Helminth Antigens Selectively Differentiate Unsensitized CD45RA +CD4+ Human T Cells in Vitro This information is current as of December 20, 2017.
Cathy Steel and Thomas B. Nutman J Immunol 1998; 160:351-360; ; http://www.jimmunol.org/content/160/1/351
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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Helminth Antigens Selectively Differentiate Unsensitized CD45RA1CD41 Human T Cells in Vitro Cathy Steel1 and Thomas B. Nutman
T
wo disparate but intriguing aspects of the immunobiology of lymphatic filariasis are that initial infection in a previously unsensitized host invariably leads to elevations of serum IgE and circulating eosinophils and that sensitization to parasite Ag can occur in utero, a process that may lead to cellular hyporesponsiveness that is parasite Ag specific and has, in one study, been shown to persist over decades (1). This lack of cellular responsiveness has been shown to be directed primarily toward the microfilariae (Mf),2 the parasite stage found in the blood circulation that represents the major depot of parasite Ag. Whether this Ag-specific hyporesponsiveness is a cause or a result of this heavy intravascular parasite burden remains to be determined, but the effect on clinical outcome is that the majority of individuals, when naturally infected, remain clinically asymptomatic (2–5). The regulatory controls on the T cell responses in these asymptomatic, but microfilaremic, individuals (and possibly in all circulating filarial Ag1 patients) (6) result in an inability to proliferate or produce IFN-g in response to parasite Ag, although the ability to produce IL-4 and IL-5 remains intact (7). A key molecule in this down-regulation of type 1 responses appears to be IL-10, whose production is not only elevated in these asymptomatic microfilaremic individuals (8) but also is preferentially induced by the microfilarial stage of the parasite (9). While many factors may be invoked for modulating the immune response to filarial parasite Ag in these asymptomatic individuals, including cross-regulatory cytokines and genetic factors, the primary and initial determinant
may be exposure to Ag in utero. As such, when infection occurs (usually in childhood before the age of 15 yr), the previous prenatal exposure has possibly conditioned the immune response such that the production of IFN-g and/or IL-2 is preferentially diminished in patent infections. Thus, the deviation of the immune system away from a type 1 response suggests that early priming events may play a critical role in determining the nature of the secondary (and long lasting) response that occurs. While many factors are known to affect the differentiation of naive T cells into mature memory cells, including the cytokine milieu (10 –17), the type of APCs (18), and the dose of Ag (19), it is possible that the antigenic composition of the parasite itself is also responsible. To examine the events that occur at the initial interface between unsensitized T cells and filarial parasite Ag (and more generally at the time of initial priming of naive T cells), we have developed an in vitro system that allows for primary sensitization of naive T cells by soluble filarial Ag, a system that has not been heretofore achieved using soluble Ag. Using soluble filarial Ag alone in the presence of accessory cells (B cells and monocytes), highly purified CD45RA1CD41 T cells from unsensitized, normal human donors were able to both proliferate and secrete type 1- and type 2-associated cytokines. These cells were able to respond on restimulation with Ag and did so with a selective array of cytokine production depending on the cytokine milieu present at the time of priming.
Materials and Methods
Purification of CD45RA1CD41 T cells Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 Received for publication June 1, 1997. Accepted for publication September 19, 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Cathy Steel, Building 4, Room 126, National Institutes of Health, Bethesda, MD 20892– 0425. E-mail address:
[email protected] 2 Abbreviations used in this paper: Mf, microfilaria; C-RPMI, RPMI, 10 mM HEPES, and 80 mg/ml gentamicin; PE, phycoerythrin; MfAg, soluble Mf antigen; SI, stimulation index; SEB, staphylococcus enterotoxin B; bright, strongly staining.
