Cell Differentiation Receptor Profiles During Naive to Effector T

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Dendritic Cells Support Sequential Reprogramming of Chemoattractant Receptor Profiles During Naive to Effector T Cell Differentiation This information is current as of September 25, 2021. Chang H. Kim, Kinya Nagata and Eugene C. Butcher J Immunol 2003; 171:152-158; ; doi: 10.4049/jimmunol.171.1.152 http://www.jimmunol.org/content/171/1/152 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Dendritic Cells Support Sequential Reprogramming of Chemoattractant Receptor Proﬁles During Naive to Effector T Cell Differentiation1

Chang H. Kim,2* Kinya Nagata,† and Eugene C. Butcher‡

T cells undergo chemokine receptor switches during activation and differentiation in secondary lymphoid tissues. Here we present evidence that dendritic cells can induce changes in T cell expression of chemokine receptors in two continuous steps. In the first switch over a 4Ð5 day period, dendritic cells up-regulate T cell expression of CXCR3 and CXCR5. Additional stimulation leads to the second switch: down-regulation of lymphoid tissue homing related CCR7 and CXCR5, and up-regulation of Th1/2 effector tissue-targeting chemoattractant receptors such as CCR4, CCR5, CXCR6, and CRTH2. We show that IL-4 and IL-12 can determine the fate of the secondary chemokine receptor switch. IL-4 enhances the generation of CCR4؉ and CRTH2؉ T cells, and Downloaded from .suppresses the generation of CXCR3؉ T cells and CCR7؊ T cells, while IL-12 suppresses the level of CCR4 in responding T cells Furthermore, IL-4 has positive effects on generation of CXCR5؉ and CCR7؉ T cells during the second switch. Our study suggests that the sequential switches in chemokine receptor expression occur during naive T cell interaction with dendritic cells. The first switch of T cell chemokine receptor expression is consistent with the fact that activated T cells migrate within lymphoid tissues for interaction with B and dendritic cells, while the second switch predicts the trafficking behavior of effector T cells away from

lymphoid tissues to effector tissue sites. The Journal of Immunology, 2003, 171: 152Ð158. http://www.jimmunol.org/

eterogeneity in lymphocyte trafficking behavior is deter- moattractant receptors induced or down-regulated? This would be mined by expression patterns of chemokine receptors valuable information in understanding changes in the trafficking H and adhesion molecules (1Ð4). Chemokines regulate behavior of the T cells undergoing activation and differentiation at leukocyte trafficking by inducing firm integrin-dependent adhesion the early vs late stages of immune responses. of blood leukocytes to endothelial cells, and by inducing direc- Dendritic cells capture Ags, mature, and up-regulate CCR7 in tional migration (chemotaxis). Leukocytes express over 20 che- inflamed tissue sites (23, 24). Mature dendritic cells migrate into mokine or chemoattractant receptors in both subset-specific and the T cell areas of secondary lymphoid tissues via afferent lym- overlapping patterns. Recently, there has been significant progress phatic vessels. Circulating naive T cells are also programmed to by guest on September 25, 2021 in characterization of chemokine receptors expressed by T cells at migrate into the T cell area of the secondary lymphoid tissues various developmental stages and functional status: immature and through a specialized endothelial layer called high endothelial mature thymocytes express different sets of chemokine receptors venules. Endothelial and other cell types in the T cell areas express (5Ð9); naive and memory effector T cells are also different in ex- specific adhesion and chemokine molecules (e.g., peripheral node pression of chemokine receptors (10Ð20). Th1 and Th2 cells are addressin and secondary lymphoid tissue chemokine (CCL21)/ distinguished from each other in expression of several chemokine EBI1-ligand chemokine (CCL19) in peripheral lymph nodes) and receptors (1, 10, 12, 13, 15, 18, 21, 22). Despite overlapping ex- recruit naive T cells into lymphoid tissues (25, 26). Dendritic cells pression patterns, chemokine receptors are often classified for sim- in the T cell area present Ag peptides to naive T cells. T cells then plicity as Th1 (CXCR3, CCR5, and CXCR6) vs Th2 (CCR4, undergo activation processes and differentiate into memory and CCR3, and CCR8) or lymphoid homing (CCR7 and CXCR5) vs effector T cell subsets with specialized functions (e.g., Th1 and non-lymphoid tissue homing (Th1 and Th2 types combined) types. Th2 cells (23, 24) or specialized gut vs skin-targeted cells (27, An important unanswered question is at which time during the 28)). differentiation of T cells is the expression of chemokine or che- Although dendritic cells have been well demonstrated to be critical in activating and differentiating T cells, it remains to be sys- tematically determined when and how different types of chemo- *Laboratory of Immunology and Hematopoiesis, Department of Veterinary Pathobi- kine receptors are regulated on T cells undergoing differentiation ology and Purdue Cancer Center, and Biochemistry and Molecular Biology Program, to memory and effector T cells in response to signals of dendritic Purdue University, West Lafayette, IN 47907; †R & D Center, BML, Saitama, Japan; ‡Laboratory of Immunology and Vascular Biology, Department of Pathology, Stan- cells. We found that dendritic cells and their cytokines play crucial ford University School of Medicine, Stanford, CA 94305, and Center for Molecular roles in sequential switching of chemokine receptors from naive to Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304 early memory, and then to effector types. These changes are important for the stage-specific migration and/or interaction of T cells Received for publication December 5, 2002. Accepted for publication April 18, 2003. with other cell types. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Materials and Methods 1 This work was supported by grants from The Eli and Edythe L. Broad Foundation, Abs and cytokines the Leukemia and Lymphoma Society (to C.H.K.), and the National Institute of Health (to E.C.B.). Abs to CD4 (RPA-T4), CD11C (B-ly6), CD45RA (HI100), CD45RO 2 Address correspondence and reprint requests to Dr. Chang H. Kim, 1243 VPTH, Purdue (UCHL1) and IFN-␥ (4S.B3) were purchased from BD PharMingen (San University, West Lafayette, IN 47907-1243. E-mail address: [email protected] Diego, CA). Anti-IL4-PE (3010.211) was purchased from BD Biosciences

