Antigen-Responsive CD4+ T Cells from C3H Mice Chronically Infected with Leishmania amazonensis Are Impaired in the Transition to an Effector
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感染与免疫杂志 2006年第3期
Immunobiology Program Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011-1250
ABSTRACT
C3HeB/FeJ mice challenged with Leishmania major develop a polarized Th1 response and subsequently heal, whereas Leishmania amazonensis challenge leads to chronic lesions with high parasite loads at 10 weeks postinfection. In this study, a comparison of draining lymph node cells from L. amazonensis- and L. major-infected mice at 10 weeks postinfection showed equivalent percentages of effector/memory phenotype CD44hi CD4+ T cells producing interleukin-2 (IL-2) and proliferating after antigen stimulation. However, these cells isolated from L. amazonensis-infected mice were not skewed toward either a Th1 or Th2 phenotype in vivo, as evidenced by their unbiased Th1/Th2 transcription factor mRNA profile. In vivo antigen stimulation with added IL-12 failed to enhance gamma interferon (IFN-) production of CD4+ T cells from L. amazonensis-infected mice. Antigen stimulation of CD4+ T cells from L. amazonensis-infected mice in vitro in the presence of IL-12 resulted in production of only 10 to 15% of the IFN- produced by T cells from L. major-infected mice under identical conditions. These results suggest that the CD4+ T-cell response during chronic L. amazonensis infection is limited during the transition from an early activated CD4+ T-cell population to an effector cell population and demonstrate that these T cells have an intrinsic defect beyond the presence or absence of IL-12 during antigen stimulation.
INTRODUCTION
Experimental infection of C3H and C57BL/6 mice with the obligate intracellular protozoan parasite Leishmania major results in a CD4+ T-cell population that produces gamma interferon (IFN-), perpetuates a Th1 response, and ultimately promotes resistance and a productive memory response (reviewed in references 19 to 21 and see reference 28). In contrast, infection of the C3HeB/FeJ, C57BL/6, or C57BL/10 strains of mice with Leishmania amazonensis results in chronic cutaneous lesions containing up to 108 parasites and low to undetectable levels of both IFN- and interleukin-4 (IL-4) in the in vitro recall responses of draining lymph node (DLN) cells (1, 13, 15). CD4+ T cells from L. amazonensis-infected mice express low levels of IL-12R2—a phenomenon that was found to be IL-4 independent (15). Additionally, the poor Th1 response associated with L. amazonensis infection has been shown to persist even in the absence of either IL-4 or IL-10 and also in the presence of exogenous IL-12 (14, 15, 26). The failure of L. amazonensis-infected mice to develop an effective Th1 response and heal their infections has prompted us to determine whether this CD4+ T-cell defect is the result of an absence of antigen (Ag)-responsive effector/memory CD4+ T cells or an inability of Ag-responsive cells to progress to a productive Th1 response.
In this study, we present evidence that the effector/memory phenotype (CD44hi) CD4+ T cells present during L. amazonensis infection exist in vivo as an unskewed T-cell population, as demonstrated by an unbiased T-bet and GATA-3 mRNA expression profile, and are only partially responsive to IL-12 both in vitro and in vivo. However, these cells are not anergic, as evidenced by their Ag responsiveness and ability to proliferate and produce IL-2 to the same extent as CD44hi CD4+ T cells from L. major-infected mice. L. major-infected mice were included in our analyses as a control for a functional Th1 effector/memory response. Moreover, CD44hi CD4+ T cells exist in equivalent percentages in both L. amazonensis- and L. major-infected C3H mice. Our results indicate that the large parasite load and nonhealing phenotype of L. amazonensis-infected mice occur in the presence of an Ag-responsive CD4+ T-cell population that is limited by an inability to progress from an early activated phenotype to an efficient effector CD4+ T-cell population. Furthermore, the data suggest that the failure of this cell population to become efficient Th1 effector cells is due to factors beyond the presence or absence of IL-12 or the ability of the cells to respond to IL-12.
MATERIALS AND METHODS
Parasites and antigens. Culture of L. amazonensis (MHOM/BR/00/LTB0016) and L. major (MHOM/IL/80/Friedlin) and preparation of parasite antigens were performed as previously described (14). In all experiments involving Ag stimulation, cells were stimulated with the matching freeze-thawed promastigote Ag. The parasite burden of infected footpads was determined using a limiting dilution assay as described previously (1) and expressed as the negative log of parasite titer.
Mice. Female C3HeB/FeJ mice (6 to 8 weeks of age) were either bred in house or obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific-pathogen-free facility. The Committee on Animal Care at Iowa State University approved all protocols involving animals. Mice were injected with 5 x 106 stationary-phase promastigotes in 50 μl phosphate-buffered saline (PBS) in the left hind footpad. Lesion size was monitored with a dial micrometer and expressed as the difference in footpad thickness between the infected and uninfected feet. Between 3 and 12 mice were pooled per group for each experiment and were sacrificed at 10 weeks postinfection. For the in vivo Ag challenge, L. amazonensis-infected mice at 10 weeks postinfection were injected in the right hind footpad with 20 μg of L. amazonensis Ag ± 0.2 μg of IL-12 (Peprotech, Rocky Hill, NJ) in a total volume of 50 μl of PBS or with 50 μl of PBS alone. L. major-infected mice at 10 weeks postinfection were injected in the right hind footpad with 20 μg of L. major Ag. Mice were sacrificed at 48 h post-Ag challenge.
Flow cytometry and proliferation assay. The memory phenotype of T cells in the DLN was assessed ex vivo using flow cytometry as described in reference 15. Cells were surface stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 (H129.19), phycoerythrin (PE)-labeled anti-CD62L (MEL-14), Cychrome-labeled CD44 (IM7), or the appropriate isotype control. All antibodies were obtained from Pharmingen (San Diego, CA) unless stated otherwise. Cells were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and data were analyzed using CellQuest software (Becton Dickinson).
To evaluate intracellular cytokines, 1 x 106 DLN cells were plated per well in a 96-well U-bottom plate with 50 μg/ml of Ag in complete tissue culture medium (CTCM; Dulbecco's modified Eagle's medium containing 4.5 mg of glucose/ml, 2 mM L-glutamine, 100 U penicillin, 100 μg streptomycin/ml, 25 mM HEPES, 0.05 μM 2-mercaptoethanol, 10% fetal bovine serum). After 18 h, cells were stimulated with phorbol myristate acetate (50 ng/ml) and ionomycin (50 ng/ml) in the presence of brefeldin A (10 μg/ml) for 6 h. Cells were harvested, washed, and stained with either FITC- or PE-labeled anti-CD4, Cychrome-labeled anti-CD44, or the appropriate isotype controls and fixed. Intracellular cytokines were assayed as described in reference 15. The antibodies used included FITC-labeled IL-2 (JES6-5H4), PE-labeled IFN- (XMG1.2), PE-labeled IL-4 (11B11), or an appropriate isotype control. Cells were acquired as described above.
Cell division was assessed by flow cytometry using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) as previously described (15). One million cells were cultured per well of a 96-well U-bottom plate with or without 50 μg/ml Ag in CTCM. After 4 days, the cells were harvested, washed, stained with PE-labeled anti-CD4 and Cychrome-labeled anti-CD44 or the appropriate isotype controls, fixed, and acquired as described above. The percentage of CD44hi CD4+ T cells present at the initiation of culture that proliferated was determined using the proliferation platform in Flowjo software (Tree Star, Ashland, OR).
