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* Laboratory of Immunology and Hematopoiesis, Department of Veterinary Pathobiology and Purdue Cancer Center, and Biochemistry and Molecular Biology Program, Purdue University, West Lafayette, IN 47907;
R & D Center, BML, Saitama, Japan;
Laboratory of Immunology and Vascular Biology, Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, and Center for Molecular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304
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Abstract |
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Introduction |
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Dendritic cells capture Ags, mature, and up-regulate CCR7 in inflamed tissue sites (23, 24). Mature dendritic cells migrate into the T cell areas of secondary lymphoid tissues via afferent lymphatic vessels. Circulating naive T cells are also programmed to migrate into the T cell area of the secondary lymphoid tissues through a specialized endothelial layer called high endothelial venules. Endothelial and other cell types in the T cell areas express specific adhesion and chemokine molecules (e.g., peripheral node addressin and secondary lymphoid tissue chemokine (CCL21)/EBI1-ligand chemokine (CCL19) in peripheral lymph nodes) and recruit naive T cells into lymphoid tissues (25, 26). Dendritic cells in the T cell area present Ag peptides to naive T cells. T cells then undergo activation processes and differentiate into memory and effector T cell subsets with specialized functions (e.g., Th1 and Th2 cells (23, 24) or specialized gut vs skin-targeted cells (27, 28)).
Although dendritic cells have been well demonstrated to be critical in activating and differentiating T cells, it remains to be systematically determined when and how different types of chemokine receptors are regulated on T cells undergoing differentiation to memory and effector T cells in response to signals of dendritic cells. We found that dendritic cells and their cytokines play crucial roles in sequential switching of chemokine receptors from naive to early memory, and then to effector types. These changes are important for the stage-specific migration and/or interaction of T cells with other cell types.
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Materials and Methods |
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Abs to CD4 (RPA-T4), CD11C (B-ly6), CD45RA (HI100), CD45RO (UCHL1) and IFN-
(4S.B3) were purchased from BD PharMingen (San Diego, CA). Anti-IL4-PE (3010.211) was purchased from BD Biosciences (San Jose, CA). Abs to CXCR6 (56811.111), CCR1 (53504.111), CCR2 (48607.121), CCR5 (45549.111), CCR6 (53103.111), CXCR3 (49801.111), and CXCR5 (51505.111) were obtained from R&D Systems (Minneapolis, MN). Abs to CCR3 (7B11) and CCR9 (GPR96-1) were purchased from Millennium Pharmaceuticals (Cambridge, MA). Abs to CCR4 (1G1) and CCR7 (7H12), and recombinant human IL-4 and IL-12 were provided by BD PharMingen.
Cell isolation and preparation
PBMC from human peripheral blood (Indiana Regional Blood Center, Indianapolis, IN) was prepared by density gradient centrifuge on Histopaque-1077 (Sigma-Aldrich, St. Louis, MO). CD4+ T cells (purity >97%) were isolated by depleting non-T cells using a magnetic bead depletion method (Miltenyi Biotec, Auburn, CA). Naive CD45RA+ CD4 T cells were further sorted by FACSVantage SE (BD Biosciences, purity >99.5%) or by CD45RO+ cell depletion by MACS. All human subject protocols were approved by the Institutional Review Board at Purdue University (West Lafayette, IN). Peripheral blood CD14+ monocytes (purity >99%) were isolated by magnetic sorting (Miltenyi Biotec). Immature dendritic cells were generated by culturing CD14+ monocytes for 5 days in RPMI 1640 medium (10% FBS), supplemented with IL-4 (1000 U/ml, BD PharMingen) and GM-CSF (50 ng/ml, R&D Systems). After the initial culture, all immature dendritic cells show a phenotype of CD14- CD11C+. Immature dendritic cells were maturated with Staphylococcus aureaus Cowan I (0.01% v/v; Calbiochem-Novabiochem, EMD Biosciences, San Diego, CA) for 48 h as described before (20, 29) for cocultures with naive T cells. In this culture system, 
10 ng/ml of IL-12 is produced by dendritic cells over a 24 h period, and IL-4 is produced in DC-T culture at low levels (0.2–1 ng/ml) during a 24 h incubation period (29)
In vitro T cell differentiation with allogeneic dendritic cells
Naive CD45RA+ CD4 T cells were cultured with dendritic cells (ratio of 10:1) for various periods of time (2–10 days) in 24-well plates in RPMI 1640 medium with 10% FBS. For cultures with IL-4 or IL-12, T cells are cultured for 5 days with mature dendritic cells at a ratio of 10:1 with IL-4 (1000 U/ml) or IL-12 (20 ng/ml, R&D Systems), followed by expansion in IL-2 (100 U/ml) for an additional 3–5 days before flow analyses of cytokines and chemokine receptors. For CFSE staining to track cell division, naive T cells were resuspended in PBS at 5 x 107/ml, and stained with 1 µM CFSE for 10 min at 37°C. After staining, cells were washed with PBS with 20% FBS 3 times to remove free CFSE, and cultured with dendritic cells.