Copyright © 1998 by The American Association of Immunologists
PBMCs from normal blood bank donors were isolated by Ficoll diatrizoate density centrifugation (20). PBMCs were then suspended at 10 3 106/ml in RPMI (BioWhittaker, Walkersville, MD), 10 mM HEPES, and 80 mg/ml gentamicin (C-RPMI) supplemented with 10% FCS (Life Technologies, Grand Island, NY) and incubated with equal volumes of FCS and 2% neuraminidase-treated (Sigma Chemical Co., St. Louis, MO) SRBCs overnight at 4°C. This suspension was subsequently relayered over Ficoll diatrizoate to separate the T cell and B cell/monocyte layers, which were then thoroughly washed following lysis of SRBCs. The partially purified T cells (;90 –95% CD31) were then further purified by negative selection for CD45RA1 T cells. T cells were suspended at 20 3 106/ml in HBSS (Biofluids, Rockville, MD), 10% FCS, and 20 mM HEPES (selection buffer) and rocked for 30 min at 4°C with a mixture containing mAbs to 0022-1767/98/$02.00
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Human filarial helminth infections are characterized by type 2 immune responses to parasite Ag that can persist for the life of the individual; one possible cause for this may be prenatal exposure to the blood-borne microfilarial (Mf) stage of the parasite. To examine the relationship between early exposure to filarial Ag and subsequent immune responsiveness, CD45RA1CD41 cells from normal unsensitized donors were stimulated in vitro with soluble microfilarial Ag (MfAg) from the filarial parasite Brugia malayi in the presence of APCs. MfAg alone induced proliferation and IFN-g and IL-5 production in unsensitized CD45RA1CD41 cells, demonstrating the ability of filarial Ags to prime naive T cells in the absence of exogenous cytokines and dendritic cells. Adding exogenous cytokine(s) (particularly IL-12 and IL-4) during priming was able to alter the MfAg-specific responses of CD45RA1CD41 cells as well as subsequent responses to Ag. Interestingly, priming solely with MfAg led to enhanced IL-5 production following Ag restimulation, suggesting that MfAg preferentially primes for type 2 responses. These data demonstrate that filarial Ags by themselves can specifically prime CD45RA1CD41 cells in vitro and do so in such a way as to deviate the immune response. The Journal of Immunology, 1998, 160: 351–360.
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PARASITE Ag-DRIVEN DIFFERENTIATION OF CD45RA1CD41 T CELLS 0.05% Tween. Plates were then blocked with PBS, 5% bovine albumin (Sigma), and 0.05% Tween for 1 h and washed again. Culture supernatants were added and incubated overnight at 4°C; plates were then washed, and IL-5 was detected with nitroiodophenyl (Sigma)-conjugated rat anti-human IL-5 (JES1–5A10; PharMingen) in PBS, 1% bovine albumin, and 0.05% Tween-20 (ELISA diluent) for 2 h at 37°C. Following washing, an antinitroiodophenyl peroxidase-conjugated Ab (J4-HRP; DNAX) in ELISA diluent was added for 1 h at 37°C. Plates were again washed and subsequently developed with a peroxidase substrate system (ABTS; Kirkegaard and Perry, Gaithersburg, MD); colorimetric development was detected at 450 nM using a microplate reader (Molecular Devices, Sunnyvale, CA). Measurements of IFN-g and IL-10 were performed similarly using the following Abs: anti-human IFN-g (Endogen, Woburn, MA) or anti-human IL-10 (JES3–9D7; PharMingen) for coating, polyclonal rabbit anti-human IFN-g (author’s laboratory) or biotin-conjugated anti-human IL-10 (JES3– 12G8; PharMingen) for detection, and peroxidase-conjugated goat antirabbit IgG or alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, West Grove, PA) for enzyme conjugation. Quantification was based on standardized curves using recombinant cytokines.
Preparation of MfAg
Because the data were generally not normally distributed, the Wilcoxon signed rank test was used for comparison of data.
A saline extract of the microfilarial stage of the parasite Brugia malayi (MfAg) was prepared as previously described (21). Briefly, Mf were collected by peritoneal lavage of infected jirds and separated from peritoneal cells by Ficoll diatrizoate density centrifugation. The Mf were then washed repeatedly in RPMI with antibiotics and cultured overnight at 37°C in 5% CO2. Worms were harvested the following day, washed with PBS, and frozen at 220°C. The frozen Mf were pulverized, sonicated, and extracted in PBS at 37°C for 4 h and then at 4°C overnight. Following centrifugation at 20,000 3 g for 30 min, the supernatant was passed through a 0.45-mm pore size filter and stored in aliquots at 270°C.