CD4+ T-cell purification. CD4+ T cells were purified from lymph nodes via either magnetic positive selection using anti-CD4 MicroBeads or magnetic depletion using a biotin-conjugated antibody cocktail and anti-biotin MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Cells were subjected to one to three passes through an AutoMACS cell sorter. The purity of the CD4+ T cells was routinely 90% or greater. CD44hi CD4+ T cells from the DLN were stained with FITC-labeled anti-CD4 and Cychrome-labeled anti-CD44 and sorted with an Epics Altra cell sorter (Beckman Coulter, Fullerton, CA). The purity of the CD44hi CD4+ T cells was routinely 90% or greater.
Polarization assay, recall responses, and ELISAs. For polarization assays, spleen cells from nave female C3HeB/FeJ mice were incubated with a lysing buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM ethylenediaminetetra-acetic acid) to lyse red blood cells. Splenocytes were treated with mitomycin C (Sigma) at a final concentration of 50 μg/ml at 37°C for 20 min and washed five times with an excess of complete media. In a 96-well U-bottom plate, each well contained 1 x 105 purified CD4+ T cells, 1 x 106 mitomycin C-treated splenocytes, and 50 μg/ml of Ag in CTCM. All cocultures were expanded for 5 days with either 2 ng/ml of IL-12 (Peprotech) and 10 μg/ml of anti-IL-4 (Pharmingen; Th1 conditions) or with no additional cytokines and antibodies (neutral conditions). Supernatants were harvested and assayed via enzyme-linked immunosorbent assay (ELISA) for IFN-; sensitivity ranged between 39 and 156 pg/ml. All ELISA antibodies were purchased from Pharmingen and used according to the manufacturer's recommendations. Ag-pulsed mitomycin C-treated splenocytes alone were cultured under polarizing conditions to determine the baseline amount of cytokine production.
For recall responses, 1 x 106 LN cells draining the site of Ag challenge of infected mice were cultured in each well of a 96-well plate with or without 50 μg/ml of Ag in CTCM. After 3 days, supernatants were assayed for IFN- via ELISA as described above.
Real-time RT-PCR. Real-time reverse transcription-PCR (RT-PCR) was performed on either CD44hi CD4+ or CD4+ T cells as described in reference 26.
Statistical procedure. Statistical analysis was performed using Statview (SAS, Cary, NC). When treatment groups were compared, the data were analyzed with the Fisher's protected least significant difference (PLSD) post hoc test. When two treatments within a group were compared, data were analyzed using a paired t test. Differences were considered significant when P was <0.05.
RESULTS
Both L. amazonensis- and L. major-infected mice have equivalent percentages of CD4+ T cells with an effector/memory phenotype. C3H mice infected with L. major develop transient lesions that subsequently heal with less than 100 parasites detectable by 10 weeks postinfection. However, L. amazonensis infection of C3H mice results in chronic lesions with up to 108 parasites detectable in the footpad at 10 weeks postinfection (Fig. 1A and B). Moreover, mice infected with L. amazonensis fail to mount an effective Th1 response and consistently demonstrate poor effector cytokine production in recall responses of the DLN. Because of these findings, we hypothesized that these mice might have a reduced percentage of effector/memory phenotype CD4+ T cells in the DLN compared to L. major-infected mice. However, an ex vivo analysis of the DLN of mice at 10 weeks postinfection revealed that both L. amazonensis- and L. major-infected mice had equivalent percentages of CD4+ T cells that were CD44hi and CD62Llo (Fig. 1C). Absolute numbers of cells per DLN averaged 59.6 x 106 for L. amazonensis-infected mice and 28.1 x 106 for L. major-infected mice, indicating that L. amazonensis-infected mice have, overall, more effector/memory phenotype CD4+ T cells at 10 weeks postinfection than do L. major-infected mice. Moreover, both groups of Leishmania-infected mice had significantly greater percentages of these T cells than uninfected control mice, indicating that this increase was associated with infection. These data indicate that the susceptibility of C3HeB/FeJ mice to L. amazonensis is not due to an absence of effector/memory phenotype CD4+ T cells in the DLN and that the percentage of effector/memory phenotype CD4+ T cells present in the DLN of L. amazonensis-infected mice is not limited in comparison to that in L. major-infected mice.
CD44hi CD4+ T cells from L. amazonensis-infected mice respond to parasite Ag by producing IL-2 and proliferating but do not have a skewed Th1/Th2 response. The CD4+ T-cell response associated with L. amazonensis infection has been previously characterized as defective in terms of proliferative responses, Ag-specific cytokine production, and chemokine and IL-12R2 mRNA expression (13, 15). Our observations of an effector/memory phenotype CD4+ T-cell population that increases in the DLN of mice chronically infected with L. amazonensis prompted us to specifically assess the functional capabilities of the CD44hi CD4+ T-cell population. After a 24-h Ag stimulation of DLN cells, no significant difference was seen between the cells isolated from either L. amazonensis- or L. major-infected mice in terms of the percentage of CD44hi CD4+ T cells that produced IL-2, as assessed by intracellular staining (Fig. 2A). In addition, the ability of CD44hi CD4+ T cells to proliferate after four days of Ag stimulation was evaluated using CFSE labeling. We found that 73% of the total CD44hi CD4+ T cells present in culture from both L. amazonensis-infected mice and L. major-infected mice proliferated (Fig. 2B). Similarly, an analysis of individual cell generations showed the percentages of CD44hi CD4+ T cells present at the initiation of culture that proliferated were equivalent between L. amazonensis- and L. major-infected mice (28.9% ± 3.7% and 33.3% ± 6.8%, respectively). CD4+ T-cell proliferation in the presence of Ag was almost exclusively from CD44hi CD4+ T cells (Fig. 2C). CD4+ T-cell IL-2 production in the presence of Ag was predominantly from this cell population as well (data not shown). Thus, CD44hi expression defines Ag-reactive CD4+ T cells with similar proliferative and IL-2-producing capabilities in both L. amazonensis- and L. major-infected mice. Although central memory and effector memory CD4+ T cells have recently been defined by CD62Lhi and CD62Llo expression, respectively, during L. major infection (28), these populations do not significantly differ between L. amazonensis- and L. major-infected mice. These results demonstrate that the CD4+ T-cell population responds to L. amazonensis infection in vivo by enhancing the percentage of cells that recognize parasite antigen and that these cells undergo at least the early events of T-cell activation, including upregulation of CD44 and proliferation. Based on these results, we were interested in determining whether there would be differences in T-cell characteristics associated with a mature Ag-responsive cell population.
Numerous studies have characterized the transcription factors T-bet and GATA-3 as master regulators of the Th1 and Th2 lineage fates, respectively, and the commitment of a T-cell population toward either phenotype is associated with an upregulation of one of these transcription factors during progression through the activation pathway (25, 29). To that end, we assayed CD44hi CD4+ T cells isolated ex vivo from the DLN of infected mice for T-bet and GATA-3 mRNA expression via real-time RT-PCR. Consistent with a Th1 effector phenotype, CD44hi CD4+ T cells from L. major-infected animals had a significant increase in T-bet expression compared to cells from uninfected animals. CD44hi CD4+ T cells from L. amazonensis-infected mice, however, expressed 51% less T-bet mRNA compared to CD44hi CD4+ T cells from L. major-infected mice (Fig. 3A), and there was no significant difference in T-bet mRNA expression between CD44hi CD4+ T cells from L. amazonensis-infected and uninfected mice. In contrast, the levels of GATA-3 mRNA expression were found to be similar between the CD44hi CD4+ T cells from both L. amazonensis- and L. major-infected mice, and this expression level was significantly lower than that of CD44hi CD4+ T cells from uninfected mice (Fig. 3B). Together, these results indicate an unbiased T helper phenotype of the CD44hi CD4+ T cells in vivo during L. amazonensis infection, as indicated by the absence of enhanced T-bet or GATA-3 mRNA expression over uninfected levels.