Analyses of surface Ags and intracellular cytokines
Chemokine receptor expression was detected using unconjugated anti-chemokine receptor mAb (or isotype-matched control mAbs), a biotinylated horse anti-mouse IgG secondary Ab (Vector Laboratories, Burlingame, CA) and streptavidin-PerCP or streptavidin-APC (BD PharMingen). Dendritic cells and T cells were distinguished by their differences in FSC and SSC or in expression of CD3. Dendritic cells have a higher SSC than that of resting and activated T cells. Freshly isolated CD4 T cells, or T cells from the cultures with dendritic cells, were first stained with Abs (to CD45RA, CD4, and/or chemokine receptors) for cell surface Ags, then activated for 4 h at 37°C with PMA (50 ng/ml) and ionomycin (1 µg/ml) in RPMI 1640 medium supplemented with penicillin/streptomycin, 10% FBS, and 10 µg/ml Monensin (Sigma-Aldrich). Activated cells were fixed and permeabilized using Cytofix/Cytoperm solution (PharMingen) and stained with isotype control Abs or mAbs to IL-4 (PE) and IFN- (FITC). Four color flow cytometry was done on a FACSCalibur (BD Biosciences) using CellQuest software, version 3.1 (BD Biosciences), or Cytomics FC500 (Beckman Coulter, Fullerton, CA), and WinMDI (v2.8) software.
Statistical analyses
Student’s t test was used. Values of p > 0.05 were considered to be significant differences.
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Results |
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We generated mature dendritic cells from peripheral monocytes, and examined whether dendritic cells can up-regulate chemokine receptors on T cells undergoing differentiation. T cells were examined for expression of chemokine receptors after 4 days in coculture with dendritic cells to see whether naive T cells that express mostly CCR7 (see Table I) can express additional receptors when they interact with dendritic cells. After the culture, various numbers of T cells expressed CCR4, CXCR3, and CXCR5 (Fig. 1), which are expressed only on memory and effector T cells in vivo (Table I). Most proliferating T cells expressed CXCR3 at this time point (Fig. 1). Approximately one-third of divided T cells expressed CXCR5, while relatively few T cells expressed CCR4 and CCR5 at this time. We also noticed in this period that as T cells undergo proliferation, some T cells (5–10%) lose CCR7.