Primary Ag stimulation of the CD45RA1 T cell population CD45RA1 T cells were placed in culture at 1 3 106/ml in C-RPMI and 10% FCS in a 48-well flat-bottom tissue culture plate (Costar, Cambridge, MA) with 25% irradiated (3000 rad; 137Cs source; Nordion, Kanata, Ontario, Canada) APCs from the B cell/monocyte layer (T cell depleted; ;1% CD31, 4% CD561). Cells were cultured in medium alone or with MfAg at 1, 5, or 10 mg/ml with or without recombinant human IL-2 (Cetus, Emeryville, CA) at 25 U/ml, IL-4 (Immunex, Seattle, WA) at 20 ng/ml, IL-10 (Genzyme, Cambridge, MA) at 50 U/ml, or IL-12 (Genetics Institute, Cambridge, MA) at 50 U/ml for 5 days at 37°C in 5% CO2, at which time supernatants were harvested for quantification of cytokine production. All reagents used for these and subsequent assays were free of endotoxin.
Restimulation of T cells with MfAg In some experiments, cells were rested for 3 days with fresh medium with or without IL-2 at 50 U/ml. Debris and nonviable cells were removed by layering over FCS and spinning at 800 rpm. Viable cells were then counted by trypan blue exclusion and recultured at 1 3 106/ml with 25% APCs and 10 mg/ml of MfAg. In some experiments, IL-2 was also added at 25 U/ml.
Proliferation and limiting dilution analysis For measurement of proliferation, purified CD45RA1 T cells were cultured with 25% irradiated APCs in 96-well U-bottom plates (Costar) at 1 3 105/well in C-RPMI supplemented with 10% AB serum (BioWhittaker) with or without MfAg for 5 days at 37°C in 5% CO2, after which 1 mCi/ well of [3H]TdR (New England Nuclear Corp., Boston, MA) was added. Cultures were harvested 18 h later, and results were expressed as stimulation indexes (SI 5 mean counts per minute of stimulated cultures/counts per minute of unstimulated (medium alone) cultures). For limiting dilution analyses, cells were cultured at varying concentrations in a similar manner. The frequency of parasite Ag-specific cells was calculated by conventional means according to the method of Lefkovits and Waldman (22) as modified by Taswell (23). The percentage of responsive wells was based on the number of wells (n 5 18 at each cell dilution) with Ag, or Ag with cytokines having proliferation values greater than the mean 1 3 SDs of the wells without Ag (n 5 6).
Quantification of cytokines by ELISA For measurement of IL-5, round-bottom plates (Immulon 2, Dynatech, Chantilly, VA) were coated with rat anti-mouse IL-5 (TRFK5; DNAX, Palo Alto, CA) at 1 mg/ml in PBS followed by washing with PBS and
Statistical analyses
Results Primary responses: proliferation CD45RA1 T cells were capable of responding to MfAg at 10 and 5 mg/ml, with SI values ranging from 2.0 to 8.5 (data not shown); there was little proliferative response at 1 mg/ml, even with the addition of IL-2 at 50 U/ml. In the absence of exogenous cytokines, the number of Ag-responsive CD45RA1 cells ranged from 1/52,000 to 1/87,000 at an Ag concentration of 10 mg/ml (Fig. 1). The frequency increased when exogenous IL-2 and IL-4 were added in addition to Ag (Fig. 1B). To exclude the possibility that MfAg was acting as a superantigen, CD45RA1 T cells and unselected T cells (all CD31) from the same individuals were stimulated with either MfAg or the superantigen Staphylococcus enterotoxin B (SEB) at various concentrations in the presence of irradiated or paraformaldehyde-fixed autologous APCs. While both CD45RA1 and total CD31 T cells responded to MfAg using normal irradiated APCs, neither T cell population proliferated in response to Ag in the presence of fixed APCs, demonstrating that MfAg requires processing to induce T cell activation and proliferation (data not shown). Interestingly, while both groups of T cells (unselected and CD45RA1) responded to SEB using irradiated autologous APCs, only unselected (all CD31) T cells were activated by SEB with fixed APCs, indicating that CD45RA1 T cells may have requirements for responses to superantigens that differ from those of more heterogeneous populations of T cells. Further, MfAg was unable to induce proliferation in unselected T cells when mismatched APCs were used (data not shown).
