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after Rechallenge with Virus In Vitro and Exhibit Decreased T Cell Alloreactivity1
Departments of Dermatology and Surgery, University of Pittsburgh, Pittsburgh, PA 15213
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when stimulated with virus in vivo and in vitro. Immunohistological staining of spleens from mice taken at different times after HSV infection revealed an early infiltration of plasmacytoid DC whereas both the myeloid DC and lymphoid-related DC had different kinetics. Upon rechallenge with virus in vitro, total splenic DCs from viral-infected mice were unable to produce IFN- when compared with DC from mice that received an initial in vivo injection with PBS. Furthermore, DC from mice that were infected with increasing doses of HSV expressed high levels of accessory and activation molecules compared with control mice. However, when cultured in vitro together with allogeneic T cells, DC from mice that had been exposed to the highest viral titers in vivo induced the lowest levels of T cell proliferation. DC exposed to PBS in vivo promoted a Th1 response upon coculture with CD4+ T cells whereas T cells cultured with DC exposed to increasing viral titers in vivo resulted in a gradually decreased Th1 response. The data suggest HSV induces DC maturation and at higher titers, exhaustion, diminishing T cell proliferation, and IFN-
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. Their existence has been known for long (6), but not their precise phenotype or origin (7). However, there is growing evidence that they play an important role in the link between innate and adaptive immunity (8). Just as the other two DC subsets (9), pDC can be expanded both in vivo and in vitro using Flt3 ligand (Flt3L) (3, 10). In fact, pDC are critically dependent of Flt3L for their development in vivo, as Flt3L-deficient mice completely lack this specific DC subset (11, 12).
pDC localization in vivo is not completely clear. One study using RAG-2-deficient mice suggests that in the steady state, pDC accumulate in the outer T cell area of the spleen (13). Myeloid DC (myDC) and lymphoid-related DCs (lyDC) have also been found in different locations in steady state (14). However, after in vivo viral challenge, cells producing IFN- can be detected in the marginal zone (MZ) of the spleen (15). In this early study, the cells were suggested to be macrophages because they expressed little MHC class II Ags and CD11c but it is possible that these cells and the pDC are the same (16, 17). It has been shown that murine CMV (MCMV) infection can severely affect DC function (18), and also that certain microbial infections can cause DC paralysis (19). In fact, many viruses are known to induce suppression of the immune system to various degrees. That specifically applies to the family of herpes virus (20, 21, 22). HSV is opportunistic and, while under normal circumstances does not pose a serious threat, may become lethal in immune-suppressed patients suffering from HIV or undergoing transplantation. To date it is not known how interaction between DC and HSV in vivo may change or affect the stimulatory properties of DC. Infection of DC in vitro with herpes virus strongly inhibits their T cell stimulatory capacity (21, 22). The present study sought to investigate 1) the tissue location of the three different DC subsets after in vivo viral challenge, 2) how in vivo viral encounter and increasing viral doses would affect DC functional properties and the subsequent T cell responses in vitro.
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Materials and Methods |
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Female BALB/c mice and C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and used at 6–10 wk of age. Animals were maintained in a pathogen-free facility according to institutional guidelines at the University of Pittsburgh Central Animal Facility (Pittsburgh, PA) under an Institutional Animal Care and Use Committee-approved protocol.
Reagents
Recombinant human (rh) Flt3L for in vivo use was the generous gift of Pharmacia (St. Louis, MO). Recombinant murine (rm) GM-CSF for in vitro use was the kind gift of Schering-Plough (Kenilworth, NJ) and rmIL-3 was purchased from R&D Systems (Minneapolis, MN). Directly fluorochrome-conjugated Abs to CD11b, CD11c, CD8, CD25, CD40, CD45R/B220, CD45RB, CD69, CD80, CD86, MHC class II, Ly-6C, and stimulatory CD40 (IgM) Abs were obtained from BD PharMingen (San Diego, CA). Magnetic microbeads directly coupled to CD11c Abs were obtained from Miltenyi Biotec (Auburn, CA). HSV (KOS-1 strain) was kindly provided by Dr. N. de Luca (University of Pittsburgh). The virus was propagated in Vero cells (American Type Culture Collection, Manassas, VA) grown to confluency and infected at 1 multiplicity of infection and concentrated over a sucrose gradient. The virus was stored in PBS 50% glycerol solution at 1010 PFU/ml at –80°C until used. Staphylococcus aureus Cowan strain I (SAC; Pansorbin) was obtained from Calbiochem (La Jolla, CA).