(San Jose, CA). Abs to CXCR6 (56811.111), CCR1 (53504.111), CCR2 CD45RA, CD4, and/or chemokine receptors) for cell surface Ags, then (48607.121), CCR5 (45549.111), CCR6 (53103.111), CXCR3 activated for4hat37¡C with PMA (50 ng/ml) and ionomycin (1 ␮g/ml) (49801.111), and CXCR5 (51505.111) were obtained from R&D Systems in RPMI 1640 medium supplemented with penicillin/streptomycin, 10% (Minneapolis, MN). Abs to CCR3 (7B11) and CCR9 (GPR96-1) were FBS, and 10 ␮g/ml Monensin (Sigma-Aldrich). Activated cells were fixed purchased from Millennium Pharmaceuticals (Cambridge, MA). Abs to and permeabilized using Cytofix/Cytoperm solution (PharMingen) and CCR4 (1G1) and CCR7 (7H12), and recombinant human IL-4 and IL-12 stained with isotype control Abs or mAbs to IL-4 (PE) and IFN-␥ (FITC). were provided by BD PharMingen. Four color flow cytometry was done on a FACSCalibur (BD Biosciences) using CellQuest software, version 3.1 (BD Biosciences), or Cytomics Cell isolation and preparation FC500 (Beckman Coulter, Fullerton, CA), and WinMDI (v2.8) software.

PBMC from human peripheral blood (Indiana Regional Blood Center, In- Statistical analyses dianapolis, IN) was prepared by density gradient centrifuge on Histopaque- ϩ 1077 (Sigma-Aldrich, St. Louis, MO). CD4 T cells (purity Ͼ97%) were Student’s t test was used. Values of p Ͼ 0.05 were considered to be sig- isolated by depleting non-T cells using a magnetic bead depletion method niﬁcant differences. (Miltenyi Biotec, Auburn, CA). Naive CD45RAϩ CD4 T cells were further sorted by FACSVantage SE (BD Biosciences, purity Ͼ99.5%) or by CD45ROϩ cell depletion by MACS. All human subject protocols were Results approved by the Institutional Review Board at Purdue University (West Dendritic cells induce chemokine receptor switch Lafayette, IN). Peripheral blood CD14ϩ monocytes (purity Ͼ99%) were isolated by magnetic sorting (Miltenyi Biotec). Immature dendritic cells We generated mature dendritic cells from peripheral monocytes, were generated by culturing CD14ϩ monocytes for 5 days in RPMI 1640 and examined whether dendritic cells can up-regulate chemokine medium (10% FBS), supplemented with IL-4 (1000 U/ml, BD PharMin- receptors on T cells undergoing differentiation. T cells were ex-

gen) and GM-CSF (50 ng/ml, R&D Systems). After the initial culture, all Downloaded from Ϫ ϩ amined for expression of chemokine receptors after 4 days in co- immature dendritic cells show a phenotype of CD14 CD11C . Immature culture with dendritic cells to see whether naive T cells that ex- dendritic cells were maturated with Staphylococcus aureaus Cowan I (0.01% v/v; Calbiochem-Novabiochem, EMD Biosciences, San Diego, press mostly CCR7 (see Table I) can express additional receptors CA) for 48 h as described before (20, 29) for cocultures with naive T cells. when they interact with dendritic cells. After the culture, various In this culture system, ϳ10 ng/ml of IL-12 is produced by dendritic cells numbers of T cells expressed CCR4, CXCR3, and CXCR5 (Fig. over a 24 h period, and IL-4 is produced in DC-T culture at low levels 1), which are expressed only on memory and effector T cells in (0.2Ð1 ng/ml) during a 24 h incubation period (29)

