TY - JOUR
AU - Baird, Margaret
AB - Abstract Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen-presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-Ed, was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA–IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA–IL-2 chimeric protein. When an antibody known to block IL-2 receptor α chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25+ BMDC. Antigen uptake and processing of HA–IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines. antigen presentation, cytokines, dendritic cells, hemagglutinin, IL-2 receptor APC antigen-presenting cell, BMDC bone marrow-derived dendritic cell, DC dendritic cell, GFP green fluorescent protein, GM-CSF granulocyte macrophage colony stimulating factor, HA hemagglutinin peptide, IL-2R IL-2 receptor, PE phytohemagglutinin, T thioredoxin, TNF tumor necrosis factor Introduction Despite the success of many vaccines, complete protection against some pathogens has proved elusive. As a result, the potential for specific cytokines to act as vaccine adjuvants, selecting for an immune response that is appropriate to remove the pathogen or neutralize its products has been under some scrutiny. Co-delivered cytokines have had variable success owing to the short half-life and toxicity associated with the high dosages required (1). Work carried out some years ago suggested that the immunogenicity of proteins could be improved if they were fused to cytokines (2). Linking an antigenic moiety and a cytokine may prolong the half-life of the cytokine, and thereby amplify its effect on the ensuing response. Chimeric vaccines such as these are delivered to the same antigen-presenting cell (APC) which appears to be important in optimizing reactivity (3). Recent work has suggested that they may have the potential to protect against pathogens, tumours and even some hyperimmune conditions. Pneumococcal surface protein A fused to IL-2 generates antibody which, when passively transferred, confers protection against challenge with S. pneumoniae (4). Idiotypic proteins fused to granulocyte macrophage colony stimulating factor (GM-CSF) have been shown to immunize mice against B cell lymphomas (5). Immunization with ovalbumin fused to IL-12 has been shown to re-direct established IL-4 responses toward IFN-γ which may be applicable in the treatment of allergies (6). These vaccines also show some promise when delivered as DNA constructs (7,8). Surprisingly little is known about the way in which fusion vaccines might operate at the cellular level. Since the fusion protein must be processed and presented it seems likely that, at least in part, they influence the response via their effects on APC. Dendritic cells (DC) are essential for the initiation of T cell-dependent immune responses and it is therefore appropriate that new strategies for improving the efficacy of vaccines be explored utilizing these cells. They may be crucial in determining the magnitude and the direction of the response at its earliest phase. DC express a number of cytokine receptors including those for IFN-γ, IL-1β, IL-4, IL-10, GM-CSF and tumor necrosis factor (TNF)-α (reviewed in 9). These transduce signals when they bind their respective ligands which can induce cell maturation, activation and migration. Chimeric vaccines incorporating these cytokines therefore have the potential to affect the response generated via this pathway. Interestingly, DC from various cultured tissues express the α chain (CD25) and the common cytokine γ chain but not the β chain of the IL-2 receptor (IL-2R) complex. The α chain has been detected in murine Langerhans cells (10), thymic, splenic and lung DC (11–17). The γ chain has been detected in murine splenic DC, Langerhans cells and bone marrow cells grown in GM-CSF (18). The α chain appears to contribute only to the binding affinity of the functional receptor, while the β and γ chains are involved in signal transduction (19). CD25 is markedly up-regulated on murine myeloid and lymphoid DC following exposure to GM-CSF, but as yet no defined biological function has