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Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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production by naive T cells through an IL-12-dependent mechanism. Dendritic cells from infected mice also support higher levels of TNF-
production by naive T cells. These same dendritic cells present parasite Ag to a malaria-specific T cell hybridoma, a finding that demonstrates that dendritic cells participate in the generation of Ag-specific immunity during infection. Our findings challenge the contention that dendritic cell function is inhibited by malaria infection. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our understanding of DC function during blood stage malaria is limited and conflicting. There has been a long-standing observation that immune responses, and in particular T cell-proliferative responses, are inhibited during acute infection (4, 5, 6, 7). Most experimental evidence supports the idea that macrophages are the host cell responsible for inhibition of T cell proliferation (8, 9, 10). Two recent reports, however, one using Plasmodium falciparum (11) and the other using Plasmodium yoelii (12), have introduced the idea that DCs may participate in the inhibition or failure of T cell responses during malaria infection. Both studies showed that when DCs are matured in vitro in the presence of infected E, they express lower levels of class II MHC and costimulatory proteins and provide poorer stimulation for T cells than control DCs. By contrast, our results (13) as well as those of Seixas et al. (14) found increased levels of class II MHC and costimulatory proteins on DCs during both P. yoelii infection and interaction with P. chabaudi, although neither study tested the ability of DCs to activate naive T cells.
Here we have directly examined the ability of DCs from infected mice to activate naive T cells using a system that allows us to mimic the initial encounter between these two cell types in vivo. We find that DCs from infected mice express high levels of costimulatory proteins and support IL-2 production as well as DCs from uninfected mice. Furthermore, these cells stimulate markedly higher levels of IFN- by responding T cells than their counterparts from uninfected mice, through an IL-12-dependent mechanism. Our results indicate that DCs from malaria-infected mice are not impaired in their ability to activate T cells and do not inhibit T cell responses during malaria infection. DCs do appear to have a central role in stimulating high levels of type 1/proinflammatory cytokines during the early stages of murine malaria infection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B10.D2 and B10.D2-DO11.10 mice, referred to as TCR-transgenic (Tg), were purchased from The Jackson Laboratory (Bar Harbor, ME) and used after a 2-wk acclimation period. B10.D2-DO11.10 mice express a TCR for OVA on CD4 T cells (15). These mice were purchased from The Jackson Laboratory and bred in our laboratory animal facility by crossing heterozygous males with nontransgenic females. Offspring were determined to be heterozygous if >90% of the CD4+ cells in the peripheral blood were positive when stained with anti-V
8.1/8.2. Mice were housed in the Painter Center, Colorado State University (Fort Collins, CO), and all experiments were approved by the Institutional Animal Care and Use Committee.
P. yoelii 17X was used as described in Ref.13 . At day 6 postinfection, when the experiments described here were conducted, the average parasitemia is 13% (n = 39). P. yoelii 17X infection is not lethal for this mouse strain, and resolves by 
day 25.
Antibodies
Abs used for purification and staining were purchased from BD Biosciences (San Jose, CA): anti-V8.1/8.2 (clone MR5-2); anti-CD4 (clone L3T4); anti-CD11c (clone HL3); anti-CD11b (clone M1/70); anti-CD40 (clone 3/23); anti-CD80 (clone 16–10A1); anti-CD86 (clone GL1); anti-CD3 (clone 145-2C11); anti-CD19 (clone 1D3); and anti-class II MHC (clone 2G9). Anti-TCR clonotype Ab (KJ1-26) was purchased from Caltag Laboratories (Burlingame, CA).