Cytokine mobilization
Mice were given daily injections of rhFlt3L at a concentration of 20 µg/ml in PBS for 10 days as indicated under an Institutional Animal Care and Use Committee-approved protocol.
Viral challenge
UV-inactivated, wild-type HSV KOS-1 strain (1–5 x 108 PFU/mice) were injected i.p. and mice were sacrificed at the times indicated. The viral titers used were based on previous data (15) resulting in IFN- production in vivo. Infected mice were housed in a biosafety level 2 facility according to institutional guidelines and under an Institutional Animal Care and Use Committee-approved protocol.
Histology
Spleens were embedded in OCT compound and kept at –80°C until used. Tissue sections (6–8 µm) were cut and fixed in ice-cold acetone and thereafter washed in PBS for 10 min. All following procedures were conducted at 25°C. Slides were incubated with 0.3% H2O2 for 5 min, washed and blocked with mixture of 1.5% mouse normal serum and 10% goat serum in PBS for 30 min. After washing, sections were further blocked using an avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). After washing, primary rat anti-mouse Abs to B220 (hybridoma supernatant TIB 120; American Type Culture Collection), CD11b, CD8 (BD PharMingen), and a Syrian hamster anti-mouse CD11c Ab (N418; Serotec, Raleigh, NC) were added and sections were incubated for 1 h. After washing, rabbit-anti rat Ig (DAKO, Glostrup, Denmark) and goat anti-Syrian hamster Ig (The Jackson Laboratory) were added for 30 min. After further washing, rat alkaline phosphatase anti-alkaline phosphatase (APAAP; DAKO) and extravidin-peroxidase (Sigma-Aldrich, St. Louis, MO) were added for 30 min. Staining with rabbit anti-rat Ig and goat anti-Syrian hamster Ig followed by rat APAAP and extravidin peroxidase was repeated. Finally, slides were developed using Fast Blue substrate (Sigma-Aldrich) and 3-amino-9-ethylcarbazol (Sigma-Aldrich), respectively.
Cell preparation
Spleens were dissected and single cell suspensions were made by passing spleens through a nylon cell strainer (Falcon; BD Biosciences, Franklin Lakes, NJ). After lysis of red cells by ammonium chloride solution, cells were incubated with Abs to block Fc interactions (CD16/CD32; BD PharMingen) for 15 min on ice. After washing, CD11c-magnetic beads (Miltenyi Biotec) were added according to the manufacturer’s instructions. After 20 min at 4°C, cells were washed and passed over a MACS column. Purity was checked by flow cytometry and found to be >97% (not shown).
FACS sorting
Murine pDC were isolated as described (3). In brief, total splenic CD11c+ DC isolated by MACS magnetic beads (Miltenyi Biotec) were stained with FITC-conjugated Abs to CD11b, PE-conjugated CD8, and CyChrome-conjugated B220. Cells were sorted using a MoFlo cell sorter (Fort Collins, CO) and dead cells and debris were excluded by forward and side scatter gating. Postsort analysis showed that cells were >97% B220+CD11c+ (data not shown).