Effector cytokine production by the Ag-responsive CD44hi CD4+ T cells was assessed after stimulating the DLN cells with Ag for 24 h and determining IFN-- or IL-4-positive cells via intracellular staining. The percentage of CD44hi CD4+ T cells producing IFN- from L. amazonensis mice was found to be 53% less than that of cells from L. major-infected mice (Fig. 3C). In addition, there was no significant difference in the percentage of IL-4-producing CD44hi CD4+ T cells from L. amazonensis-infected mice compared to cells from L. major-infected mice. As with IL-2 production and proliferation, effector cytokine production in the presence of Ag was almost exclusively from the CD44hi CD4+ T-cell population, thus reinforcing that these cells constitute the Ag-specific CD4+ T-cell population (data not shown). The ratio of IFN--producing cells to IL-4-producing cells was greater than 10 to 1 for the CD44hi CD4+ population derived from L. major-infected mice. Although L. amazonensis-infected mice had more IFN--producing than IL-4-producing CD44hi CD4+ T cells (3 to 1), the ratio was not as skewed toward a Th1 response as that of cells from L. major-infected mice. This 3-to-1 ratio reflects the absence of a productive Th1 immune response in L. amazonensis-infected mice rather than an enhanced Th2 phenotype. Collectively, these intracellular staining results closely reflect the Th1/Th2 transcription factor mRNA expression profile and indicate that the Ag-responsive CD4+ T cells associated with chronic L. amazonensis infection exist in vivo as an unskewed population.
Despite in vivo Ag responsiveness, CD4+ T cells present in L. amazonensis-infected mice exhibit limited IL-12 responsiveness. Considering that IL-12 expression is necessary for the development and maintenance of a CD4+ Th1 phenotype in vivo during L. major infection (reviewed in reference 20) and that IL-12 production has been shown to be limited during L. amazonensis infection (13, 15, 26), we determined if the CD4+ T-cell population of mice chronically infected with L. amazonensis could respond to IL-12 in vivo in the presence of Ag. Since a delayed-type hypersensitivity reaction has long been utilized as a technique to evaluate memory CD4+ Th1 cell responses in vivo (reviewed in reference 18), we infected mice in the left hind footpad with L. amazonensis for 10 weeks and then injected the right hind footpad with either L. amazonensis Ag, Ag plus IL-12, or PBS. At 48 h post-Ag challenge, there was a significant increase in the percent of CD44hi CD4+ T cells present in the lymph node (LN) draining the site of Ag challenge over the PBS-injected controls in L. amazonensis-infected mice (Fig. 4A), again demonstrating that CD44hi CD4+ T cells do respond to Ag in vivo. However, the recall responses of the LN cells draining the site of Ag challenge showed no significant enhancement in IFN- production regardless of the presence or absence of IL-12 at the time of Ag challenge (Fig. 4B). In contrast, high levels of IFN- were obtained from the recall responses of L. major-infected mice challenged with L. major Ag. To determine if the CD4+ T cells present in L. amazonensis-infected mice responded to the IL-12 treatment by altering Th1/Th2 transcription factor gene expression, real-time RT-PCR was used to analyze T-bet and GATA-3 mRNA expression in CD4+ T cells purified from the LN draining the site of Ag challenge. The presence of IL-12 at the time of in vivo restimulation did significantly enhance the T-bet/GATA-3 mRNA ratio over Ag-challenged L. amazonensis-infected mice, but it was still significantly less than the ratio observed in L. major-infected mice challenged with Ag (Fig. 4C). Data are expressed as the ratio of T-bet to GATA-3 mRNA as a previous study has shown that the relative expression of T-bet and GATA-3, rather than the expression of either transcription factor alone, was found to be more representative of the Th1/Th2 cytokine balance in a mixed population of cells (5). The more abundant GATA-3 mRNA expression than T-bet in the LN draining the site of Ag challenge results in a T-bet/GATA-3 mRNA ratio of less than 1. However, this phenomenon is true for all samples, including the CD4+ T cells from L. major-infected mice that have a productive Th1 response. These results indicate that CD4+ T cells from L. amazonensis-infected mice can respond to Ag and IL-12 in vivo by enhancing accumulation. Additionally, an enhanced T-bet/GATA-3 mRNA ratio in CD4+ T cells draining the site of Ag challenge from L. amazonensis-infected mice is observed when IL-12 is present at the time of in vivo Ag stimulation, although it is unclear as to how IL-12 is influencing T-bet mRNA in these experiments. Taken together, our data indicate that the absence of IL-12 in vivo during Ag stimulation is not the sole reason for inefficient IFN- production from CD4+ T cells present during L. amazonensis infection.
CD4+ T cells from L. amazonensis-infected mice have limited responsiveness to IL-12 in vitro. Since other cell types, including APCs, may influence the function of CD4+ T cells, we wanted to determine if APCs from noninfected mice with or without IL-12 could promote a Th1 effector phenotype in T cells comparable to that of T cells derived from L. major-infected mice in vitro. We purified CD4+ T cells from the DLN of infected mice and cocultured those cells with Ag-pulsed, mitomycin C-treated splenocytes from nave mice for 5 days; the production of effector cytokines was then quantified via ELISA. Under neutral conditions (no polarizing cytokines or antibodies), CD4+ T cells from L. amazonensis-infected mice produced significantly less IFN- than those cells from L. major infected mice (Fig. 5A). Under Th1 conditions (anti-IL-4 and recombinant IL-12 [rIL-12]), CD4+ T cells from L. amazonensis-infected mice did respond to IL-12 by enhancing their production of IFN- in comparison to neutral condition values. Despite equivalent absolute numbers of CD44hi CD4+ T cells present in both L. amazonensis and L. major cultures, the CD4+ T cells from L. amazonensis-infected mice consistently produced only 10 to 15% of the IFN- produced by the CD4+ T cells purified from L. major-infected mice stimulated under identical conditions (Fig. 5B). These data indicate that although CD4+ T cells from L. amazonensis-infected mice can respond to IL-12 in vitro, intrinsic defects prevent them from developing a Th1 phenotype equivalent to that of CD4+ T cells derived from L. major-infected mice.
DISCUSSION
In this study, we extend our understanding of the dysfunctional immune response associated with the high parasite load and persistent lesion that characterizes the chronic stage of L. amazonensis infection in immunocompetent mice. Our data indicate that the immune response consists of an Ag-responsive CD4+ T-cell population that expresses CD44, proliferates, and produces IL-2 as well as their counterparts from L. major-infected mice (Fig. 1C and 2). However, the CD44hi CD4+ T-cell population from L. amazonensis-infected mice has an unskewed effector phenotype, as reflected by low levels of both Th1/Th2 transcription factor mRNA expression, IFN- and IL-4 protein expression (Fig. 3), and a limited responsiveness to IL-12 (Fig. 4 and 5).