Purification of cellular subsets
DCs were purified from the pooled spleens of three to five mice by positive selection with anti-CD11c. Initially, we used both directly conjugated anti-CD11c beads (clone N418; Miltenyi Biotec, Auburn, CA) as well as biotinylated anti-CD11c (clone HL3) followed by streptavidin coupled to microbeads (Miltenyi Biotec). Cytokine production was similar with either purification, with slightly higher purity and yield using the indirect method. Therefore, this method was used for all experiments in this report. Purification was conducted according to the manufacturer. Of note, the separation buffer was PBS, 0.5% BSA, 0.2 mM EDTA. Once the purified cell populations were obtained, they were subjected to several washes in medium without EDTA to remove this compound. The CD11c- (flowthrough) population was then used for positive selection with biotinylated anti-CD11b and streptavidin-conjugated microbeads. The purity of each population was routinely between 75 and 85% determined by flow cytometry. The CD11b+ population contained 30% class II negative, Gr-1+ cells, regardless of the infection status of the host. These cells likely represent neutrophils (which can be seen in cytospin preparations of these cells). Cells were kept in the separation buffer on ice until they were used and were then placed in culture together with T cells and Ag within 8–10 h of sacrifice.
CD4+ T cells were positively selected from the spleens of TCR-Tg mice. Spleen cells were passed first over Sephadex G-10 (16), and the nonbinding cells used for positive selection with anti-CD4 biotin and streptavidin-conjugated microbeads. Purity was generally >90%.
Cell sorting
DC subpopulations (CD11c+b- and CD11c+b+) were sorted by FACS as follows. CD11c+ cells were enriched as described above. The cells were then stained with anti-CD3 and anti-CD19 conjugated to PE, anti-CD11c conjugated to biotin, followed by streptavidin-PE-CY5 (BD Biosciences) and CD11b conjugated to FITC. Cells were sorted on a MoFlo (DakoCytomation, Fort Collins, CO) with technical assistance kindly provided by DakoCytomation. CD3- and CD19-positive cells were outgated, and CD11c+b- and CD11c+b+ were sorted into separate populations. In both uninfected and infected mice, there were 4 times as many CD11c+b+ cells as CD11c+b- cells in the population before sorting. DC and T cells were cultured at a 1:1 ratio (105 of each cell type).
Cell culture conditions
Cell cultures were conducted as described in Ref.13 . Briefly, cells were cultured in round-bottom 96-well plates. DCs (3 x 105) and T cells (1–2 x 105) were used in each well, together with 1 mg/ml OVA. Lower concentrations of OVA resulted in proportionally lower production of each cytokine measured. Titration of DC numbers showed that as the numbers were decreased (to 104 DC with 105 T cells), IL-2, IFN-, and TNF- production decreased proportionally in the presence of both uninfected DC (nDC) and infected DC (iDC) (not shown).
The OVA (Sigma-Aldrich, St. Louis, MO; grade V) was not endotoxin free. The addition of OVA to DC cultures without T cells resulted in TNF- production (but no other cytokine) that was twice the spontaneous release. By contrast, the deliberate addition of LPS to DC cultures resulted in an 8- to 10-fold increase in TNF-. The magnitude of both effects (OVA and LPS) was similar in DC from infected and uninfected mice.
When macrophages were added to cell cultures to look for inhibitory activity, 3 x 105 macrophages were added to DCs and T cells. All cultures were conducted in triplicate.
Cytokine assays
IL-2, IFN-, IL-10, and IL-12 p40 and p70 cytokine ELISAs were conducted using reagents from BD Biosciences as described (13). Cytokine bead assays to quantify IL-2, IFN-, TNF-, IL-4, and IL-5 were obtained from BD Biosciences and used as described by the manufacturer. Beads were analyzed on a Coulter Epics XL, using FL2 and FL4 for acquisition. Cytokine quantities obtained by ELISA were similar to those obtained with the bead assay.
Costimulatory molecule staining
Whole spleen cells were isolated from either naive or day 6-infected mice. Cells were plated out into 96-well plates and blocked with anti-CD32 for 30 min at 4°C. To each well were added 2 x 106 cells. After the block, the cells were stained with anti-CD3 and anti-CD19 conjugated to biotin, as well as anti-CD11c-APC and either anti-CD80, CD86, CD40, or an isotype control all conjugated to FITC. Streptavidin APC-Cy7 or Alexa Fluor 350 (Molecular Probes, Eugene, OR) was used to gate out the CD3 and CD19+ lymphocytes. The expression of costimulatory proteins on the CD11c+ population was then determined using a Cyan flow cytometer (DakoCytomation).
