after immunization with peptides corresponding to HLA-A2 motifs from human p53. An alpha/beta T-cell receptor was cloned from such CTL which exhibited a moderately high affinity to the human p53(149-157) peptide. In this report, we investigated the possibility of using a recombinant tumor-specific TCR for antigen-specific elimination of cells that express the specific MHC-peptide complex. To this end, we constructed a functional single-chain Fv fragment from the cloned TCR and fused it to a very potent cytotoxic molecule, a truncated form of Pseudomonas exotoxin A (PE38). The p53 TCR scFv-P38 fusion protein was generated by in vitro refolding from bacterially-expressed inclusion bodies, and was found to be functional by its ability to bind antigen-presenting cells (APC) which express the specific p53-derived peptide. Moreover, we have shown that the p53-specific TCR scFv-PE38 molecule specifically kills APC in a peptide-dependent manner. These results represent the first time that a TCR-derived recombinant single-chain Fv fragment has been used as a targeting moiety to deliver a cytotoxic effector molecule to cells and has been able to mediate the efficient killing of the particular cell population that expresses the specific MHC/peptide complex. Similarly to antibody-based targeting approaches, TCR with tumor cell specificity represent attractive candidates for generating new, very specific targeting moieties for various modes of cancer immunotherapy.     

Autophagy induction and autophagic cell death in effector T cells

The population size of the T cells is tightly regulated. The T cell number drastically increases in response to their specific antigens. Upon antigen clearance, the T cell number decreases over time. Apoptosis, also called type I programmed cell death, plays an important role in eliminating T cells. The role of autophagic cell death, also called type II programmed cell death, is unclear in T cells. Our recent work demonstrated that autophagy is induced in both Th1 and Th2 cells. Both TCR signaling and IL-2 increase autophagy in T cells, and JNK MAP kinases are required for the induction of autophagy in T cells, whereas caspases and mTOR inhibit autophagy in T cells. Autophagy is required for mediating growth factor withdrawal-dependent cell death in T cells. Here, we hypothesize that autophagic cell death plays an important role in T cell homeostasis.