Cell culture
All cell cultures were performed in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 5% FCS (Life Technologies), 2 mM L-glutamine, penicillin, streptomycin, and 2 mM HEPES buffer (Life Technologies). DCs were cultured for 24 h in the absence or presence of SAC (2 µg/ml), HSV (10 PFU/cell), CD40 mAb (10 µg/ml) in duplicates in 96-well round-bottom plates at 50,000 DC/well. Supernatants were harvested and frozen until analyzed by ELISA. For DC maturation, isolated CD11c+ DC were cultured at 1 x 106/ml in 24-well plates in the presence of rmGM-CSF (500 U/ml) and/or CD40 mAb (10 µg/ml). After 24 h, DC were washed, stained with fluorochrome-labeled Abs, and analyzed using a FACScan (BD Biosciences).
T cell stimulation
Serial dilutions of irradiated DCs (2000 rad), either freshly isolated or preactivated for 24 h with rmGM-CSF and CD40 Abs (10 µg/ml), or CD40 Abs and rmIL-3, were cultured for 4 days together with 2 x 105 allogeneic CD4+ or CD8+ T cells in round-bottom, 96-well plates at a final volume of 200 µl. [3H]Thymidine (1 µCi/well) was added during the last 18 h of culture. Proliferation was measured in a beta-scintillation counter.
T cell differentiation
Allogeneic, splenic CD4+ or CD8+ T cells were cultured together with irradiated DCs (2000 rad), either freshly isolated or preactivated for 24 h with rmGM-CSF and CD40 Abs (10 µg/ml) at a 5:1 T cell to DC ratio in round-bottom, 96-well plates at a final volume of 200 µl/well. After 6 days, cells were washed and transferred to new round-bottom 96-well plates. PMA (10 ng/ml; Sigma-Aldrich) and 1 µg/ml ionomycin (Sigma-Aldrich) were added and plates were further incubated for 2 days. Supernatants were harvested and frozen until analyzed for cytokine production.
ELISA
ELISA was performed using standard procedures. In brief, for detection of IFN-2, 96-well flat-bottom plates (Costar, Corning, NY) were coated with 1 µg/ml rat-anti-mouse IFN-
(4E-A1 (IgG1); Seikagaku America, Falmouth, MA) overnight at 4°C. After blocking with 3% BSA in PBS-Tween 20 for 1 h at 37°C, plates were washed and rmIFN-2 (a kind gift from Schering-Plough that was titrated in parallel against a known rmIFN-2 standard provided by the National Institute of Allergy and Infectious Diseases Reference Reagent Repository) and culture supernatants were added and plates were incubated at 4°C overnight. After washing, rabbit anti-mouse IFN-2 Ab (PBL Laboratories, Piscataway, NJ) was added and plates were incubated at room temperature for 2 h. After washing, biotinylated donkey anti-rabbit Abs were added for 1 h at room temperature. Extravidine-peroxidase (Sigma-Aldrich) was added and plates were further incubated for 1 h. After washing, plates were developed using TMB substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stopped by addition of 1 M sulfuric acid. For determination of IL-10, IFN- or IL-12 (p40) and IL-12 (p70), plates were coated with 5 µg/ml rat anti-mouse IL-10, IFN-, IL-12 (p40/70), or IL-12 (p70) (all BD PharMingen) in PBS overnight. After blocking with BSA, supernatants and standard (rmIFN-, rmIL-10, or IL-12; R&D Systems) were added. After incubation overnight at 4°C, specific biotinylated IL-10, IFN-, or IL-12 p40/p70 Abs were added and plates were incubated for 1 h at room temperature. After washing, extravidin-peroxidase (Sigma-Aldrich) was added and plates were further incubated for 1 h. Finally, plates were developed using TMB substrate as above. For determination of IL-4, a minikit from Endogen (Pierce, Rockford, IL) was used according to the manufacturer’s description. Plates were incubated with extravidin peroxidase for 1 h and developed using TMB substrate.