Phenotype changes The low frequency of MfAg-responsive cells, as demonstrated above by the precursor frequencies, was also evident by the small percentage of cells that exhibited a change in phenotype from CD45RA1RO2 to CD45RA2RO1. In three individual experiments, two of three individuals showed a demonstrable, but small, shift to the CD45RO1 phenotype following stimulation with MfAg and a 3-day rest period with rIL-2. In the two patients who responded to Ag with a phenotypic change, an average of 82% of cells retained the CD45RA1RO2 phenotype compared with 86% in those cells stimulated with medium alone. Three percent of cells changed to the CD45RA2RO1 phenotype compared with 1% of cells in medium. In addition, 6% of cells demonstrated a shift to
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CD8 (B9.8; B. Malissen, CIML, Marseilles, France), CD14 (63D3; American Type Culture Collection, Rockville, MD), CD16 (3G8; J. Jaffe, Hahnemann University, Philadelphia, PA), CD19 (FMC63; H. Zola, Woman’s and Children’s Hospital, Adelaide, Australia), HLA-DR (IVA12; American Type Culture Collection), CD56 (PharMingen, San Diego, CA), glycophorin (10F7; American Type Culture Collection), and CD45RO (UCHL1; P. Beverly, University College, London, U.K.). Cells were then washed three times in selection buffer, resuspended with goat anti-mouse IgG magnetic beads (PerSeptive Diagnostics, Cambridge, MA) at 50 beads/cell, and again rocked at 4°C for 30 min. Beads were removed twice by magnetic separation, and nonselected cells were washed and counted. These cells were combined with goat anti-mouse IgG magnetic beads (Dynal, Oslo, Norway) at five beads per cell for an additional 30 min at 4°C followed by two magnetic separations. Following the two negative selections, approximately 1 to 3% of the initial PBMCs were recovered as CD45RA1CD41 cells. This remaining population was approximately 99 to 100% CD45RA1 T cells as measured by flow cytometry, with 2 to 3% of cells being CD45RA1ROdim. To phenotype cells, Ab reagents used for flow cytometry were: FITC-conjugated CD45RA and phycoerythrin (PE)conjugated CD45RO (Immunotech, Westbrook, ME), FITC- and PE-conjugated CD3 (Immunotech), and FITC- and PE-conjugated mouse IgG1 (Caltag, San Francisco, CA) as isotype controls.
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FIGURE 2. Production of IFN-g ( left panel ) and IL-5 ( right panel ) by CD45RA1 T cells to IL-2 at 25 U/ml and to MfAg (at 10 mg/ml) plus IL-2. Each line represents the response from cells of a separate individual ( n 5 11).
the double-positive CD45RA1RObright phenotype compared with 3% in cells stimulated with media alone. In a single individual in whom there were sufficient numbers of cells to be restimulated for 5 days with MfAg, 29% of cells exhibited the CD45RA2RO1 phenotype, with a corresponding loss of 32% of CD45RA1RO2 cells. Repeated stimulation with MfAg eventually caused a complete shift to the CD45RA2RO1 phenotype (data not shown).
Primary cytokine responses MfAg by itself induced low levels of IFN-g (geometric mean net production 5 5.8 pg/ml; range 5 0 –250) and IL-5 (1.8 pg/ml;
range 5 0 – 63.4) in 7 of the 11 individuals studied (data not shown). Priming with MfAg in the presence of exogenous IL-2 caused an increase in the production of IFN-g by the CD45RA1 T cells in 8 of 11 individuals (geometric mean 5 868.3 pg/ml) over that produced in response to IL-2 alone (geometric mean 5 639.1 pg/ml; P 5 0.041; Fig. 2). In contrast, IL-5 production (used as a prototypical type 2 response indicator) was variable, increasing in 6 of 11 individuals when primed with both Ag and IL-2 (geometric mean 5 13.8; with IL-2 alone, 8.7 pg/ml; P 5 0.086). The effect of the cytokine milieu present at the time of priming with Ag was then assessed by comparing the production of IFN-g and IL-5 in response to MfAg with IL-2, IL-4, or the combination
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FIGURE 1. The frequency of MfAg-responsive CD45RA1 T cells by limiting dilution analysis. A, The frequency (F0) of responsive cells in one individual to different Ag concentrations. B, The frequency of responsive cells in an individual to Ag alone or to Ag with IL-2 plus IL-4 added.