The ability of Ag-responsive CD44hi CD4+ T cells from L. amazonensis-infected mice to proliferate as efficiently as cells derived from L. major-infected animals was surprising, as previous works have indicated a suppressed proliferative response in comparative studies (13, 15). These differences may be a result of assessing proliferation at different time points during infection, whereas the current studies were performed exclusively during the established chronic phase of disease. Other factors that may influence the proliferative response of these cells could include Ag preparation and the amount of Ag used in restimulation. The phenomenon of cell death could also account for these different observations as a loss of live cells in culture would not influence the results of our analysis using CFSE labeling but would lead to decreased tritiated thymidine incorporation.
This current report of an uncommitted CD4+ T-cell phenotype during L. amazonensis infection complements previous studies in which a mixed Th1/Th2 response was observed throughout the course of L. amazonensis infection (1, 12). Our studies specifically characterize an Ag-responsive subpopulation of CD4+ T cells from L. amazonensis-infected mice with low mRNA expression levels of the Th1 transcription factor, T-bet, in comparison to similar cells from L. major-infected mice. These findings describe a specific defect in the T-cell activation pathway that can account for the previously described inefficient IFN- production and low levels of IL-12R2 mRNA expression observed from CD4+ T cells during L. amazonensis infection (see references 13 and 15 and see Results). Moreover, we show that CD44hi CD4+ T cells from L. amazonensis- and L. major-infected mice both express similar levels of GATA-3 mRNA and IL-4 intracellular staining, indicating that this T-cell population is also not biased toward a Th2 response. Interestingly, CD44hi CD4+ T cells from uninfected mice express more GATA-3 mRNA than those cells from L. amazonensis-infected mice (Fig. 3B). These observations are consistent with the phenomenon that GATA-3 mRNA transcripts are high in nave cells and then either decrease as cells polarize toward a Th1 phenotype or remain high if a Th2 phenotype is developed (8). Nonetheless, relatively low levels of GATA-3 and T-bet mRNA expression indicate that CD44hi CD4+ T cells from L. amazonensis-infected mice are a population that has not committed to either a Th1 or Th2 phenotype in vivo.
Previous work has indicated that mice infected with L. amazonensis fail to increase the number of IL-12-producing cells as compared to uninfected controls (15). To compensate for this deficit, we restimulated CD4+ T cells in vivo by injecting both IL-12 and Ag into the contralateral (uninfected) footpad of chronically infected mice to test the ability of this uncommitted cell population to differentiate toward a Th1 population during antigen stimulation in the presence of IL-12. We observed a response to Ag by the CD4+ T-cell population, as indicated by an increased percentage of CD44hi CD4+ T cells in the lymph node draining the site of Ag challenge compared to that in PBS-injected controls (Fig. 4A). Despite observing an enhanced T-bet/GATA-3 mRNA ratio in the CD4+ T cells of L. amazonensis-infected mice challenged with Ag in the presence of IL-12, the production of IFN- upon in vitro Ag stimulation was unchanged (Fig. 4B). One in vivo administration of IL-12 may not be sufficient to promote the development of a population of Th1 CD4+ T cells, and previous work has suggested that CD4+ T cells from C3H mice with an acute L. amazonensis infection may be unable to respond to IL-12 due to low levels of IL-12R2 mRNA expression (15). However, the limited in vitro IL-12 responsiveness observed in this study and the successful development of CD4+ T cells with a Th1 phenotype by repeated administration of IL-12 with Ag-pulsed bone marrow-derived dendritic cells to mice chronically infected with L. amazonensis indicate that the CD4+ T-cell population present during chronic L. amazonensis infection is able to respond to IL-12 to some extent (26). The relatively low levels of IFN- production both in vitro and in vivo in response to IL-12 suggest that transition to a Th1 phenotypic cell population is limited by intrinsic defects in the CD4+ T cells rather than simply the presence or absence of IL-12 during T-cell activation.
Much attention has been given to the negative role of T regulatory (Treg) cells in infectious disease, where Treg cells limit productive immune responses and promote pathogen persistence (reviewed in references 4 and 17). However, recent work has shown that Treg cells can limit the immunopathogenesis of L. amazonensis infection, although that beneficial effect is transitory (11). Previous work has shown that Treg cells are necessary for the establishment of a chronic L. major infection with accompanying low parasite load and that Treg function was dependent on IL-10 (3). However, IL-10 knockout mice infected with L. amazonensis still develop a chronic infection with a relatively high parasite load and poor cytokine production from the recall responses during chronic infection, suggesting that T-cell-derived IL-10 is not entirely responsible for limiting an effective immune response (14). Blocking transforming growth factor (TGF-) in BALB/c mice infected with L. amazonensis has been reported to facilitate healing (2). However, the effects of TGF- are often conflicting, and this may be due in part to its opposing effects on Th1 development in various mouse strains (9). Blocking TGF- in vitro fails to enhance IFN- production in CD4+ T cells isolated from L. amazonensis-infected C3H mice (15; A. E. Ramer and D. E. Jones, unpublished observations). These observations, along with the fact that the CD44hi CD4+ T-cell population from L. amazonensis-infected mice proliferates as readily as those isolated from L. major-infected animals, indicate it is unlikely that Treg cells, IL-10, or TGF- is preferentially limiting the Th1 phenotype of CD4+ T cells during L. amazonensis infection.
We believe that the limited effector functions of CD4+ T cells present in L. amazonensis-infected mice may result from a combination of priming by immature or semimature dendritic cells and the presence of high antigen load. Chronic L. amazonensis infection is characterized by the absence of a robust inflammatory response, as evidenced by decreased IL-12 production and decreased mRNA expression of multiple inflammatory mediators and a high parasite load (13, 15, 26). Recent work has shown that inappropriately primed dendritic cells in vivo can support CD4+ T-cell clonal expansion but cannot prime an effector response (23). Indeed, some persistent infections, including human immunodeficiency virus, are thought to limit dendritic cell maturation and thus induce peripheral tolerance due to Ag capture and presentation by immature dendritic cells (24). In addition, activation of CD4+ T cells in the presence of high viral load or providing multiple Ag stimulations to CD4+ T cells results in diminished CD4+ effector responses (6, 10). Dysfunctional CD8+ T-cell responses have also been described in chronic lymphocytic choriomeningitis virus and Trypanosoma cruzi infections of mice, as these cells are activated yet exhibit attenuated IFN- production and cytotoxic activity (16, 27). With these studies in mind, we suggest that chronic L. amazonensis infection could result from parasite resistance to macrophage killing, which creates a persistent, high-Ag load that impairs CD4+ T-cell effector functions (7, 22). In turn, these dysfunctional T cells are incapable of promoting effective macrophage activation and subsequent parasite elimination.
Altogether, our data indicate that mice with chronic L. amazonensis infections do possess an Ag-responsive CD44hi CD4+ T-cell population that can proliferate and produce IL-2 but is impaired in the ability to efficiently produce IFN-. These CD44hi CD4+ T cells have an unbiased pattern of Th1/Th2 transcription factor mRNA expression in vivo and cannot be effectively polarized toward a Th1 phenotype either in vitro or in vivo in the presence of IL-12. Our observations implicate an impaired, unskewed CD44hi CD4+ T-cell population as a factor contributing to the chronicity of L. amazonensis infection.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI48357 and the Biotechnology Council and College of Veterinary Medicine at Iowa State University.
We thank Dennis Byrne for his technical assistance and Christine Petersen for her critical reading of the manuscript.
Present address: Department of Medicine, Division of Hematology/Oncology, University of California—San Francisco, San Francisco, CA 94143.