Intracellular staining for cytokines
Cells were incubated at a 1:2 APC:T cell ratio in round-bottom, 96-well plates in complete medium for 6 days. For intracellular staining of IL-4 or IFN-, cells were washed and restimulated with PMA and ionomycin (Sigma-Aldrich) for 7 h, and brefeldin A (10 µg/ml) was added during the last 4 h of culture. Surface staining of cells was performed using CyChrome-conjugated Abs to CD4. After washing, cells were fixed in 2% PFA for 20 min and then permeabilized for 30 min in PBS containing 0.55% saponin, 5% FCS and 2 mM HEPES. PE-conjugated rat anti-mouse IL-4 Abs or FITC-conjugated rat anti-mouse-IFN- Abs (5 µg/ml) were added and cells were further incubated for 30 min. After washing, cells were analyzed by flow cytometry.
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Mice were given daily injections of Flt3L for 10 days. On the day of the last Flt3L injection, mice were given a single i.p. dose of HSV (108 PFU/mice) and 6–24 h later, spleens were harvested and prepared for immunohistology. pDCs are defined as CD11c+B220+ and negative for CD11b and CD8. lyDC are CD11c+CD8+ and CD11b and B220-negative. Finally, myDC are CD11c+CD11b+ and negative for CD8 and B220. Already after 6 h, pDC could be found in the edges of the T cell area and in the MZ (Fig. 1a). A few myDC could also be detected in the MZ, but more pronounced was the infiltration of macrophages (Fig. 1b). Scattered lyDC located mostly in the red pulp with only a few present in the T cell area (Fig. 1c). Twelve hours post-HSV infection, more pDC had accumulated (Fig. 1d). Using a higher magnification, 12 h after HSV infection (Fig. 1j), splenic CD11c+B220+ pDC were clearly localized in the MZ and in the outer T cell area. At this point, lyDC could be found in the T cell zone (Fig. 1f). At 24 h post-HSV challenge, pDC could still be found in the MZ and outer T cell area (Fig. 1g), but myDC were mostly absent as were macrophages (Fig. 1h). However, the number of lyDC was increased along with a strong infiltration of CD8+ T cells (Fig. 1i).
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Mice treated for 10 days with Flt3L were infected with HSV (1 x 108 PFU/mice) on the last days of Flt3L injections. Serum was collected at 6 and 24 h postinfection and IFN- levels were determined. As shown in Fig. 2a, most IFN- could be detected early (6 h postinfection), and by 24 h the amount of IFN- had reached levels comparable to control mice given PBS (not shown).
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levels were determined. DCs that had been exposed to virus in vivo for 24 h were unable to respond to rechallenge with virus in vitro (Fig. 2b) although they could still produce IFN- when exposed to HSV for a short period in vivo. The precise mechanism for this unresponsiveness or paralysis is not known. Mice that received PBS in vivo could mount a response to rechallenge with virus in vitro (Fig. 2c), both when DCs were isolated early after infection (6 h) and after 24 h. The DCs were viable as determined by trypan blue exclusion (not shown). However, one possibility could be that the cells had differentiated in response to virus in vivo and in their more mature state would be unable to produce IFN-. FACS analysis of DC from mice exposed to HSV in vivo for 6 h show a slight up-regulation of MHC class I and II as well as of CD25 (Fig. 2d), compared with DC from control animals (Fig. 2e). The difference was more pronounced at 24 h (Fig. 3c). Moreover, the DC yields were not significantly different when comparing DC harvested after 6 or 24 h (Table I).