been ascribed to this molecule since in IL-2Rα null mice, neither the maturation of DC subsets nor their capacity to present alloantigen is affected (20). Current research in our laboratory is focused on the optimization of protective immune responses using chimeric vaccines. Here we describe experiments in which we have investigated DC-mediated processing and presentation of a chimeric protein comprising a well-defined, class II-restricted T cell epitope from influenza hemagglutinin (HA) fused to IL-2 (T–HA–IL-2). On the basis of our results we suggest that the marked increase in DC-mediated T cell activation recorded in response to T–HA–IL-2 may be attributable to enhanced antigen uptake via CD25-mediated endocytosis. Methods Animals Specific pathogen-free BALB/c mice (6–10 weeks old) were obtained from the Department of Animal Laboratory Sciences, University of Otago, New Zealand. Media, cytokines and antibodies Bone marrow cells were cultured in DMEM supplemented with l-glutamine, streptomycin, penicillin, 50 μM 2-mercaptoethanol and 5% FCS (cDMEM). The A5 T cell hybridoma cell line was cultured in IMDM supplemented with streptomycin, penicillin, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 0.5 mg/ml hygromycin B and 5% FCS. All medium and supplements were purchased from Life Technologies (Auckland, New Zealand). Recombinant murine GM-CSF was purchased from R & D Systems (Minneapolis, MN). The phycoerythrin (PE)-conjugated AMT-13 mAb (rat anti-mouse IL-2Rα chain, CD25; IgG2a) was purchased from Sigma (Castle Hill, NSW, Australia). The isotype control antibody PE-conjugated rat IgG2a isotype antibody, 3C7 mAb (rat anti-mouse IL-2Rα chain, CD25; IgG2b) and rat IgG2b isotype control antibody were all purchased from PharMingen (San Diego, CA). Antigens The HA peptide (110–119) SFERFEIFPK from influenza virus A/PR/8/34 (synthesized by Macromolecular Resources, Colorado State University) was used as a test antigen. Constructs were made encoding this peptide linked to IL-2, IL-4 or IFN-γ and thioredoxin (T). Mouse RNA was isolated from splenocytes stimulated with concanavalin A and converted into cDNA using Superscript II according to the manufacturer's instructions (Life Technologies). IL-2, IL-4 and IFN-γ were amplified from mouse cDNA using PCR. The 5′ primer included sequences specific for not only murine cytokine but for HA peptide, shown in bold. The murine IL-2-specific primers were (upstream) 5′-C AAG TGA TCA TCT TTT GAA CGT TTC GAA ATC TTC CCG AAA GGA TCC GCA CCC ACT TCA AGC TCC-3′, (downstream) 5′-ATA TGT CGA CGA ATT CTT ATT GAG GGC TTG TTG AGA T-3′. The murine-specific IL-4 primers were (upstream) 5′-A TTT GGA TCC TCT TTT GAA CGT TTC GAA ATC TTC CCG AAA CAT ATC CAC GGA TGC GAC AAA AAT-3′, (downstream) 5′-T ATA AAG CTT CTA CGA GTA ATC CAT TTG CAT GAT–3′. The murine-specific IFN-γ primers were (upstream) 5′-A TTA GGA TCC TCT TTT GAA CGT TTC GAA ATC TTC CCG AAA GAG TAC TGC CAC GGC ACA GTC ATT GAA-3′, (downstream) 5′-A TAT AAG CTT TCA GCA GCG ACT CCT TTT CCG CTT-3′. For constructs containing just the cytokine the upstream primer did not contain the HA sequence. A construct containing just the HA peptide (110–119) and one with additional flanking sequences (94–131) were made by PCR from a DNA sequence of HA from influenza virus A/PR/8/34 kindly provided by Alistair Ramsay (University of Newcastle, Newcastle, NSW, Australia). The HA(110–119)-specific primers were (upstream) 5′-GAA TCC TCT TTT GAA CGT TTC GAA GAA ATC TTC CCG AAA TAA GCT T-3′, (downstream) 5′-CCT AGG AGA AAA CTT GCA AAG CTT TAG AAG GGC TTT ATT CGA A-3′. The HA(94–131)-specific primers were (upstream) 5′- GGA TCC GAT TTC ATC GAC TAT GAG GAG-3′, (downstream) 5′-A AGC TTA TCC TTT GGT TGT GTT GTG GTT-3′. The resulting PCR products were digested with BclI and SalI for the IL-2 constructs and BamHI and HindIII for all the remaining constructs, and then cloned into the thioredoxin fusion vector pET 32a (Novagen, Madison, WI). The constructs were expressed in Escherichia coli BL21(DE3)pLysS and the recombinant protein purified over a Ni-NTA-Agarose (Qiagen, Hilden, Germany) column. The