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of IL-2 and IL-4 to the production in the presence of these cytokines alone (Fig. 3). While MfAg increased production of IFN-g in 8 of 11 individuals (same individuals as in Fig. 2) in the presence of exogenous IL-2 (median 5 277 pg/ml), when IL-4 was present either alone or in combination with IL-2, IFN-g production was absent or diminished. This suggests that IL-4 predominates over IL-2 if present at the time of priming with Ag. IL-5 production was enhanced by MfAg, particularly if IL-4 was also present (Fig. 3). As seen, in the presence of IL-2 at priming, MfAg enhanced IL-5 production in 6 of 11 individuals (same individuals as in Fig. 2; median 5 8.2 pg/ml); 7 of 9 individuals showed increased IL-5 production in the presence of IL-4 (median 5 18.7 pg/ml; P 5 0.018), and 7 of 10 showed increased production when IL-4 was used in conjunction with IL-2 (median 5 13.4 pg/ml; P 5 0.066).
IL-10 priming and production Because filarial Ags and, in particular, the Mf and infective larval stage (L3) Ags induce in vitro IL-10 secretion in naturally infected individuals, and because this IL-10 induction may influence a patient’s response upon subsequent exposure to Ag, CD45RA1 T cells were primed in the presence of MfAg with or without IL-10 in the presence or the absence of IL-2 or IL-4 (Fig. 4). Priming in the presence of IL-10 greatly reduced the production of both IFN-g and IL-5 compared with that seen in response to MfAg alone and in response to Ag plus IL-2 or IL-4. Although the mechanism for this down-regulation of cytokine production by IL-10 was not specifically addressed here, toxicity did not appear to be the reason for
this decrease in production, as cell number and viability were no different in cultures containing IL-10 from those in cultures without IL-10. Although production of IL-10 by CD45RA1 naive T cells could not be separated from that produced by the APCs in these experiments, IL-10 production in response to MfAg under the various priming conditions was nevertheless examined (Table I). Unexpectedly, primary stimulation of the CD45RA1 cells by MfAg induced a down-regulation of IL-10 in five of six individuals compared with the production by cells in medium alone; however, this difference was not significant (P 5 0.12). The addition of IL-2 during priming with MfAg, however, did increase IL-10 production over that produced by cells stimulated with IL-2 alone (P 5 0.04; Table I). No differences, however, were observed when IL-4 and MfAg were used in priming compared with priming with IL-4 alone (P 5 0.72). When both IL-2 and IL-4 were present at priming in concert with MfAg, four of five individuals demonstrated an increase in IL-10 production over that in response to the combination of cytokines alone, although the levels were not significantly different (P 5 0.08).
Priming with IL-12 In three individual experiments, the use of IL-12 at priming enhanced IFN-g production; this production was augmented in two of three individuals with the addition of MfAg (Fig. 5). When IL-2 was added to IL-12, the levels of IFN-g increased over those produced in response to IL-12 alone and again were enhanced in two
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FIGURE 3. Net production of IFN-g ( left panel ) and IL-5 ( right panel ) by CD45RA1 T cells to MfAg in the presence of cytokines during priming. Net production was calculated as the production of IFN-g and IL-5 in response to MfAg in the presence of cytokines minus the production in the presence of cytokine(s) alone. Each dot represents one individual, and the shaded area represents the level at which there were either no differences or diminished production in the absence of Ag compared with that in the presence of Ag.
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Table I. IL-10 production as measured in primary cultures (n 5 6) of CD45RA1 T cells with irradiated B cells and monocytes
Priming Condition
Geometric Mean (pg/mL)
Median (pg/mL)
Range (pg/mL)
Media MfAg IL-2 IL-2 1 MfAga IL-4 IL-4 1 MfAg IL-2 1 IL-4 IL-2 1 IL-4 1 MfAg
45.2 34.9 48.2 76.1 37.3 28.7 160.0 230.4
60.6 36.8 66.5 109.8 47.1 33.5 159.5 156.6
0.0–90.0 17.5–53.7 0.0–124 21.7–169.2 0.0–136.6 0.0–115.4 104.5–299.0 148.8–494.5
a
Significantly different from IL-2 alone (p , 0.04).
of three individuals with the addition of MfAg. There was virtually no IL-5 production in the presence of IL-12 priming, and the addition of IL-2 did not change this. IL-4, as shown in previous experiments, however, was capable of inducing IL-5 in all three individuals (shown with the addition of IL-12; Fig. 5, bottom panel). In two of three individuals, this production was again enhanced with the addition of MfAg. There was very little IFN-g production in the presence of IL-4, despite the addition of either IL-2 or IL-2 plus MfAg. When IL-4 was used in combination with IL-12, IFN-g production increased in both individuals tested. Interestingly and unexpectedly, however, when priming was performed in the presence of both IL-4 and IL-12 with MfAg, the highest levels of IL-5 were produced.