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Flt3L-treated mice were challenged with different doses (i.e., 1 x 108 "low", 2 x 108 "medium", or 5 x 108 "high" PFU per mouse) and splenic CD11c+ DC were prepared and analyzed by FACS after 24 h. Staining for CD11c, CD11b, and B220 revealed a significant increase in the number of pDC and CD11c+CD8+ lyDC at high viral dose, whereas the percentage of myeloid, CD11c+CD11b+ DC decreased (Fig. 3a). However, when calculating the absolute number of infiltrating DCs, only the increase in pDC was found to be significant (Fig. 3b). Ly-6C, an Ag expressed on pDC, was significantly up-regulated compared with PBS control mice (Fig. 3c). Ly-6C expression is known to be induced by IFN- on CD8+ T cells (23), and high serum levels of IFN- are likely the reason for the increased expression seen here. Both CD80 and CD86 expression increased with increased viral titer (Fig. 3c), as did MHC class II (I-Ad) and CD25, a marker that is expressed on both activated murine and human DC (24, 25). In addition, the expression of MHC class I (H2kd) increased further. Because these activation markers are also known to increase with maturation, total CD11c+ DC were cultured overnight in the presence of GM-CSF + CD40 Abs. This resulted in increased expression of accessory molecules on DC from all groups (PBS, low and high virus titer) reaching almost comparable, high levels (Fig. 4). Control cultures showed that in contrast to human DCs, murine DC appeared to mature well in the presence of GM-CSF alone and the addition of CD40 mAbs did not significantly augment the expression of CD25, CD80, CD86, or MHC class II (data not shown). In accordance with this, CD40 Abs alone induced only a modest increase in the expression of accessory molecules (not shown).
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Freshly isolated, irradiated (2000 rad) CD11c+ DC from Flt3L-treated mice that received PBS, low or high dose HSV were cultured for 4 days together with allogeneic CD4+ or CD8+ T cells. Proliferation was measured by [3H]thymidine incorporation during the last 18 h of culture. Interestingly, DC exposed to virus in vivo induced lower T cell response in vitro compared with PBS control DC, and this correlated inversely to the increased expression of accessory molecules so that at higher viral load, T cell proliferation was at its lowest (Fig. 5a). The same pattern could be seen if the DC was matured overnight in vitro in the presence of GM-CSF and CD40 mAb. However, the basic level of proliferation for all groups of DCs was increased (Fig. 5b).
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CD11c+ DC that had been matured with GM-CSF + CD40 mAb for 24 h were cultured together with total CD4+ or CD8+ T cells for 6 days. T cells were washed and put back in culture for 48 h in the presence of PMA and ionomycin. Supernatants were collected and assessed for their content of IFN-, IL-4, and IL-10. Cultures with DC from mice that had been given a low dose virus showed a significant decrease in the level of IFN- produced by CD4+ T cells as compared with PBS controls (Fig. 6a). IL-4 and IL-10 levels did not change, resulting in a net Th2-type T cell response when measured by ELISA. Cultures with DC that had been exposed to the highest viral dose in vivo showed an overall decreased capacity to activate T cells in vitro (Fig. 6a). CD8+ T cells were also affected, in that their levels of IFN- decreased at a high viral titer (Fig. 6b). Intracellular staining for IL-4 and IFN- did not reveal as clear-cut results as when measuring secreted cytokines, and the frequency of IFN--producing CD4+ T cells remained comparably high (Fig. 6c). The differences when measuring intracellular vs secreted cytokines require further study.
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To study specifically the T cell responses induced by the pDC subset, these cells were isolated to high purity (>97%) by FACS sorting (CD11clowB220+CD8–). pDCs were matured by culture overnight with CD40 mAb and IL-3, thereafter washed, irradiated, and cultured together with total CD4+ T cells for 6 days. T cells were further washed and replated together with PMA and ionomycin for 48 h. Culture supernatants were collected and analyzed for their content of IFN-, IL-4, and IL-10. Mature pDC induced a strong Th2 response (Fig. 6d) which would compare with the cytokine profile induced by their human counterpart. However, freshly isolated pDC did not induce significant T cell cytokine production (IFN-, IL-4, or IL-10) as would be expected, because these cells do not induce strong T cell proliferation (Ref.3 and data not shown).