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ABSTRACT
C3HeB/FeJ mice challenged with Leishmania major develop a polarized Th1 response and subsequently heal, whereas Leishmania amazonensis challenge leads to chronic lesions with high parasite loads at 10 weeks postinfection. In this study, a comparison of draining lymph node cells from L. amazonensis- and L. major-infected mice at 10 weeks postinfection showed equivalent percentages of effector/memory phenotype CD44hi CD4+ T cells producing interleukin-2 (IL-2) and proliferating after antigen stimulation. However, these cells isolated from L. amazonensis-infected mice were not skewed toward either a Th1 or Th2 phenotype in vivo, as evidenced by their unbiased Th1/Th2 transcription factor mRNA profile. In vivo antigen stimulation with added IL-12 failed to enhance gamma interferon (IFN-) production of CD4+ T cells from L. amazonensis-infected mice. Antigen stimulation of CD4+ T cells from L. amazonensis-infected mice in vitro in the presence of IL-12 resulted in production of only 10 to 15% of the IFN- produced by T cells from L. major-infected mice under identical conditions. These results suggest that the CD4+ T-cell response during chronic L. amazonensis infection is limited during the transition from an early activated CD4+ T-cell population to an effector cell population and demonstrate that these T cells have an intrinsic defect beyond the presence or absence of IL-12 during antigen stimulation.
INTRODUCTION
Experimental infection of C3H and C57BL/6 mice with the obligate intracellular protozoan parasite Leishmania major results in a CD4+ T-cell population that produces gamma interferon (IFN-), perpetuates a Th1 response, and ultimately promotes resistance and a productive memory response (reviewed in references 19 to 21 and see reference 28). In contrast, infection of the C3HeB/FeJ, C57BL/6, or C57BL/10 strains of mice with Leishmania amazonensis results in chronic cutaneous lesions containing up to 108 parasites and low to undetectable levels of both IFN- and interleukin-4 (IL-4) in the in vitro recall responses of draining lymph node (DLN) cells (1, 13, 15). CD4+ T cells from L. amazonensis-infected mice express low levels of IL-12R2—a phenomenon that was found to be IL-4 independent (15). Additionally, the poor Th1 response associated with L. amazonensis infection has been shown to persist even in the absence of either IL-4 or IL-10 and also in the presence of exogenous IL-12 (14, 15, 26). The failure of L. amazonensis-infected mice to develop an effective Th1 response and heal their infections has prompted us to determine whether this CD4+ T-cell defect is the result of an absence of antigen (Ag)-responsive effector/memory CD4+ T cells or an inability of Ag-responsive cells to progress to a productive Th1 response.
In this study, we present evidence that the effector/memory phenotype (CD44hi) CD4+ T cells present during L. amazonensis infection exist in vivo as an unskewed T-cell population, as demonstrated by an unbiased T-bet and GATA-3 mRNA expression profile, and are only partially responsive to IL-12 both in vitro and in vivo. However, these cells are not anergic, as evidenced by their Ag responsiveness and ability to proliferate and produce IL-2 to the same extent as CD44hi CD4+ T cells from L. major-infected mice. L. major-infected mice were included in our analyses as a control for a functional Th1 effector/memory response. Moreover, CD44hi CD4+ T cells exist in equivalent percentages in both L. amazonensis- and L. major-infected C3H mice. Our results indicate that the large parasite load and nonhealing phenotype of L. amazonensis-infected mice occur in the presence of an Ag-responsive CD4+ T-cell population that is limited by an inability to progress from an early activated phenotype to an efficient effector CD4+ T-cell population. Furthermore, the data suggest that the failure of this cell population to become efficient Th1 effector cells is due to factors beyond the presence or absence of IL-12 or the ability of the cells to respond to IL-12.
MATERIALS AND METHODS
Parasites and antigens. Culture of L. amazonensis (MHOM/BR/00/LTB0016) and L. major (MHOM/IL/80/Friedlin) and preparation of parasite antigens were performed as previously described (14). In all experiments involving Ag stimulation, cells were stimulated with the matching freeze-thawed promastigote Ag. The parasite burden of infected footpads was determined using a limiting dilution assay as described previously (1) and expressed as the negative log of parasite titer.
Mice. Female C3HeB/FeJ mice (6 to 8 weeks of age) were either bred in house or obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific-pathogen-free facility. The Committee on Animal Care at Iowa State University approved all protocols involving animals. Mice were injected with 5 x 106 stationary-phase promastigotes in 50 μl phosphate-buffered saline (PBS) in the left hind footpad. Lesion size was monitored with a dial micrometer and expressed as the difference in footpad thickness between the infected and uninfected feet. Between 3 and 12 mice were pooled per group for each experiment and were sacrificed at 10 weeks postinfection. For the in vivo Ag challenge, L. amazonensis-infected mice at 10 weeks postinfection were injected in the right hind footpad with 20 μg of L. amazonensis Ag ± 0.2 μg of IL-12 (Peprotech, Rocky Hill, NJ) in a total volume of 50 μl of PBS or with 50 μl of PBS alone. L. major-infected mice at 10 weeks postinfection were injected in the right hind footpad with 20 μg of L. major Ag. Mice were sacrificed at 48 h post-Ag challenge.
Flow cytometry and proliferation assay. The memory phenotype of T cells in the DLN was assessed ex vivo using flow cytometry as described in reference 15. Cells were surface stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 (H129.19), phycoerythrin (PE)-labeled anti-CD62L (MEL-14), Cychrome-labeled CD44 (IM7), or the appropriate isotype control. All antibodies were obtained from Pharmingen (San Diego, CA) unless stated otherwise. Cells were acquired on a FACScan flow cytometer (Becton Dickinson, San Jose, CA), and data were analyzed using CellQuest software (Becton Dickinson).
To evaluate intracellular cytokines, 1 x 106 DLN cells were plated per well in a 96-well U-bottom plate with 50 μg/ml of Ag in complete tissue culture medium (CTCM; Dulbecco's modified Eagle's medium containing 4.5 mg of glucose/ml, 2 mM L-glutamine, 100 U penicillin, 100 μg streptomycin/ml, 25 mM HEPES, 0.05 μM 2-mercaptoethanol, 10% fetal bovine serum). After 18 h, cells were stimulated with phorbol myristate acetate (50 ng/ml) and ionomycin (50 ng/ml) in the presence of brefeldin A (10 μg/ml) for 6 h. Cells were harvested, washed, and stained with either FITC- or PE-labeled anti-CD4, Cychrome-labeled anti-CD44, or the appropriate isotype controls and fixed. Intracellular cytokines were assayed as described in reference 15. The antibodies used included FITC-labeled IL-2 (JES6-5H4), PE-labeled IFN- (XMG1.2), PE-labeled IL-4 (11B11), or an appropriate isotype control. Cells were acquired as described above.
Cell division was assessed by flow cytometry using the dye carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) as previously described (15). One million cells were cultured per well of a 96-well U-bottom plate with or without 50 μg/ml Ag in CTCM. After 4 days, the cells were harvested, washed, stained with PE-labeled anti-CD4 and Cychrome-labeled anti-CD44 or the appropriate isotype controls, fixed, and acquired as described above. The percentage of CD44hi CD4+ T cells present at the initiation of culture that proliferated was determined using the proliferation platform in Flowjo software (Tree Star, Ashland, OR).