T cell hybridoma production
CD11c+ cells and T cells were purified from infected mice (day 6). These were cocultured for 5 days. Cells were washed, and recultured with DCs from an additional group of infected mice (also at day 6 postinfection). After 2 days of restimulation, a fusion was conducted with the T cell hybridoma partner BWZ (17). This hybridoma carries the LACZ gene under control of the IL-2 promoter and was used with the hope of obtaining hybridomas with responses that could be measured by LACZ activity. IL-2 production proved to be more consistent, however, and that is what is reported. Hybridomas were screened for expression of CD3 and then for their ability to produce IL-2 in response to parasitized E cultured with uninfected spleen cells. These assays were conducted with 3 x 105 APCs, 5 x 105 hybridomas, and 107 E with a 10% parasitemia. Supernatants were harvested after 24 h and assayed for IL-2.
IL-12 inhibition studies
Anti IL-12 studies were conducted to measure the dependence of IFN- production on IL-12. The IL-12-neutralizing Ab was obtained from Caltag Laboratories (clone C17.8). The Ab reacts with the p40 subunit of IL-12. Titration of the Ab showed nearly complete inhibition of IL-12-dependent IFN- production at 5 µg/ml and inhibition to a slightly lesser amount occurred at a 10-fold lower dilution (data not shown). Cells were isolated from naive or infected mice as described above and cultured with 5, 0.5, or 0 µg/ml anti-IL-12. The 0-µg/ml group contained 5 µg/ml isotype control.
Statistical analysis
Student’s t test was used to calculate p values and to compare cytokine levels between treatment groups within an individual experiment. Individual experiments in which cytokines were measured using DCs from uninfected and infected mice generally used pooled spleen cells from three to five mice, resulting in two treatment groups. To analyze cytokine levels between multiple experiments, a mixed effects model was used (18). Using this model, we were able to directly compare the differences in cytokine levels between treatments across multiple experiments and obtain p values for these differences. The value of this approach was that we could measure multiple independent infections. All statistical analyses were made using the SAS system software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The focus of these studies was to determine how DCs guide T cell responses to malaria. Specifically, we wanted to understand how DCs influence the early phases of the immunity, when the T cell response is initiated. In our previous studies (13), we established that TCR-Tg T cells, when transferred to infected mice, responded to Ag (OVA) with high levels of IFN- and low levels of IL-2, reflecting the behavior described for malaria-specific T cells at early stages of infection. Therefore, we chose to use this system to dissect APC function in vitro.
To confirm that DCs are the cells primarily responsible for the activation of naive T cells, we purified CD11c+ DCs from uninfected and infected mice (nDCs and iDCs, respectively). We purified these cells directly through positive magnetic selection based on the expression of surface markers, rather than differential adherence and culture methods. This was done because the latter protocols are known to affect the maturation state and function of both DCs and macrophages (19, 20, 21, 22). Macrophage-enriched populations were positively selected from the CD11c-negative cells using anti-CD11b. We then asked which of these populations was able to support the activation of naive T cells.
As expected, the DC population contained all of the T cell accessory capacity for IL-2 and IFN- production in both naive and infected mice (Fig. 1). The CD11b+c- cells alone supported small amounts of TNF-, but no IFN- or IL-2 from naive T cells. In addition, highly purified B cells from either uninfected or acutely infected mice had minimal capacity to support IL-2 production by naive T cells (data not shown).
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Nominal T cell activation requires the expression of costimulatory proteins on the surface of DCs and presentation of Ag by MHC. With the exception of our previous study (13), the expression of costimulatory proteins on DCs isolated directly from infected mice has not been reported, although expression of these proteins after 24 h of culture was described by Ocana-Morganer et al. (12). We measured the expression of CD80, CD86, and CD40 on nDCs and iDCs. A higher percentage of DCs from infected mice express CD80 and CD40 (Fig. 2). Similarly significant differences can be seen when using mean fluorescence intensity to assess the level of CD80 expression. Expression of CD86 was low and did not differ between nDC and iDC (not shown). We have also found previously that class II expression is strongly up-regulated on all cell types during infection (13). These changes begin as early as day 3 postinfection (not shown).