protein concentration was measured by using the BioRad protein assay (BioRad, Auckland, New Zealand). Therefore the recombinant protein had thioredoxin at the N-terminus of HA peptide and IL-2, IL-4 or IFN-γ at the C-terminus (T–HA110–119, T–HA94–131, T–HA–IL-2, T–HA–IL-4, T–HA–IFN-γ). Generation of APC A modified method of Inaba et al. (21) was used for the isolation of bone marrow precursors. Briefly, the bone marrow was flushed from femurs and tibias with 5% FCS in PBS, the red blood cells were lysed with ammonium chloride, and the remaining cells washed 3 times. Cells were plated at 2×106 cells/ml in six-well plates and cultured in medium containing 20 ng/ml GM-CSF. The cultures were fed on day 4 and on day 6 by replacing ~75% of the medium. On days 6–7 non-adherent cells were harvested as bone marrow-derived DC (BMDC). BMDC cultured in GM-CSF show an immature phenotype and are capable of taking up both soluble and particulate antigen (22). Approximately 50–60% of the cells are MHC class II+, 40–50% CD80+ and 25–35% CD86+. Resident macrophages were harvested from BALB/c mice by peritoneal lavage using 5% FCS, 5 U/ml heparin in PBS and then used on the same day. Assay for T cell activation T cell activation was measured using the A5 cell line which was derived from the 14-3-d T cell hybridoma expressing TCR specific for the influenza HA peptide 110–120 presented in the context of MHC class II I-Ed molecules (23). These cells have been transfected with a construct containing a triple NF-AT promotor linked to green fluorescent protein (GFP). Upon activation these cells produce GFP which can be detected by flow cytometry. A5 cells were subcultured 1:10 the day before use. To induce activation 2×105 A5 cells and 1×105 BMDC or peritoneal macrophages were added to 12-mm diameter, round-bottomed polystyrene tubes and pulsed overnight with varying concentrations of HA peptide, T–HA chimeric proteins or thioredoxin alone. Assays were performed using cDMEM. The cells were washed once and analyzed by flow cytometry using a FACSCalibur (Becton Dickinson, Mountain View, CA). A minimum of 10,000 events per sample were analyzed using CellQuest software. Fluorescence data was collected with logarithmic amplification. Analysis of the role of BMDC in T cell activation using fusion proteins BMDC were exposed to antigen for 2 h at 37°C, washed twice and mixed with A5 cells overnight. Before and after antigen exposure BMDC were treated with 1% paraformaldehyde for 1 min at room temperature and washed twice with PBS. BMDC alone and BMDC together with A5 cells were exposed to antigen overnight and then stained with PE-conjugated AMT-13 for 40 min on ice (1/200 dilution). Untreated peritoneal macrophages and A5 cells were stained in the same way. The degree of expression of CD25 (IL-2Rα) was then measured by flow cytometry. To test the effect of blocking CD25 on T cell activation a mAb known to block receptor function was used (24). BMDC and A5 cells were incubated separately for 40 min on ice with 3C7 mAb or isotype-matched control at 5 μg/ml. The BMDC were then exposed to antigen for 2 h at 37°C. The BMDC and A5 cells were then mixed together, washed 3 times, and incubated overnight. The cells were washed once and analyzed by flow cytometry. Results Linking IL-2 to the HA antigen greatly increased the activation of A5 cells compared to HA antigen alone A5 cells and BMDC were mixed in a 2:1 ratio and incubated with antigen overnight. Activation of A5 cells by antigen resulted in induction of GFP which was detected by flow cytometry. All forms of the HA antigen caused activation of the A5 cells, i.e. HA peptide alone and the chimeric proteins: T–HA110–119, T–HA94–131, T–HA–IL-2, T–HA–IL-4 and T–HA-IFN-γ (Fig. 1). The percent of GFP+ A5 cells and their mean fluorescence increased with increasing antigen concentration (Figs 1 and 2). The theoretical maximum level of A5 activation was 66%, although in practise the actual maximum was slightly lower and usually between 50–60%. When the percent GFP+ cells was maximal then mean fluorescence was