CD4+ T-cell purification. CD4+ T cells were purified from lymph nodes via either magnetic positive selection using anti-CD4 MicroBeads or magnetic depletion using a biotin-conjugated antibody cocktail and anti-biotin MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's protocol. Cells were subjected to one to three passes through an AutoMACS cell sorter. The purity of the CD4+ T cells was routinely 90% or greater. CD44hi CD4+ T cells from the DLN were stained with FITC-labeled anti-CD4 and Cychrome-labeled anti-CD44 and sorted with an Epics Altra cell sorter (Beckman Coulter, Fullerton, CA). The purity of the CD44hi CD4+ T cells was routinely 90% or greater.
Polarization assay, recall responses, and ELISAs. For polarization assays, spleen cells from nave female C3HeB/FeJ mice were incubated with a lysing buffer (0.15 M ammonium chloride, 10 mM potassium bicarbonate, and 0.1 mM ethylenediaminetetra-acetic acid) to lyse red blood cells. Splenocytes were treated with mitomycin C (Sigma) at a final concentration of 50 μg/ml at 37°C for 20 min and washed five times with an excess of complete media. In a 96-well U-bottom plate, each well contained 1 x 105 purified CD4+ T cells, 1 x 106 mitomycin C-treated splenocytes, and 50 μg/ml of Ag in CTCM. All cocultures were expanded for 5 days with either 2 ng/ml of IL-12 (Peprotech) and 10 μg/ml of anti-IL-4 (Pharmingen; Th1 conditions) or with no additional cytokines and antibodies (neutral conditions). Supernatants were harvested and assayed via enzyme-linked immunosorbent assay (ELISA) for IFN-; sensitivity ranged between 39 and 156 pg/ml. All ELISA antibodies were purchased from Pharmingen and used according to the manufacturer's recommendations. Ag-pulsed mitomycin C-treated splenocytes alone were cultured under polarizing conditions to determine the baseline amount of cytokine production.
For recall responses, 1 x 106 LN cells draining the site of Ag challenge of infected mice were cultured in each well of a 96-well plate with or without 50 μg/ml of Ag in CTCM. After 3 days, supernatants were assayed for IFN- via ELISA as described above.
Real-time RT-PCR. Real-time reverse transcription-PCR (RT-PCR) was performed on either CD44hi CD4+ or CD4+ T cells as described in reference 26.
Statistical procedure. Statistical analysis was performed using Statview (SAS, Cary, NC). When treatment groups were compared, the data were analyzed with the Fisher's protected least significant difference (PLSD) post hoc test. When two treatments within a group were compared, data were analyzed using a paired t test. Differences were considered significant when P was <0.05.
RESULTS
Both L. amazonensis- and L. major-infected mice have equivalent percentages of CD4+ T cells with an effector/memory phenotype. C3H mice infected with L. major develop transient lesions that subsequently heal with less than 100 parasites detectable by 10 weeks postinfection. However, L. amazonensis infection of C3H mice results in chronic lesions with up to 108 parasites detectable in the footpad at 10 weeks postinfection (Fig. 1A and B). Moreover, mice infected with L. amazonensis fail to mount an effective Th1 response and consistently demonstrate poor effector cytokine production in recall responses of the DLN. Because of these findings, we hypothesized that these mice might have a reduced percentage of effector/memory phenotype CD4+ T cells in the DLN compared to L. major-infected mice. However, an ex vivo analysis of the DLN of mice at 10 weeks postinfection revealed that both L. amazonensis- and L. major-infected mice had equivalent percentages of CD4+ T cells that were CD44hi and CD62Llo (Fig. 1C). Absolute numbers of cells per DLN averaged 59.6 x 106 for L. amazonensis-infected mice and 28.1 x 106 for L. major-infected mice, indicating that L. amazonensis-infected mice have, overall, more effector/memory phenotype CD4+ T cells at 10 weeks postinfection than do L. major-infected mice. Moreover, both groups of Leishmania-infected mice had significantly greater percentages of these T cells than uninfected control mice, indicating that this increase was associated with infection. These data indicate that the susceptibility of C3HeB/FeJ mice to L. amazonensis is not due to an absence of effector/memory phenotype CD4+ T cells in the DLN and that the percentage of effector/memory phenotype CD4+ T cells present in the DLN of L. amazonensis-infected mice is not limited in comparison to that in L. major-infected mice.
CD44hi CD4+ T cells from L. amazonensis-infected mice respond to parasite Ag by producing IL-2 and proliferating but do not have a skewed Th1/Th2 response. The CD4+ T-cell response associated with L. amazonensis infection has been previously characterized as defective in terms of proliferative responses, Ag-specific cytokine production, and chemokine and IL-12R2 mRNA expression (13, 15). Our observations of an effector/memory phenotype CD4+ T-cell population that increases in the DLN of mice chronically infected with L. amazonensis prompted us to specifically assess the functional capabilities of the CD44hi CD4+ T-cell population. After a 24-h Ag stimulation of DLN cells, no significant difference was seen between the cells isolated from either L. amazonensis- or L. major-infected mice in terms of the percentage of CD44hi CD4+ T cells that produced IL-2, as assessed by intracellular staining (Fig. 2A). In addition, the ability of CD44hi CD4+ T cells to proliferate after four days of Ag stimulation was evaluated using CFSE labeling. We found that 73% of the total CD44hi CD4+ T cells present in culture from both L. amazonensis-infected mice and L. major-infected mice proliferated (Fig. 2B). Similarly, an analysis of individual cell generations showed the percentages of CD44hi CD4+ T cells present at the initiation of culture that proliferated were equivalent between L. amazonensis- and L. major-infected mice (28.9% ± 3.7% and 33.3% ± 6.8%, respectively). CD4+ T-cell proliferation in the presence of Ag was almost exclusively from CD44hi CD4+ T cells (Fig. 2C). CD4+ T-cell IL-2 production in the presence of Ag was predominantly from this cell population as well (data not shown). Thus, CD44hi expression defines Ag-reactive CD4+ T cells with similar proliferative and IL-2-producing capabilities in both L. amazonensis- and L. major-infected mice. Although central memory and effector memory CD4+ T cells have recently been defined by CD62Lhi and CD62Llo expression, respectively, during L. major infection (28), these populations do not significantly differ between L. amazonensis- and L. major-infected mice. These results demonstrate that the CD4+ T-cell population responds to L. amazonensis infection in vivo by enhancing the percentage of cells that recognize parasite antigen and that these cells undergo at least the early events of T-cell activation, including upregulation of CD44 and proliferation. Based on these results, we were interested in determining whether there would be differences in T-cell characteristics associated with a mature Ag-responsive cell population.
Numerous studies have characterized the transcription factors T-bet and GATA-3 as master regulators of the Th1 and Th2 lineage fates, respectively, and the commitment of a T-cell population toward either phenotype is associated with an upregulation of one of these transcription factors during progression through the activation pathway (25, 29). To that end, we assayed CD44hi CD4+ T cells isolated ex vivo from the DLN of infected mice for T-bet and GATA-3 mRNA expression via real-time RT-PCR. Consistent with a Th1 effector phenotype, CD44hi CD4+ T cells from L. major-infected animals had a significant increase in T-bet expression compared to cells from uninfected animals. CD44hi CD4+ T cells from L. amazonensis-infected mice, however, expressed 51% less T-bet mRNA compared to CD44hi CD4+ T cells from L. major-infected mice (Fig. 3A), and there was no significant difference in T-bet mRNA expression between CD44hi CD4+ T cells from L. amazonensis-infected and uninfected mice. In contrast, the levels of GATA-3 mRNA expression were found to be similar between the CD44hi CD4+ T cells from both L. amazonensis- and L. major-infected mice, and this expression level was significantly lower than that of CD44hi CD4+ T cells from uninfected mice (Fig. 3B). Together, these results indicate an unbiased T helper phenotype of the CD44hi CD4+ T cells in vivo during L. amazonensis infection, as indicated by the absence of enhanced T-bet or GATA-3 mRNA expression over uninfected levels.