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We then compared the ability of iDCs and nDCs to support production of IL-2, type 1 and proinflammatory cytokines (IFN- and TNF-), and type 2 and anti-inflammatory cytokines (IL-4, IL-5, and IL-10) by naive T cells in an extensive series of experiments. The amount of IL-4 and IL-5 detected was at or below the limits of detection (15 pg/ml for each) in all assays regardless of the source of DCs. This was true even when cells were stimulated for 5 days and then restimulated with fresh Ag and APC (whole spleen cells) for 2 additional days (not shown). IL-10 was below the limits of detection in three of six assays. In the others, the presence of DCs from infected mice stimulated low amounts of IL-10 in the presence of Ag, and none without Ag added (data not shown).
iDCs supported high levels of IL-2, IFN-, and TNF-. As shown in Table I, cytokine production in this system was dependent on the addition of T cells and Ag, because production of all cytokines was nominal without these elements (data represent one of two similar experiments). A series of eight experiments was performed in which IL-2 production was measured after 24 and 72 h of culture. iDCs and nDCs did not differ in their ability to support IL-2 production when measured at either time point. By contrast, IFN- was consistently higher when iDCs were used as APC (p < 0.05). TNF- also tended to be higher in the presence of iDCs, but this comparison did not achieve significance (p = 0.08). These data are summarized in Table II. Our findings indicate that iDCs are equally effective at T cell activation as nDCs but that they support a higher level of proinflammatory/type 1 cytokines by T cells.
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After 5 days of culture, there were equivalent numbers of viable cells between cultures with nDCs and iDCs. Spleen cells from uninfected mice were added as a source of fresh APCs, and cytokine secretion was measured after 24 of culture with OVA. We found that iDCs and nDCs not only supported equivalent amounts of IL-2 but also retained the pattern of elevated IFN- and TNF- secretion (Fig. 3). These findings, together with our observations that iDCs express high levels costimulatory proteins (Fig. 2) as well as class II MHC (13), provide strong support for the contention that DCs from infected mice are fully functional APCs.
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To rule out the possibility that a small contaminating population of cells copurified with DCs was influencing cytokine production, we sorted purified CD11c+ cells. CD3+ and CD19+ cells were stained with the same fluorochrome and negatively selected during the process. In addition, CD11c-b+ cells were negatively selected to remove macrophages and neutrophils. The CD11c+ population was subdivided into CD11c+b- and CD11c+b+ populations (Fig. 4A). These two subpopulations represent the major subsets of murine DCs; the myeloid (CD11c+b+) and the CD11c+b- population that contains the lymphoid (CD8+) and plasmacytoid (Gr1+/B220+) subpopulations.
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, and TNF- than the same population from uninfected mice (Fig. 4B), confirming the behavior observed when DC were purified by positive selection with magnetic beads. To make this comparison directly, CD11c+b- and b+ cells were mixed together and cultured with T cells and OVA (in the proportions found in the presort population). The results again confirmed that DCs from infected mice are fully functional in their ability to support cytokine production by responding T cells. A second experiment comparing sorted CD11c+b+ nDC and iDC yielded similar results.
DCs from infected mice induce IFN- production through IL-12
Ocana-Morgener et al. (12) showed that DCs from infected mice were refractory to LPS stimulation of IL-12 production. This result presents a paradox for our finding that DCs (which were purified using a similar protocol) support high levels of IFN- (Table II). The major stimulus for IFN- production is IL-12, although an IL-12-independent pathway for IFN- production has been described for P. berghei (23). Therefore, we measured IL-12 p40 and p70. IL-12 p40 was significantly higher in iDC than in nDC in three of three experiments, one of which is shown in Fig. 5A. IL-12 p70 was undetectable by ELISA at 24 or 72 h (lower limits of detection, 20 pg/ml). We then determined whether IFN- production in our system was dependent on IL-12 by adding anti-IL-12 to cultures with iDCs plus naive T cells. The anti p40 Ab effectively abrogated the detection of IL-12 p40 (Fig. 4A) and reduced the production of IFN- to <5% of control. Furthermore, IFN- production depended on the presence of Ag (Table I), indicating that contaminating NK cells in the DC preparation could not account for the observed IFN-. This finding indicates that biologically relevant concentrations of IL-12 were present even though IL-12 p70 was not detectable.