a more sensitive indicator of A5 activation as it maintained its dose responsiveness (Fig. 2). The presence of the HA antigen was absolutely necessary for A5 cell activation since exposure to 200 nM T–IL-2, T–IL-4, T–IFN-γ, thioredoxin or medium alone resulted in <5% of A5 cells becoming activated. This result also shows that the A5 cells were not activated by bacterial products, such as lipopolysaccharide, that may be present in the fusion protein preparations. Since all fusion proteins were prepared in the same way, any contaminating bacterial products would be present in all the different preparations. The concentration of antigen required to activate A5 cells depended on the form of the antigen available to the APC. When BMDC and A5 cells were exposed to HA peptide then cell activation was detected over a concentration range of 3–50 μM (Fig. 1). When the peptide was produced as a fusion protein with thioredoxin, T–HA110–119, then less antigen was required for cell activation, i.e. 75–600 nM (Fig. 1). This trend continued when additional flanking sequences were added to the HA peptide, i.e. T–HA94–131, and when IL-2, IL-4 and IFN-γ were linked to the HA(110–119) antigen. In these forms an antigen concentration of 1–150 nM was able to activate A5 cells (Fig. 1). However, at concentrations <10 nM the T–HA–IL-2 fusion protein was by far the most effective at activating A5 cells. Thus, the T–HA–IL-2 form of the HA antigen was capable of activating A5 cells at 1000-fold less concentration than the HA peptide when presented by BMDC. Linking the antigen to another cytokine, e.g. IL-4, was better than the T–HA or peptide alone but not as effective as linking the antigen to IL-2. The type of APC used was also important with respect to the presentation of the chimeric proteins. When resident peritoneal macrophages were used instead of BMDC then a slightly different pattern of A5 activation was seen. T–HA–IL-2 and T–HA–IL-4 gave similar levels of activation, and were more effective at lower concentrations than T–HA94–131 and T–HA-IFN-γ (Fig. 3). All these chimeric proteins were more effective than T–HA110–119 as seen with BMDC. Adding the IL-2 and HA antigen separately was not as effective at activating A5 cells as linking the two in a chimeric protein To investigate whether the physical linkage of the cytokine and antigen was necessary for this increased sensitivity we compared A5 cell activation by T–HA–IL-2 to activation by T–HA and IL-2 added separately. BMDC and A5 cells were exposed to three different concentrations of T–HA in the presence of three different concentrations T–IL-2. Adding IL-2 separately did increase cell activation by T–HA but was not as effective as linking the two in the fusion protein, T–HA–IL-2 (Fig. 4). T–IL-2 alone did not activate the A5 cells (< 1% GFP+), whereas between 19 and 54% of A5 cells were GFP+ in the presence of BMDC incubated with T–HA–IL-2 and T–HA alone and in combination with T–IL-2. Paraformaldehyde inhibited the ability of BMDC to activate A5 cells To investigate whether antigen processing was necessary for A5 cell activation, BMDC were treated with paraformaldehyde before and after antigen exposure. The antigen concentration used was selected to give similar levels of A5 activation by the three different antigens. Treatment of BMDC with paraformaldehyde decreased A5 cell activation by HA peptide, T–HA and T–HA–IL-2 (Fig. 5). This shows that antigen processing was required for the presentation of the HA epitope from both T–HA and T–HA–IL-2 and for the peptide itself. Inclusion of an antibody blocking CD25 inhibited T cell activation via BMDC Even though IL-2 alone did not activate A5 cells, it was possible that the IL-2 component of the antigen was having a direct effect on the APC and A5 cells. To investigate this possibility we measured IL-2Rα (CD25) expression on both BMDC and A5 cells before and after antigen exposure. The antigen concentration used was selected to give similar levels of A5 activation by the two different antigens. Expression of CD25 could not be detected on unstimulated A5 