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To determine the exact kinetics of chemokine receptor modulation, we next examined frequencies of chemokine receptor-expressing T cells at multiple time points over an 8 day period. Three groups of chemokine receptors were identified based upon changes in their expression patterns (Fig. 2). The first group contains chemokine receptors (CCR4, CCR5, CXCR3, and CXCR6) that went up continuously over time (Fig. 2A). The second group of chemokine receptors (CCR7 and CXCR4) was down-regulated over time (Fig. 2B). The last group contains CXCR5 that was initially up-regulated during the first 4–5 days and down-regulated thereafter (Fig. 2C). Also, it was notable that CXCR3 and CXCR5 appeared earlier than CCR4 and other chemokine receptors. In this regard,Schaerli et al. have reported that CXCR5 is transiently expressed on superantigen-activated T cells (30). There were notable differences in frequencies of T cells expressing each chemokine receptor. Among the chemokine receptors examined, the frequency of CXCR3+ T cells reached over 90% after 8 days in this culture system. The frequency of CCR4+ T cells was often very high (50%) as well. Frequencies of the T cells expressing CCR1, CCR2, CCR5, CCR6, CCR9, CXCR6, and CRTH2 remained low (2–15%, Fig. 2A).
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Dendritic cells secrete IL-4 and IL-12, which regulate T cell polarization into Th1 and Th2 cells. We examined whether these two cytokines had any effect on the switch of T cell chemokine receptors by dendritic cells. Naive CD4 T cells were cultured with dendritic cells for 5 days in the presence of IL-4 or IL-12 followed by further expansion in IL-2 for 3–5 days. In the absence of any exogenous cytokines, 40% T cells differentiated in the culture were Th1 cells, while few were Th2 cells (Fig. 4A). When IL-4 was added, more Th2 cells and fewer Th1 cells were formed (Fig. 4A). In the presence of exogenous IL-12, more Th1 cells and almost no Th2 cells were generated. Next, we examined the T cell expression of chemokine receptors in these different cytokine situations. IL-4 added to the culture resulted in generation of more CCR4+ and CRTH2+ T cells, but decreased the frequencies of CXCR3 and CCR5 expression (Fig. 4B). In contrast, IL-12 reduced CCR4 and CRTH2, and enhanced CCR5 expression on T cells. IL-12 and IL-4 not only decrease the numbers of T cells expressing CCR4 and CXCR3, but also the expression levels of CCR4 and CXCR3 respectively (Fig. 4C). Interestingly, more T cells retained, or expressed, CXCR5 and CCR7 after culture with exogenous IL-4. CCR3, expressed on eosinophils (reviewed in Ref. 21), has been a controversial chemokine receptor regarding its association with Th2 cells in general (31, 32). In this regard, IL-4 (or IL-12) did not enhance CCR3 expression on activated T cells in our study (not shown). Together, these results suggest the regulation of many chemokine receptors during dendritic cell-mediated T cell proliferation can be dramatically altered by IL-4 and IL-12, without requiring neutralizing Abs or extreme Th1/2 polarizing conditions. Naive T cells were also cultured with IL-4 or IL-12 in the absence of dendritic cells for up to 8 days. After the culture, IL-4 or IL-12 did not change the chemokine receptor (CCR2–7, CXCR3–6, and CRTH2) expression profile of naive T cells (data not shown), suggesting that IL-4 and IL-12 are effective in changing T cell chemokine receptor expression only when T cells undergo activation.
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As summarized in Table I, different T cell subsets at distinct developmental stages in vivo express different combinations of chemokine receptors, suggesting that T cells undergo changes in expression of chemokine receptors during peripheral differentiation processes. One of the key changes in chemokine receptor expression during naive T cell differentiation to effector T cells in vivo is the appearance of CCR7-, CCR4+(expressed by 90% of Th2), and CXCR3+(expressed by 95% of Th1 cells) T cells (Table I). Many CXCR5+ T cells (25%) are found among total memory T cell pool, but their frequencies are reduced in Th1 and Th2 populations (10%), suggesting either a transient appearance of this type of cells before they become Th1/2 cells or that the expression of CXCR5 is limited to non-polarized T cells. Overall, the dendritic cell:T cell coculture system faithfully reproduces many aspects of the in vivo switches in chemokine/chemoattractant receptor expression.