DC exposed to virus in vivo produce little or no IL-12
Lymphoid-related, CD8+ DC have been shown to induce a Th1-type T cell response both in vitro and in vivo, and this can be directly correlated to their ability to produce IL-12 (26, 27). To see whether the reduced Th1 response seen here is related to IL-12 production, CD11c+ DC were isolated from Flt3L-treated mice that been exposed either to HSV for 24 h or from mice given PBS. DCs were cultured in the presence or absence of SAC, CD40 Abs, or LPS for 24 h and supernatants were collected and analyzed for their content of IL-12 (p40 and p70). As shown in Fig. 6e, DC from mice exposed to virus produced less IL-12 than control animals. As expected, only very low levels of IL-12 (p70) could be detected, using CpG (oligonucleotide) 1558, but IL-12 (p40) was produced in high amounts after stimulation with both LPS and CD40 Abs.
DCs have also been found to have the capacity to produce IL-2 (28, 29), a major cytokine implied in T cell proliferation. To examine the possibility that the decrease in T cell proliferation was due to lack of DC IL-2 production, splenic CD11c+ DC from HSV or control mice were activated in vitro. However, irrespective of the stimuli used, no IL-2 could be found in supernatants from the different conditions tested, as determined by proliferation of the IL-2-sensitive cell-line HT-2 (data not shown).
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Discussion |
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production after rechallenge of CD11c+ DC in vitro. The data shown here confirm previous studies showing that IFN- production has very rapid kinetics, and disappears after 
24 h (32, 33).
The MZ localization of several cell types capable of producing some IFN- adds to the difficulty in distinguishing in situ which cell would be the highest producer. However, it now appears unlikely that the major burst of viral-induced IFN- derives from macrophages, although it is possible that these may contribute (15).
HSV infection of human DC in vitro results in decreased T cell proliferation, slight up-regulation of accessory molecules (21, 22), and in a dose-dependent down-modulation and degradation of CD83 (22). It also results in decreased DC cytokine production (21). The present study is in support of this conclusion and further extends the findings to be true when DCs are exposed to virus in vivo.
Because pDC and lyDC are thought to have a regulatory rather than stimulatory capacity, this shift in balance of the different DC subsets after viral infection may affect the outcome of the T cell response. The precise mechanisms whereby pDC and lyDC perform their regulatory function on T cells remain elusive. They do express lower levels of accessory molecules than myDC, but also possibly express Ags that promote T cell anergy or induce a regulatory T cell phenotype. If data presented in this study hold true also for in vivo T cell responses, they may be of importance for instance in HIV infection, where an increasing viral dose would first decrease the Th1 response and finally deplete the DC pool (34, 35). If pDC promote a Th2 response in vivo they may also play a role in diseases like allergy and asthma, where mature pDC could aggravate the disease (36, 37). In contrast, a Th2 bias would be an important feature when using DC in allotransplantation (38) where Th2-inducing DC would be more likely to promote graft tolerance. Therefore, it will be important to determine pDC-T cell interactions in vivo.
Use of different types of virus may result in different immune responses. MCMV have been shown to induce DC paralysis (18), resulting in the lack of IL-12 and IL-2 production and down-regulation of MHC class II and CD86. The up-regulation of MHC class I found in this study is also contrary to the subversive effect of MCMV (39). It is possible that Flt3L treatment may have a favorable effect in maintaining MHC expression. The data suggests that infection by a harmful virus such as MCMV or a high dose HSV results in DC paralysis that can completely subvert the immune response. DC paralysis has also been demonstrated after infection with Toxoplasma gondii (19) but in that case, isolated DCs were still able to respond by IL-12 production to antigenic restimulation in vitro. Thus, the subversive effect of viruses on DCs seems to be more severe, and depending on type and dose of virus may lead to complete paralysis. Moreover, different viruses may target different cell types (16, 17) that in turn may interact differently with T cells. As shown in this study, CD8+ T cell cytokine secretion was affected, as their IFN- production was decreased when cocultured with DC exposed to high, but not low, viral concentrations.