cells (Table 1). After antigen activation the level of CD25 expression on GFP+ A5 cells was very low, suggesting that the T–HA–IL-2 construct had little or no direct affect on receptor expression of these cells (Table 1). Furthermore, activation by both T–HA and T–HA–IL-2 resulted in similar levels of CD25 expression, suggesting that expression was not mediated directly by IL-2. BMDC expressed low levels of CD25 and expression was increased with exposure to T–HA and T–HA–IL-2 (Table 1), T–HA94–131, T–HA–IL-4 and T–HA-IFN-γ (data not shown). This suggested that the IL-2 component of the antigen was not directly responsible for CD25 up-regulation but was probably indicative of DC maturation which occurs after antigen uptake (11,13,15). However, BMDC express much higher levels of CD25 compared to A5 cells which suggests that this receptor may have a role in antigen uptake and presentation of T–HA–IL-2 by BMDC. Expression of CD25 could not be detected on resident peritoneal macrophages (0.2% positive, 5.5 mean fluorescence; isotype control 0.3% positive, 5.6 mean fluorescence). The role of CD25 was further investigated by treating BMDC and A5 cells separately with an antibody known to block the receptor. BMDC were exposed to antigen for 2 h, in the presence of antibody, before being added to the A5 cells and incubated overnight. Activation of A5 cells by T–HA–IL-2 was decreased when BMDC were treated with CD25 antibodies but not when A5 cells were so treated. No such decrease was observed in the presence of the control antibody or when T–HA94–131 was used as the antigen (Fig. 6). Discussion We have shown that the form of the antigen supplied to the APC is important in determining the T cell response. Peptide was the least effective form of the HA antigen, whereas fusing the peptide to thioredoxin and cytokines was more effective than peptide alone in activating T cells. The presence of thioredoxin and cytokines may have enhanced the effectiveness of the HA antigen by providing epitope protection, by enhancing antigen uptake or by enhancing the subsequent processing of the HA antigen. By flanking both ends of the HA epitope the thioredoxin and cytokine may protect the epitope from destructive degradation allowing it to be more efficiently processed into free peptide and presented to the T cell. This explanation is also consistent with the intermediate stimulatory capacity of the T–HA fusion protein where only one of the two ends of the peptide is protected. However, this explanation cannot account for the enhanced T cell activation by T–HA–IL-2 when presented by BMDC, and by both T–HA–IL-2 and T–HA–IL-4 when presented by resident peritoneal macrophages. Antigen processing of the fusion proteins was clearly required because fixing BMDC dramatically impaired their ability to stimulate T cells. However, paraformaldehyde treatment affected the processing of both T–HA and T–HA–IL-2 to a similar degree. This implies that enhanced antigen uptake may be largely responsible for the difference in effectiveness of these two forms of the HA antigen. Indeed, preliminary experiments using labeled fusion proteins show that uptake of T–HA–IL-2 is higher than that of T–HA (unpublished observations). An interesting difference emerged with the T–HA–IL-2 and T–HA–IL-4 fusion proteins showing that the APC was also important. Investigations into why T–HA–IL-2 and T–HA–IL-4 fusion proteins were the most effective form of the antigen at activating T cells when presented by resident peritoneal macrophages are currently underway. In this paper we have concentrated on trying to explain why the T–HA–IL-2 fusion protein was the most effective form of the antigen at activating T cells when presented by BMDC. Our investigations suggest this difference was probably associated with CD25 expression on BMDC rather than on the responding T cell. A5 cells do not express CD25 and it seems unlikely that the small increase in CD25 expression seen on activated A5 cells could be responsible for the large difference in antigen sensitivity we detected. On the other hand, BMDC do