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Discussion |
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-inducible protein 10/CXCL10) is produced by mature dendritic cells, and is critical for retention of the T cells in lymph nodes (35). Activated B cells produce chemokines such as thymus and activation regulated chemokine and MDC, and may attract CCR4-expressing T cells (36, 37). Thymus and activation regulated chemokine/CCL17 and MDC/CCL22 are also produced from dendritic cells during immune responses (37, 38). Therefore, the rapid up-regulation of CXCR3, CXCR5, and CCR4 in the first switch occurring within the first 5 days of T cell activation may play an important role in the collaboration of dendritic cells and B lymphocytes with early activated T cells in initiation of the adaptive immune responses. Dendritic cells exist in many different forms with different functions. Depending on their phenotype, origin, and types and doses of Ags, dendritic cells are heterogeneous in production of cytokines and in their effects on T cell polarization (23, 24, 39, 40). We have simplified these variable factors of dendritic cells in T cell polarization by adding exogenous IL-4 or IL-12 to the culture system. Our data suggest that IL-4 and IL-12 are critical factors that govern the second chemokine receptor switch to type 1 vs 2. IL-4 boosts the generation of CCR4+ and cells expressing a Th2 associated G-protein-coupled receptor CRTH2 (41), while it suppresses the generation of CXCR3+ (Fig. 4) and CXCR6+ T cells as reported previously (20). Conversely, IL-12 significantly reduces the levels of CCR4 expression during the secondary switch. Additional levels of in vivo control undoubtedly exist as well, potentially including tissue-specific dendritic cell effects on homing chemokine receptor expression, which can be induced, or selected, within two cell divisions during the naive to memory transition in lymphoid tissues (28).
Primary immune responses usually take 7–12 days to generate memory and effector T cells. Some memory T cells retain their homing property to lymphoid tissues by retaining the expression of CCR7 and L-selectin, while other memory and effector T cells lose the expression of these molecules and gain the expression of other chemokine (and adhesion) receptors including, but not limited to, CCR5, CXCR6, CRTH2, CXCR3, and/or CCR4 to migrate to inflammatory tissue sites (1, 3, 21, 42). Therefore, the second switch in chemokine receptor expression is consistent with this later stage of T cell differentiation and migration for effector functions.
IL-4 supports general humoral immune responses and also steers B cell Ab class switching to IgE production, effects which are mediated through its action at different stages of lymphocyte development and through different cell types (43, 44, 45). Our data demonstrates that IL-4 plays a positive role in the expression of CXCR5 and/or CCR7 on CD4 T cells (Fig. 4B and C). It has been reported that CXCR5+ T cells in tonsils have efficient B cell helping activity (17, 19, 46). Therefore, the positive role of IL-4 in expression of CXCR5 may be another mechanism by which IL-4 promotes humoral immune responses through regulation of T cell migration to B cell areas. Because down-regulation of CCR7 is thought to be required for localization to follicles, the effect of IL-4 is complex, and likely to depend on interaction with other factors to drive final T cell differentiation into a bona fide follicle homing T helper cell.
Despite the fact that there are many CCR2+ (28% of memory CD4) or CCR6+ (46% of memory CD4) T cells in circulation (18), the culture system we used for this study failed to up-regulate chemokine receptors such as CCR2 and CCR6 on more than a few percent of cells. CCR2 is expressed by many Th1 cells (42%) and plays important roles in Th1-mediated immune responses and host defense against intracellular pathogens (47, 48, 49, 50). CCR6 is implicated in allergic pulmonary inflammation and delayed type hypersensitivity (51, 52). This suggests that other factors that are necessary for the induction of CCR2 and CCR6 are missing in the culture system. Further investigation to find precise conditions, in which T cells with defined migratory capacities and functions can be generated ex vivo, would be extremely valuable in many types of T cell therapies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Chang H. Kim, 1243 VPTH, Purdue University, West Lafayette, IN 47907-1243. E-mail address: chkim{at}purdue.edu
Received for publication December 5, 2002. Accepted for publication April 18, 2003.
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