Secondary cytokine responses CD45RA1CD41 T cells originally primed with MfAg alone or in medium alone were rested for 3 days and then restimulated with MfAg. When cells had been primed with MfAg, the subsequent IFN-g production in the secondary cultures was increased in only
one of seven individuals from cells primed in medium alone (Fig. 6). In contrast, IL-5 production increased in five of seven individuals, indicating that cells primed in the presence of filarial Ag alone, without any exogenous cytokine, deviate toward a type 2 phenotype on restimulation with the Ag. This finding occurred regardless of exogenous IL-2 being added during the secondary stimulation (not shown). The pattern of secondary cytokine production differed depending on the nature of the cytokine milieu present at the time of CD45RA1 cell priming. Thus, if Ag priming occurred in the presence of IL-2, IFN-g production increased in three of five individuals (Fig. 7). In contrast, there was little IL-5 production under these same priming conditions, with IL-5 production in four of five individuals either decreasing or showing no change. In four individual experiments in which IL-4 was present at priming with Ag, IFN-g decreased in all individuals compared with production by those cells primed with IL-4 alone (Fig. 7). In contrast, IL-5 levels were higher when cells were primed with Ag in the presence of IL-4 than when they were primed with Ag in the presence of IL-2. Interestingly, priming in the presence of IL-4 alone, without Ag, produced significantly higher IL-5 production (P 5 0.043) upon contact with Ag than did priming in the presence of IL-2 alone.
Discussion A system for in vitro priming of CD45RA1CD41 naive human T cells by soluble helminth Ag has been developed and used to demonstrate that these Ags selectively differentiate CD41CD45RA1 cells. To date, it has been difficult to prime naive T cells with soluble Ag, with most previous in vitro studies having used direct stimulation with anti-CD3 plus a second signal (such as those delivered through anti-CD28 (24, 25), anti-CD70 (26), CD32-transfected fibroblasts (27, 28), mitogens (29, 30), or various exogenous cytokines (15, 31–34)) or by using cell-bound alloantigen (35–38)
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FIGURE 4. Effect of IL-10 on the production of IFN-g ( top panels ) and IL-5 ( bottom panels ) by CD45RA1 T cells from two individuals. Cells were primed with MfAg alone or in the presence of IL-2 or IL-4, with or without IL-10.
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or bacterial superantigens (39, 40). Alternatively, the study of Aginduced differentiation of naive cells has been accomplished using TCR transgenic mice (37, 41– 44). We have demonstrated that using a soluble parasite Ag alone in the presence of APCs, highly purified naive T cells from unsensitized normal human donors were able to both proliferate and secrete type 1- and type 2-associated cytokines. In addition, these cells continued to respond to secondary stimulation with Ag. Naive T cells responded to MfAg in a dose-dependent fashion without the use of exogenous cytokines; the very low frequencies of responsive cells, as demonstrated by the phenotypic changes and precursor frequencies, indicates that this probably was not a recall response to cross-reactive Ags. Additionally, although the MfAg preparation consists of a crude mixture of proteins, the Ag preparation did not appear to have any superantigen or mitogenic effects, as the responses required processing by autologous APCs (data not shown). Of interest, priming could be accomplished in the presence of primarily B cells and monocytes rather than dendritic cells, a situation not paralleled in murine systems in which B cells have been shown to be unable to prime naive T cells in vivo, although they were able to present Ag to activated T cells (45). In contrast, cultured Langerhans cells have been shown to be capable of priming naive T cells to soluble Ag (35) and, unlike B cells or monocytes, were also successful in inducing significant primary in vitro stimulation of hapten-specific T cells (46). Further, it has been suggested that naive cells primed in the presence of unprimed B cells,
but not dendritic cells, should lead to tolerance (47), a hypothesis not supported by the present study using human cells. Mehta-Damani and colleagues have been able to generate Agspecific human T cell lines from naive CD41 cells, but only when dendritic cells were used as APCs during the priming; macrophages were unable to elicit a response by T cells regardless of the number of APCs used (48). Since the number of APCs used in our study was similar in range to that used by Mehta-Damani, and since macrophages are considered better Ag presenters than either B cells or monocytes, the fact that we were able to generate a response in naive cells may be related to the nature of the Ag used. In two studies in mice, for example, dendritic cells were shown to be more efficient at presenting peptides to naive cells, whereas B cells were necessary for priming to the protein forms of the same Ags (49, 50). Whatever the mechanism, MfAg is able, as a soluble extract, to prime cells under the conditions used in this study. Indeed, in preliminary experiments, several other helminth Ags (e.g., Toxocara, Schistosoma, and Onchocerca) were also able to prime CD45RA1CD41 T cells to produce both IL-5 and IFN-g, suggesting that helminth Ags will be particularly effective in eliciting responses from naive T cells (data not shown). Of note, soluble adult Brugia male Ag (from a stage devoid of Mf), while capable of priming for both IFN-g and IL-5, induced IFN-g at levels 6 to 100 times that produced by MfAg. A relatively high level of IFN-g was also observed in a single experiment using a soluble extract of the nonhelminth intracellular parasite Toxoplasma gondii.