Effector cytokine production by the Ag-responsive CD44hi CD4+ T cells was assessed after stimulating the DLN cells with Ag for 24 h and determining IFN-- or IL-4-positive cells via intracellular staining. The percentage of CD44hi CD4+ T cells producing IFN- from L. amazonensis mice was found to be 53% less than that of cells from L. major-infected mice (Fig. 3C). In addition, there was no significant difference in the percentage of IL-4-producing CD44hi CD4+ T cells from L. amazonensis-infected mice compared to cells from L. major-infected mice. As with IL-2 production and proliferation, effector cytokine production in the presence of Ag was almost exclusively from the CD44hi CD4+ T-cell population, thus reinforcing that these cells constitute the Ag-specific CD4+ T-cell population (data not shown). The ratio of IFN--producing cells to IL-4-producing cells was greater than 10 to 1 for the CD44hi CD4+ population derived from L. major-infected mice. Although L. amazonensis-infected mice had more IFN--producing than IL-4-producing CD44hi CD4+ T cells (3 to 1), the ratio was not as skewed toward a Th1 response as that of cells from L. major-infected mice. This 3-to-1 ratio reflects the absence of a productive Th1 immune response in L. amazonensis-infected mice rather than an enhanced Th2 phenotype. Collectively, these intracellular staining results closely reflect the Th1/Th2 transcription factor mRNA expression profile and indicate that the Ag-responsive CD4+ T cells associated with chronic L. amazonensis infection exist in vivo as an unskewed population.
Despite in vivo Ag responsiveness, CD4+ T cells present in L. amazonensis-infected mice exhibit limited IL-12 responsiveness. Considering that IL-12 expression is necessary for the development and maintenance of a CD4+ Th1 phenotype in vivo during L. major infection (reviewed in reference 20) and that IL-12 production has been shown to be limited during L. amazonensis infection (13, 15, 26), we determined if the CD4+ T-cell population of mice chronically infected with L. amazonensis could respond to IL-12 in vivo in the presence of Ag. Since a delayed-type hypersensitivity reaction has long been utilized as a technique to evaluate memory CD4+ Th1 cell responses in vivo (reviewed in reference 18), we infected mice in the left hind footpad with L. amazonensis for 10 weeks and then injected the right hind footpad with either L. amazonensis Ag, Ag plus IL-12, or PBS. At 48 h post-Ag challenge, there was a significant increase in the percent of CD44hi CD4+ T cells present in the lymph node (LN) draining the site of Ag challenge over the PBS-injected controls in L. amazonensis-infected mice (Fig. 4A), again demonstrating that CD44hi CD4+ T cells do respond to Ag in vivo. However, the recall responses of the LN cells draining the site of Ag challenge showed no significant enhancement in IFN- production regardless of the presence or absence of IL-12 at the time of Ag challenge (Fig. 4B). In contrast, high levels of IFN- were obtained from the recall responses of L. major-infected mice challenged with L. major Ag. To determine if the CD4+ T cells present in L. amazonensis-infected mice responded to the IL-12 treatment by altering Th1/Th2 transcription factor gene expression, real-time RT-PCR was used to analyze T-bet and GATA-3 mRNA expression in CD4+ T cells purified from the LN draining the site of Ag challenge. The presence of IL-12 at the time of in vivo restimulation did significantly enhance the T-bet/GATA-3 mRNA ratio over Ag-challenged L. amazonensis-infected mice, but it was still significantly less than the ratio observed in L. major-infected mice challenged with Ag (Fig. 4C). Data are expressed as the ratio of T-bet to GATA-3 mRNA as a previous study has shown that the relative expression of T-bet and GATA-3, rather than the expression of either transcription factor alone, was found to be more representative of the Th1/Th2 cytokine balance in a mixed population of cells (5). The more abundant GATA-3 mRNA expression than T-bet in the LN draining the site of Ag challenge results in a T-bet/GATA-3 mRNA ratio of less than 1. However, this phenomenon is true for all samples, including the CD4+ T cells from L. major-infected mice that have a productive Th1 response. These results indicate that CD4+ T cells from L. amazonensis-infected mice can respond to Ag and IL-12 in vivo by enhancing accumulation. Additionally, an enhanced T-bet/GATA-3 mRNA ratio in CD4+ T cells draining the site of Ag challenge from L. amazonensis-infected mice is observed when IL-12 is present at the time of in vivo Ag stimulation, although it is unclear as to how IL-12 is influencing T-bet mRNA in these experiments. Taken together, our data indicate that the absence of IL-12 in vivo during Ag stimulation is not the sole reason for inefficient IFN- production from CD4+ T cells present during L. amazonensis infection.
CD4+ T cells from L. amazonensis-infected mice have limited responsiveness to IL-12 in vitro. Since other cell types, including APCs, may influence the function of CD4+ T cells, we wanted to determine if APCs from noninfected mice with or without IL-12 could promote a Th1 effector phenotype in T cells comparable to that of T cells derived from L. major-infected mice in vitro. We purified CD4+ T cells from the DLN of infected mice and cocultured those cells with Ag-pulsed, mitomycin C-treated splenocytes from nave mice for 5 days; the production of effector cytokines was then quantified via ELISA. Under neutral conditions (no polarizing cytokines or antibodies), CD4+ T cells from L. amazonensis-infected mice produced significantly less IFN- than those cells from L. major infected mice (Fig. 5A). Under Th1 conditions (anti-IL-4 and recombinant IL-12 [rIL-12]), CD4+ T cells from L. amazonensis-infected mice did respond to IL-12 by enhancing their production of IFN- in comparison to neutral condition values. Despite equivalent absolute numbers of CD44hi CD4+ T cells present in both L. amazonensis and L. major cultures, the CD4+ T cells from L. amazonensis-infected mice consistently produced only 10 to 15% of the IFN- produced by the CD4+ T cells purified from L. major-infected mice stimulated under identical conditions (Fig. 5B). These data indicate that although CD4+ T cells from L. amazonensis-infected mice can respond to IL-12 in vitro, intrinsic defects prevent them from developing a Th1 phenotype equivalent to that of CD4+ T cells derived from L. major-infected mice.
DISCUSSION
In this study, we extend our understanding of the dysfunctional immune response associated with the high parasite load and persistent lesion that characterizes the chronic stage of L. amazonensis infection in immunocompetent mice. Our data indicate that the immune response consists of an Ag-responsive CD4+ T-cell population that expresses CD44, proliferates, and produces IL-2 as well as their counterparts from L. major-infected mice (Fig. 1C and 2). However, the CD44hi CD4+ T-cell population from L. amazonensis-infected mice has an unskewed effector phenotype, as reflected by low levels of both Th1/Th2 transcription factor mRNA expression, IFN- and IL-4 protein expression (Fig. 3), and a limited responsiveness to IL-12 (Fig. 4 and 5).