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To establish that the DCs used in these experiments are capable of presenting parasite Ag and thus are a population of cells relevant to immune responses to P. yoelii infection, we examined their ability to activate a P. yoelii-specific T cell hybridoma. The hybridoma was generated by immunization with iDCs, and then restimulation in vitro as described in Materials and Methods. Fig. 6A shows that the hybridoma recognizes parasite Ag, and not an autoantigen present on spleen cells or RBC, because it produced IL-2 only when cultured with parasitized E.
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CD11b+ cells from the spleen inhibit IL-2 production but not IFN- or TNF-
Our experiments revealed that DCs from infected mice are unlikely players in the inhibition of T cell responses described. In previous work, we found that depletion of CD11b+ cells from the spleens of infected mice abrogated IL-2-inhibitory capacity (13). To directly determine how this subset of cells influences cytokine production, we added CD11b+c- cells to cultures with naive TCR-Tg T cells and DCs. This population is enriched for macrophages as assessed by cytologic examination and contains no primary T cell-stimulatory capacity (Fig. 7). The addition of these cells from both uninfected and infected mice tended diminish IL-2 production, a finding consistent with the literature (24). The macrophage-enriched population from infected mice, however, suppressed IL-2 production substantially more than those from uninfected mice. This effect was noted whether the DCs were derived from uninfected (Fig. 6) or infected mice (not shown). IL-2 inhibition was statistically significant in four of four experiments. The effect of these cells on IFN- production and TNF- production was variable, leading to either slight enhancement (two experiments) or no difference (one experiment). The degree of parasitemia was similar in all three experiments, as was the degree of purity of the APC subsets.
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or TNF- suggests that the DC-T cell axis is primarily responsible for the production of proinflammatory cytokines early in malaria infection. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The experimental approach that we chose was to isolate DCs from infected mice and study their function after purification by positive selection, without a period of culture. In this way their function in vitro would most closely reflect their function in vivo. The ability of these DCs to activate a homogeneous population of naive T cells was then assessed to recreate the initial encounter between DCs and naive T cells. We find that DCs taken directly from infected mice, which have matured in vivo rather than in vitro, 1) produce biologically relevant levels of IL-12, 2) support proliferation and IL-2 production by naive T cells to the same extent as DCs from uninfected mice, 3) support substantially higher levels of IFN- by responding T cells, and 4) in this and as we demonstrated in previous studies (13) DCs from infected mice express greater levels of class II, CD40, and CD80 than DCs from uninfected mice. To establish that the DC population was relevant to malaria infection, we produced a T cell hybridoma specific for a P. yoelii-derived Ag (the Ag has not yet been identified). DCs purified directly from infected mice stimulated high levels of IL-2 production by the hybridoma, without the addition of additional parasite Ag, indicating that the purified DCs present parasite Ag in vivo.
Two recent reports have suggested that human or murine DC grown in GM-CSF and IL-4, when exposed to malaria-infected E in vitro or in vivo and then matured with LPS, have diminished T cell stimulatory capacity (11, 12). In our studies, DC taken directly from infected mice showed no impairment of their ability to activate T cells. Although it is possible that differences in the responding T cell populations may account for some of the discrepancies between this report and previous ones, we favor the idea that the different signals to which the DC are exposed during development explain the results. When DC are matured in vitro with the use of LPS, they mature without T cell ligation of CD40. A recent study by Straw et al. (29) found that even when DCs are exposed to infectious stimuli provided by a protozoan pathogen (Toxoplasma gondii) in vivo, they will not exhibit a mature phenotype in the absence of T cell signaling through CD40-CD40L interactions. Reis e Sousa and colleagues (20, 30) found that CD40 ligation was required for maximal stimulation of cytokine production by DCs, even in the presence of potent microbial stimuli. Thus, the phenotype and responsiveness of DCs grown in vitro without direct T cell signaling may be different from that of DCs grown in vivo. We hypothesize that when T cells are present during DC development and maturation, the signals they provide to DCs overcome the inhibitory effects of infected E. Other factors that could influence DC maturation, such as Ag uptake through Fc or C receptors, are also not recapitulated in in vitro culture systems. Therefore, we suggest that the function of DCs taken directly from an infected environment more closely resembles their phenotype in vivo.