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FIGURE 5. Effects of IL-12 on the production of IFN-g ( left panels ) and IL-5 ( right panels ) by CD45RA1 T cells from three individuals. Priming conditions are indicated on the left, and the numbers at the top right of each panel indicate the patient number. ND, not done.
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While MfAg alone was able to elicit a response in most individuals, as illustrated by both Ag-specific proliferation and cytokine production, the addition of exogenous cytokines to the cultures clearly altered the nature of the response. It has been shown from many studies that the cytokine milieu, particularly at the time of priming, influences the nature of the subsequent immune response (10 –17, 33, 51). In the present study, for example, when exogenous IL-2 was used in combination with MfAg, the production of IFN-g was enhanced in nearly all individuals, whereas IL-4 favored the production of IL-5. IL-4, like IL-2, has long been considered a T cell growth factor; however, its potential polarizing effect with regard to CD41 T cell differentiation has been more recently elucidated. While IL-2 has been shown to induce both type 1 and type 2 cytokines (16), the presence of IL-4 favors the type 2 cells (14, 18, 41) and appears to down-regulate IFN-g (16, 51, 52). From our data, the effect of IL-4, either alone or in combination with MfAg or IL-2, on the development of CD45RA1 cells was apparent through analysis of both primary and secondary IFN-g and IL-5 production (Figs. 3 and 7). The addition of IL-4 exogenously with Ag strongly diminished primary IFN-g production and enhanced IL-5 production even when IL-2 was present at priming. This IFN-g down-regulation continued upon restimulation with Ag, even though IL-4 was not used in the secondary stimulation. It has been theorized, at least from data observed using transgenic mice, that the effect of IL-4 occurs early in the activation and priming stages and has a lesser effect on activated cells (52); in contrast, it has also been demonstrated that in human neonatal cells, while the induction of the type 1 phenotype appears to be stable, induction of a type 2 phenotype requires more constant exposure to IL-4 (29, 53). Our
data suggest that the presence of MfAg perhaps stabilizes the type 2 response not only by inducing IL-4 but also by diminishing IFN-g. Thus, having a sustained source of IL-4 skews the cellular response toward a type 2 (and away from a type 1) response, a finding given credence by a study in mice demonstrating that sustained exposure to IL-4 through diffusion chambers enhances IL-4 production while simultaneously decreasing IFN-g production (13). Similar to results obtained in this investigation, several studies have indicated that exogenous IL-4 at the time of priming leads to diminished IFN-g production and/or the production of increased levels of IL-4 or IL-5 (13, 16, 18, 41, 51, 52). Other studies, however, have shown that the addition of IL-4, either alone (32) or in combination with neutralizing anti-IL-2 Ab (17), is not sufficient for the induction of IL-4. Because IL-5 (rather than IL-4) was used as the type 2 indicator cytokine, and because IL-4 and IL-5 may be regulated independently (54), this might explain the differences between the findings in the present study and those of other studies. Our data do indicate, however, that IL-2 and IL-4 together have an additive effect, particularly in the diminution of IFN-g production. IL-12, like IL-4, has been implicated in the differentiation of naive T cells into mature effector CD41 T cells. Most notable is the finding of enhanced IFN-g production when cells are primed with IL-12 (14, 31, 51, 55), although not all this IFN-g production is IL-12 dependent (56). The findings from this study also demonstrated that priming in the presence of exogenous IL-12 leads not only to enhanced IFN-g but also to decreased levels of IL-5. It was apparent that IL-2 worked synergistically with IL-12 to polarize the response toward a type 1 phenotype. Whether this was
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FIGURE 6. Secondary production of IFN-g ( left panel ) and IL-5 ( right panel ) in seven individuals. CD45RA1 T cells were primed in medium alone (gray bars) or in the presence of MfAg (black bars) for 5 days. Cells were then rested in the presence of IL-2 for 3 days and subsequently restimulated with MfAg.