express CD25 and the level of expression increased with antigen exposure. The low-affinity form of the IL-2R, comprising α and γ chains in the absence of the β chain, has been detected on DC isolated from different sources, i.e. murine spleen, thymus, lymph nodes and skin (10–18). The relatively low level of expression we observed on BMDC compared to DC extracted from lymphoid tissues can be explained with reference to the heterogeneous nature of DC and the different stages of maturation of DC derived from different tissues. Indeed the proportion of CD25+ cells is consistent with that reported by Lutz et al. (25), where DC were also generated under the influence of GM-CSF. Immature DC can internalize soluble exogenous antigens by both fluid-phase uptake through macropinocytosis or by receptor-mediated uptake (reviewed in 26–28) via the mannose receptor, DEC 205 (29), and Fc receptors (30,31). The antigens are delivered to endocytic/lysosomal compartments for processing and loading on to MHC class II molecules. We suggest that CD25 on DC may also be involved in receptor-mediated antigen uptake. If the same antigen can be taken up by both macropinocytosis and receptor-mediated uptake then the density of antigen on the cell surface is likely to be greater. This will result in greater T cell activation. It has been demonstrated that mannose receptor-mediated antigen uptake was 100-fold more efficient at antigen presentation to T cells (32). The efficiency of receptor-mediated uptake in turn depends on whether the receptor is used once like the Fc receptors (33) or whether it is constitutively recycled like the mannose receptor (34). It is not known whether CD25 is constitutively recycled in DC. However, in T cells the α, β and γ chains of the IL-2R have been shown to localize to different subcellular compartments and undergo different fates. Following IL-2 binding, the α chain was found in an early endosomal compartment and subsequently recycled to the plasma membrane, whereas both the β and γ chains were found in the late endosomal compartment and subsequently degraded (35,36). If CD25 acts in a similar manner to the mannose receptor then even a low of expression could have a marked effect on antigen uptake. The physical linkage of the peptide and cytokine was required for the enhanced activity, suggesting that delivery at the same time and to the same APC was important. The importance of co-delivery has been reported by others using DNA constructs—those encoding antigen together with cytokine were found to be more effective immunogens than DNA encoding either the antigen or the cytokine alone (7,37). Previous work by others using IL-2Rα null mice suggested that this chain was not involved in DC differentiation since these mice had a full complement of cells. Neither could this be implicated in the presentation of alloantigen since APC from these mice were capable of stimulating mixed lymphocyte responses that were indistinguishable in magnitude from those generated using stimulator cells from wild-type mice. The authors did point out that it was possible that CD25 may play a role in antigen acquisition. We suggest that our results support a model in which antigen fused to IL-2 is taken up by DC more efficiently using receptor-mediated endocytosis, resulting in greater antigen density on the cell surface or possibly a greater degree of DC activation. This in turn is reflected in enhanced stimulatory capacity. This suggestion is supported by two findings. Firstly, that delivery of equivalent amounts of IL-2 and the peptide separately failed to increase T cell activation to the same degree and secondly, that blocking CD25 on DC with an antibody during antigen exposure resulted in a significant decrease in T cell activation. It has been reported that the absence of the β chain precludes signal transduction through the IL-2R, resulting in a non-functional receptor on DC (19,20). However, a recent report by Fukao and Koyasu (16) has demonstrated that mature murine splenic DC are capable of responding to IL-2, arguing for a functional IL-2R. IL-2 