In previous work, we showed that the spleens of infected mice produce a substance that inhibits IL-2 production without direct cell-cell contact (13). The substance was distinct from NO, PGE2, TGF, or IL-10, and removing CD11b+ cells from the spleen cell preparation could eliminate inhibition. By blocking indoleamine 2,3-dioxygenase function in vitro, we were also able to rule out the possibility that indoleamine 2,3-dioxygenase activation in macrophages and subsequent tryptophan catabolism plays an important role in IL-2 suppression (our unpublished results). Here, although we have yet to isolate the inhibitory factor involved, we demonstrate that purified CD11b+CD11c- cells are key players in this inhibition of IL-2 production, but have little effect on IFN- or TNF-. These in vitro observations are consistent with our in vivo findings that T cells maturing in an infected environment undergo limited division but produce high levels of IFN- (13). Although a large body of earlier literature indicates that macrophages from malaria-infected individuals have immunosuppressive properties, there is a surprising paucity of studies in which macrophages are shown to be directly suppressive to T cell proliferation (25, 31). We cannot exclude the possibility that CD11b+Gr-1+ cells, which are likely to be neutrophils, also participate in the suppression of IL-2 production, because these cells can mediate IL-2 suppression through direct cell contact (32) and the production of soluble factors (33). Ongoing studies in our laboratory are addressing this question.
Our results have implications for the progression of immune responses to malaria pathogens, as well as to other Ags encountered during malaria infection, such as those derived from concurrent viral infections or vaccines. During the acute phase of infection, naive T cells recruited to the immune response may be stimulated to produce IFN- and TNF-. Regulation of these cytokines can be crucial in determining the balance between protection and pathologic responses. Although the T cells in our system were responding to unrelated Ag, other studies have established that the presence of a pathogen influences the nature of the T cell response, even when the Ag is not derived from the pathogen. For example, Chen and Jenkins (34) showed that TCR-Tg T cells specific for OVA (similar to those used here but on a BALB/c background) responded with high IL-2 and IFN- to both OVA expressed by Escherichia coli, and an OVA-containing protein injected together with E. coli that did not express OVA. The response was qualitatively and quantitatively different when the OVA-containing protein was given without coinjection of E. coli. Thus, the immune response to a pathogen can mold the response to third-party Ags, and the response to third-party Ags can be used to model pathogen-specific immune responses.
Our finding that IL-2 production was inhibited by macrophages from infected mice may mean that the expansion of Th1 cells early in the immune response is limited, allowing for an outgrowth of Th2 cells. The observation that the cytokine response is dominated by type 1/proinflammatory cytokines early in infection, and type 2 cytokines later is consistent with our results (our unpublished observations with P. yoelii and Refs.35 and36). Although such a well-defined switch has not been documented in human malaria, the acquisition of immunity to disease (but not infection) may be the result of a more gradual shift in the ratio of pro- to anti-inflammatory cytokines by T cells (3). A greater understanding of the mechanisms that mediate this shift in mice may help us better understand similar phenomena in human malaria.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anne C. Avery, Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523-1671. E-mail address: anne.avery{at}colostate.edu
3 Abbreviations used in this paper: DC, dendritic cell; nDC, uninfected DC; iDC, infected DC; Tg, transgenic.
Received for publication May 23, 2003. Accepted for publication October 17, 2003.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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interferon responses in lethal and nonlethal murine blood-stage malaria. Infect. Immun. 65:1593.[Abstract]
1 in severe childhood malaria: relationship of cytokine balance with disease severity. J. Infect. Dis. 182:988.[Medline]
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). Values were obtained by gating out lymphocytes using anti-CD3 and anti-CD19 and then gating on CD11c+ cells. The percentage of cells expressing the indicated marker when compared with an isotype control is indicated. *, Significant difference between iDCs and nDCs.