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due to increased numbers of cells producing IFN-g or to increased production within a single cell has not yet been determined, although the latter is less likely. Most unexpected was the observation of extremely high production of IL-5 when T cells were primed in the presence of IL-4 together with IL-12, production that was further enhanced with soluble MfAg. Indeed, the level of IL-5 produced under these conditions surpassed that seen in all other priming conditions studied. Since IL-12 by itself normally down-regulates type 2 cytokine production, the mechanism underlying the synergism (with regard to IL-5 production) seen when IL-4 and IL-12 are present together remains to be elucidated. In a human in vitro system that primed CD41 and CD81 T cell clones for IL-4 production, IL-12 when used in conjunction with IL-4 was shown to induce maximal amounts of IL-4 (57). Further, in a study in unsensitized mice, the use of IL-12 and IL-4 together at priming (in an Ag-independent system) showed increased production of IL-4 (compared with IL-4 alone), but only on restimulation (14). It is conceivable that IL-12 is expanding the type 2 precursors responding to the exogenous IL-4 added, although it is generally thought to only affect type 1 cells in this way. Since IL-4 is normally the dominant cytokine when used with IL-12 (14), it is possible that these naive cells are responding initially to the IL-4 but are then expanding or, alternatively, increasing their individual levels of IL-5 production. It has also been proposed that priming in the presence of IL-4 plus
IL-12 selectively expands a small IL-4-producing population of CD312CD41 cells (29). Filarial infections are particularly relevant to the study of CD41 subset differentiation. In both lymphatic filariasis and onchocerciasis (the two most common forms of filarial infection), the majority of infected individuals have immune responses that are deviated away from a type 1 response (7, 8) and have, as a result, an inability to clear the parasites such that they are constantly and chronically exposed to Mf and MfAg. This is in contrast to those who have cleared their Mf, whose immune responses are generally mixed (type 1 and type 2). In this study, priming with MfAg alone, without exogenous cytokines, followed by restimulation with Ag enhanced the type 2 response and decreased IFN-g levels. In utero exposure to MfAgs circulating in the mother’s blood presumably acts in a similar fashion, such that type 2 responses are induced when the subject is subsequently infected naturally later in life. Because IL-10 has been implicated in the down-regulation of type 1 responses in actively infected individuals (7, 8), the levels of IL-10 produced by CD41CD45RA1 cells were examined in response to MfAg. Interestingly, these cells had a trend toward lower levels of IL-10 production in response to MfAg, possibly indicating a different means of regulation in unexposed naive individuals than in those with active infection who are constantly exposed to MfAg. Addition of exogenous IL-10 down-regulated
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FIGURE 7. Effects of cytokine priming on secondary production of IFN-g ( top panels ) and IL-5 ( bottom panels ) in five individuals. CD45RA1 T cells were primed in the presence of IL-2 or IL-4 in the presence or the absence of MfAg. Cells were then rested for 3 days in the presence of IL-2 and subsequently restimulated with MfAg plus IL-2. The numbers at the top right of each panel represent the patient number. ND, not done.
The Journal of Immunology
Acknowledgments We thank Drs. Alan Sher, Guy Delespesse, and Dragana Jankovic for helpful suggestions, and Ms. Brenda Rae Marshall for editorial assistance.
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