augmented the production of IFN-γ by these cells when they were stimulated with IL-12 or by CD40 and MHC class II cross-linking. Furthermore, this enhancing effect was blocked by CD25 antibodies. The role of the β chain in this process remains uncertain since the authors failed to detect IL-2Rβ chain on the cell surface but did detect IL-2Rβ chain mRNA. This finding opens the possibility that T–HA–IL-2 could have other affects on DC function by signaling directly through the IL-2R. The physiological relevance of antigen acquisition via the CD25 is unclear. It is not inconceivable that some viruses express receptors homologous to mammalian CD25. Various pox viruses have been shown to express a range of cytokine receptor homologues. These may block the delivery of cytokines detrimental to virus survival (38). Many of these receptors are soluble but a few, such as vaccinia virus TNFR, are found in membrane-bound form (39). This suggests a mechanism for host counter-attack since binding IL-2 to a surface receptor such as this could render a virus susceptible to uptake via CD25 on the APC surface. Alternatively, CD25-mediated antigen acquisition may simply be an unexpected use of a receptor designed for other physiological purposes. For instance, there is some evidence to suggest that IL-2 may be important in the development of DC from progenitor cells in human cord blood (40), although this does not seem to be the case in mice. Irrespective of these speculations, there remains the exciting possibility that our results may be exploited in the development of new vaccination strategies by targeting fusion proteins to receptors on APC. Table 1. T–HA–IL-2 is not acting directly on A5 cells to cause activation Antigen activation of A5 cells  CD25 expression on  Antigen  GFP+ A5 cells  GFP+ A5 cells  BMDC    %  Mean fluorescence  %  %  Mean fluorescence  aMean fluorescence of all A5 cells.  600 nM T–HA  60.1  1678.8  2.6 (0.1)  20.7 (0.5)  52.1 (10.7)  300 nM T–HA  58.3  1352.2  1.5  17.9  47.1  150 nM T–HA  50.8  918.1  1.2  15.6  41.9  10 nM T–HA–IL-2  57.0  2149.1  2.9 (0.2)  21.7 (1.6)  54.1 (15.7)  5 nM T–HA–IL-2  58.0  2206.9  2.3  10.0  33.3  2.5 nM T–HA–IL-2  52.2  1928.4  1.7  12.0  36.6  None  1.0  [11.5]a  0.0 (0.0)  2.9 (0.8)  37.7 (17.0)  Antigen activation of A5 cells  CD25 expression on  Antigen  GFP+ A5 cells  GFP+ A5 cells  BMDC    %  Mean fluorescence  %  %  Mean fluorescence  aMean fluorescence of all A5 cells.  600 nM T–HA  60.1  1678.8  2.6 (0.1)  20.7 (0.5)  52.1 (10.7)  300 nM T–HA  58.3  1352.2  1.5  17.9  47.1  150 nM T–HA  50.8  918.1  1.2  15.6  41.9  10 nM T–HA–IL-2  57.0  2149.1  2.9 (0.2)  21.7 (1.6)  54.1 (15.7)  5 nM T–HA–IL-2  58.0  2206.9  2.3  10.0  33.3  2.5 nM T–HA–IL-2  52.2  1928.4  1.7  12.0  36.6  None  1.0  [11.5]a  0.0 (0.0)  2.9 (0.8)  37.7 (17.0)  View Large Fig. 1. View largeDownload slide Activation of A5 cells by BMDC and different forms of HA antigen. A5 cells (2×105) and BMDC (1×105) were incubated overnight with varying concentrations of antigen. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 1. View largeDownload slide Activation of A5 cells by BMDC and different forms of HA antigen. A5 cells (2×105) and BMDC (1×105) were incubated overnight with varying concentrations of antigen. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 2. View largeDownload slide Activation of A5 cells by T–HA–IL-2. A5 cells (2×105) and BMDC (1×105) were incubated overnight with varying concentrations of T–HA–IL-2. Induction of GFP was analyzed by flow cytometry and the results are given as the percent GFP+ cells and the mean fluorescence of the GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 2. View largeDownload slide Activation of A5 cells by T–HA–IL-2. A5 cells (2×105) and BMDC (1×105) were incubated overnight with varying concentrations of T–HA–IL-2. Induction of GFP was analyzed by flow cytometry and the results are given as the percent GFP+ cells and the mean fluorescence of the GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 3. View largeDownload slide Activation of A5 cells by peritoneal macrophages and different forms of HA antigen. A5 cells (2×105) and resident peritoneal macrophages (1×105) were incubated overnight with varying concentrations of antigen. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 3. View largeDownload slide Activation of A5 cells by peritoneal macrophages and different forms of HA antigen. A5 cells (2×105) and resident peritoneal macrophages (1×105) were incubated overnight with varying concentrations of antigen. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 4. View largeDownload slide Linking IL-2 to HA peptide is more effective at activating A5 cells than adding them separately. A5 cells (2×105) and BMDC (1×105) were incubated overnight with (A) T–IL-2 alone, (B) T–HA–IL-2 and T–IL-2 alone or with T–HA and T–IL-2 together. Induction of GFP was analyzed by flow cytometry. The results are given as the mean fluorescence of (A) all the cells or (B) GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 4. View largeDownload slide Linking IL-2 to HA peptide is more effective at activating A5 cells than adding them separately. A5 cells (2×105) and BMDC (1×105) were incubated overnight with (A) T–IL-2 alone, (B) T–HA–IL-2 and T–IL-2 alone or with T–HA and T–IL-2 together. Induction of GFP was analyzed by flow cytometry. The results are given as the mean fluorescence of (A) all the cells or (B) GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 5. View largeDownload slide Treatment of BMDC with paraformaldehyde prevents presentation of HA to A5 cells. BMDC (1×105) were fixed with 1% paraformaldehyde before and after antigen exposure for 2 h at 37°C. A5 cells (2×105) were added and incubated overnight. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 5. View largeDownload slide Treatment of BMDC with paraformaldehyde prevents presentation of HA to A5 cells. BMDC (1×105) were fixed with 1% paraformaldehyde before and after antigen exposure for 2 h at 37°C. A5 cells (2×105) were added and incubated overnight. Induction of GFP was analyzed by flow cytometry. The results are given as the percent GFP+ cells. Three experiments were performed and the results of one representative experiment are shown here. Fig. 6. View largeDownload slide Addition of anti-CD25 antibodies decreases activation of A5 cells by T–HA–IL-2. A5 cells (2×105) and BMDC cells (1×105) were incubated separately with 3C7 or isotype-matched control antibody for 30 min at 4°C. BMDC were then incubated with 50 nM of antigen for 2 h at 37°C. BMDC and A5 cells were washed, added together and incubated overnight. Induction of GFP was analyzed by flow cytometry. The percent GFP+ cells in the presence of the 3C7 mAb was calculated with respect to percent GFP+ cells in the presence of the isotype-matched control which was assigned a value of 100%. Three experiments were performed and the results of one representative experiment are shown here. Fig. 6. View largeDownload slide Addition of anti-CD25 antibodies decreases activation of A5 cells by T–HA–IL-2. A5 cells (2×105) and BMDC cells (1×105) were incubated separately with 3C7 or isotype-matched control antibody for 30 min at 4°C. BMDC were then incubated with 50 nM of antigen for 2 h at 37°C. BMDC and A5 cells were washed, added together and incubated overnight. Induction of GFP was analyzed by flow cytometry. The percent GFP+ cells in the presence of the 3C7 mAb was calculated with respect to percent GFP+ cells in the presence of the isotype-matched control which was assigned a value of 100%. Three experiments were performed and the results of one representative experiment are shown here. 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TI - IL-2 linked to a peptide from influenza hemagglutinin enhances T cell activation by affecting the antigen-presentation function of bone marrow-derived dendritic cells
JO - International Immunology
DO - 10.1093/intimm/13.6.713
DA - 2001-06-01
UR - https://www.deepdyve.com/lp/oxford-university-press/il-2-linked-to-a-peptide-from-influenza-hemagglutinin-enhances-t-cell-b3Nfp0R7yM
SP - 713
EP - 721
VL - 13
IS - 6
DP - DeepDyve
ER -