The ability of Ag-responsive CD44hi CD4+ T cells from L. amazonensis-infected mice to proliferate as efficiently as cells derived from L. major-infected animals was surprising, as previous works have indicated a suppressed proliferative response in comparative studies (13, 15). These differences may be a result of assessing proliferation at different time points during infection, whereas the current studies were performed exclusively during the established chronic phase of disease. Other factors that may influence the proliferative response of these cells could include Ag preparation and the amount of Ag used in restimulation. The phenomenon of cell death could also account for these different observations as a loss of live cells in culture would not influence the results of our analysis using CFSE labeling but would lead to decreased tritiated thymidine incorporation.
This current report of an uncommitted CD4+ T-cell phenotype during L. amazonensis infection complements previous studies in which a mixed Th1/Th2 response was observed throughout the course of L. amazonensis infection (1, 12). Our studies specifically characterize an Ag-responsive subpopulation of CD4+ T cells from L. amazonensis-infected mice with low mRNA expression levels of the Th1 transcription factor, T-bet, in comparison to similar cells from L. major-infected mice. These findings describe a specific defect in the T-cell activation pathway that can account for the previously described inefficient IFN- production and low levels of IL-12R2 mRNA expression observed from CD4+ T cells during L. amazonensis infection (see references 13 and 15 and see Results). Moreover, we show that CD44hi CD4+ T cells from L. amazonensis- and L. major-infected mice both express similar levels of GATA-3 mRNA and IL-4 intracellular staining, indicating that this T-cell population is also not biased toward a Th2 response. Interestingly, CD44hi CD4+ T cells from uninfected mice express more GATA-3 mRNA than those cells from L. amazonensis-infected mice (Fig. 3B). These observations are consistent with the phenomenon that GATA-3 mRNA transcripts are high in nave cells and then either decrease as cells polarize toward a Th1 phenotype or remain high if a Th2 phenotype is developed (8). Nonetheless, relatively low levels of GATA-3 and T-bet mRNA expression indicate that CD44hi CD4+ T cells from L. amazonensis-infected mice are a population that has not committed to either a Th1 or Th2 phenotype in vivo.
Previous work has indicated that mice infected with L. amazonensis fail to increase the number of IL-12-producing cells as compared to uninfected controls (15). To compensate for this deficit, we restimulated CD4+ T cells in vivo by injecting both IL-12 and Ag into the contralateral (uninfected) footpad of chronically infected mice to test the ability of this uncommitted cell population to differentiate toward a Th1 population during antigen stimulation in the presence of IL-12. We observed a response to Ag by the CD4+ T-cell population, as indicated by an increased percentage of CD44hi CD4+ T cells in the lymph node draining the site of Ag challenge compared to that in PBS-injected controls (Fig. 4A). Despite observing an enhanced T-bet/GATA-3 mRNA ratio in the CD4+ T cells of L. amazonensis-infected mice challenged with Ag in the presence of IL-12, the production of IFN- upon in vitro Ag stimulation was unchanged (Fig. 4B). One in vivo administration of IL-12 may not be sufficient to promote the development of a population of Th1 CD4+ T cells, and previous work has suggested that CD4+ T cells from C3H mice with an acute L. amazonensis infection may be unable to respond to IL-12 due to low levels of IL-12R2 mRNA expression (15). However, the limited in vitro IL-12 responsiveness observed in this study and the successful development of CD4+ T cells with a Th1 phenotype by repeated administration of IL-12 with Ag-pulsed bone marrow-derived dendritic cells to mice chronically infected with L. amazonensis indicate that the CD4+ T-cell population present during chronic L. amazonensis infection is able to respond to IL-12 to some extent (26). The relatively low levels of IFN- production both in vitro and in vivo in response to IL-12 suggest that transition to a Th1 phenotypic cell population is limited by intrinsic defects in the CD4+ T cells rather than simply the presence or absence of IL-12 during T-cell activation.
Much attention has been given to the negative role of T regulatory (Treg) cells in infectious disease, where Treg cells limit productive immune responses and promote pathogen persistence (reviewed in references 4 and 17). However, recent work has shown that Treg cells can limit the immunopathogenesis of L. amazonensis infection, although that beneficial effect is transitory (11). Previous work has shown that Treg cells are necessary for the establishment of a chronic L. major infection with accompanying low parasite load and that Treg function was dependent on IL-10 (3). However, IL-10 knockout mice infected with L. amazonensis still develop a chronic infection with a relatively high parasite load and poor cytokine production from the recall responses during chronic infection, suggesting that T-cell-derived IL-10 is not entirely responsible for limiting an effective immune response (14). Blocking transforming growth factor (TGF-) in BALB/c mice infected with L. amazonensis has been reported to facilitate healing (2). However, the effects of TGF- are often conflicting, and this may be due in part to its opposing effects on Th1 development in various mouse strains (9). Blocking TGF- in vitro fails to enhance IFN- production in CD4+ T cells isolated from L. amazonensis-infected C3H mice (15; A. E. Ramer and D. E. Jones, unpublished observations). These observations, along with the fact that the CD44hi CD4+ T-cell population from L. amazonensis-infected mice proliferates as readily as those isolated from L. major-infected animals, indicate it is unlikely that Treg cells, IL-10, or TGF- is preferentially limiting the Th1 phenotype of CD4+ T cells during L. amazonensis infection.
We believe that the limited effector functions of CD4+ T cells present in L. amazonensis-infected mice may result from a combination of priming by immature or semimature dendritic cells and the presence of high antigen load. Chronic L. amazonensis infection is characterized by the absence of a robust inflammatory response, as evidenced by decreased IL-12 production and decreased mRNA expression of multiple inflammatory mediators and a high parasite load (13, 15, 26). Recent work has shown that inappropriately primed dendritic cells in vivo can support CD4+ T-cell clonal expansion but cannot prime an effector response (23). Indeed, some persistent infections, including human immunodeficiency virus, are thought to limit dendritic cell maturation and thus induce peripheral tolerance due to Ag capture and presentation by immature dendritic cells (24). In addition, activation of CD4+ T cells in the presence of high viral load or providing multiple Ag stimulations to CD4+ T cells results in diminished CD4+ effector responses (6, 10). Dysfunctional CD8+ T-cell responses have also been described in chronic lymphocytic choriomeningitis virus and Trypanosoma cruzi infections of mice, as these cells are activated yet exhibit attenuated IFN- production and cytotoxic activity (16, 27). With these studies in mind, we suggest that chronic L. amazonensis infection could result from parasite resistance to macrophage killing, which creates a persistent, high-Ag load that impairs CD4+ T-cell effector functions (7, 22). In turn, these dysfunctional T cells are incapable of promoting effective macrophage activation and subsequent parasite elimination.
Altogether, our data indicate that mice with chronic L. amazonensis infections do possess an Ag-responsive CD44hi CD4+ T-cell population that can proliferate and produce IL-2 but is impaired in the ability to efficiently produce IFN-. These CD44hi CD4+ T cells have an unbiased pattern of Th1/Th2 transcription factor mRNA expression in vivo and cannot be effectively polarized toward a Th1 phenotype either in vitro or in vivo in the presence of IL-12. Our observations implicate an impaired, unskewed CD44hi CD4+ T-cell population as a factor contributing to the chronicity of L. amazonensis infection.
ACKNOWLEDGMENTS
This work was supported by NIH grant AI48357 and the Biotechnology Council and College of Veterinary Medicine at Iowa State University.
We thank Dennis Byrne for his technical assistance and Christine Petersen for her critical reading of the manuscript.
Present address: Department of Medicine, Division of Hematology/Oncology, University of California—San Francisco, San Francisco, CA 94143.
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