CD11c+ DCs exposed to HSV in vivo do not produce much IL-12 (p70) when rechallenged in vitro, which may give a Th2 response by default. IL-12 production by CD8+ DC has been shown to be instrumental to drive a Th1 response (40, 41, 42, 43). pDCs, although capable of IL-12 production, do not produce IL-12 after stimulation with CD40 mAbs (3, 4, 5) which would mimic the interaction with activated T cells. Thus, the pDC and lyDC infiltrating the spleen of infected mice (Figs. 1 and 3a) do no longer produce IFN- and IL-12, resulting in a decreased Th1 response. In addition, increasing viral titers induced the expression of CD86, an Ag found to be important for induction of IL-4 production by T cells (44).
The concept of cellular exhaustion (45, 46) may in part apply to the data presented here. pDC only produce IFN- as precursors, and once mature they cannot respond to virus by IFN- production. Thus, as suggested in this study and as shown by others (47, 48), pre-pDC may induce a Th1-type T cell response after viral challenge. However, prolonged stimulation or high dose of virus will exhaust DC production of cytokines promoting a Th1 response. The type and the dose of Ag may determine the outcome of the immune response, Th1 or Th2, rather than DC1-DC2 as separate lineages (48). More clear-cut T cell polarization may be obtained by sorting the DC subsets from infected mice and determining separately their respective contribution to T cell cytokine production. Subsequent B cell responses may also provide information on the nature of the CD4+ T cell response that can strengthen the data shown here.
BALB/c mice are susceptible to live HSV and pretreatment of mice with Flt3L has been shown to reduce virus latency (49) and promote innate immunity in neonatal mice (50), a fact attributed to increased numbers of DCs and NK cells. Thus, this suggests a function for Flt3L in preventing viral infection, in addition to its role in inhibiting tumor growth (51). Although not studied here, interactions between DC and NK cells may play an important part in antiviral immunity (52). Both IL-12 and IFN- may act to enhance NK cell IFN- production and cytotoxicity (53, 54), and the CD8+ DC subset has been found to expand NK cells by an IL-18-dependent mechanism (55).
In summary, HSV promote several activation pathways of DCs that affect both CD4+ and CD8+ T cell stimulation. The data presented here suggest that in vivo, not only the nature of the Ag or adjuvant, but also the dose and the length of exposure, are all important for the outcome of the T cell response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pia Björck, Departments of Dermatology and Surgery, University of Pittsburgh, 190 Lothrop Street, 145 Lothrop Hall, Pittsburgh, PA 15213. E-mail address: bjorckp{at}msx.upmc.edu
3 Abbreviations used in this paper: DC, dendritic cell; pDC, plasmacytoid DC; Flt3L, Flt3 ligand; myDC, myeloid DC; lyDC, lymphoid-related DC; MZ, marginal zone; MCMV, murine CMV; rh, recombinant human; rm, recombinant murine; SAC, Staphylococcus aureus Cowan strain I; APAAP, alkaline phosphatase anti-alkaline phosphatase.
Received for publication December 12, 2003. Accepted for publication February 13, 2004.
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G.-X. Yang, Z.-X. Lian, K. Kikuchi, Y.-J. Liu, A. A. Ansari, S. Ikehara, and M. E. Gershwin CD4- Plasmacytoid Dendritic Cells (pDCs) Migrate in Lymph Nodes by CpG Inoculation and Represent a Potent Functional Subset of pDCs J. Immunol., March 15, 2005; 174(6): 3197 - 3203. [Abstract] [Full Text] [PDF]
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K. McKenna, A.-S. Beignon, and N. Bhardwaj Plasmacytoid Dendritic Cells: Linking Innate and Adaptive Immunity J. Virol., January 1, 2005; 79(1): 17 - 27. [Full Text] [PDF]
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), SAC (2 µg/ml) (
), or medium (
); culture supernatants were collected after 24, 48, or 72 h and analyzed for IFN-
; PBS DC-CD8+ T cells, 
; HSVlow DC-CD4+ T cells, 
; HSVhigh DC and CD8+ T cells, 
.