vivo (Table I). Most proliferating T cells expressed CXCR3 at this http://www.jimmunol.org/ In vitro T cell differentiation with allogeneic dendritic cells time point (Fig. 1). Approximately one-third of divided T cells ϩ expressed CXCR5, while relatively few T cells expressed CCR4 Naive CD45RA CD4 T cells were cultured with dendritic cells (ratio of 10:1) for various periods of time (2Ð10 days) in 24-well plates in RPMI and CCR5 at this time. We also noticed in this period that as T 1640 medium with 10% FBS. For cultures with IL-4 or IL-12, T cells are cells undergo proliferation, some T cells (5Ð10%) lose CCR7. 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 Chemokine receptors on developing T cells are modulated at IL-2 (100 U/ml) for an additional 3Ð5 days before flow analyses of cyto- different time points kines and chemokine receptors. For CFSE staining to track cell division, naive T cells were resuspended in PBS at 5 ϫ 107/ml, and stained with 1 To determine the exact kinetics of chemokine receptor modulation, ␮M CFSE for 10 min at 37¡C. After staining, cells were washed with PBS we next examined frequencies of chemokine receptor-expressing T by guest on September 25, 2021 with 20% FBS 3 times to remove free CFSE, and cultured with dendritic cells. cells at multiple time points over an 8 day period. Three groups of chemokine receptors were identified based upon changes in their Analyses of surface Ags and intracellular cytokines expression patterns (Fig. 2). The first group contains chemokine Chemokine receptor expression was detected using unconjugated anti- receptors (CCR4, CCR5, CXCR3, and CXCR6) that went up con- chemokine receptor mAb (or isotype-matched control mAbs), a biotinyl- tinuously over time (Fig. 2A). The second group of chemokine ated horse anti-mouse IgG secondary Ab (Vector Laboratories, Burlin- receptors (CCR7 and CXCR4) was down-regulated over time (Fig. game, CA) and streptavidin-PerCP or streptavidin-APC (BD PharMingen). 2B). The last group contains CXCR5 that was initially up-regu- 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 lated during the first 4Ð5 days and down-regulated thereafter (Fig. that of resting and activated T cells. Freshly isolated CD4 T cells, or T cells 2C). Also, it was notable that CXCR3 and CXCR5 appeared ear- from the cultures with dendritic cells, were first stained with Abs (to lier than CCR4 and other chemokine receptors. In this regard,

Table I. Summary of chemoattractant receptor expression by naive, memory, and Th1/2 effector T cells in circulationa

Chemokine Receptor Naive CD4 T Cellsb Memory CD4 Cellsc Th1 Cellsd Th2 Cellse

CCR2 0.95 Ϯ 0.73 22.5 Ϯ 7.5 42.5 Ϯ 3.6 18.7 Ϯ 11 CCR3 0.61 Ϯ 0.22 2.7 Ϯ 1.1 1.6 Ϯ 1.3 3.5 Ϯ 1.8 CCR4 0.92 Ϯ 0.82 31.0 Ϯ 2.9 25.9 Ϯ 11.7 94.5 ؎ 4.8 CCR5 1.25 Ϯ 1.2 25.4 Ϯ 12.9 48.6 ؎ 16.5 10.6 Ϯ 7.3 CCR6 0.95 Ϯ 0.90 48.5 Ϯ 7.9 39.9 Ϯ 9.5 11.8 Ϯ 4.9 CCR7 98.9 ؎ 1.2 83.5 ؎ 5.1 60.9 Ϯ 7.5 57.0 Ϯ 8.3 CXCR3 3.3 Ϯ 1.0 41.5 Ϯ 11.2 89.7 ؎ 9.5 16.6 Ϯ 6.9 CXCR5 0.98 Ϯ 0.97 25 ؎ 4.5 13.1 Ϯ 7.1 10.9 Ϯ 7.7 CXCR6 1.8 Ϯ 0.88 10.25 Ϯ 6.1 14.5 ؎ 5.3 1.9 Ϯ 1.5 CRTH2 0.27 Ϯ 0.1 2.2 Ϯ 0.64 0.75 Ϯ 0.6 37.2 ؎ 4.9

a Data (% positive cells in each subset) are averages Ϯ SD of at least ﬁve experiments using freshly isolated peripheral blood CD4 T cells from different donors. Some data are from Refs. 10 and 23. b CD45RAϩ or CD45ROϪ CD4 T cells in blood. c CD45RAϩ or CD45ROϩ CD4 T cells in blood. d IFN-␥ϩ IL-4Ϫ CD4 T cells upon activation with PMA and ionomycin. e IL-4ϩIFN-␥Ϫ CD4 T cells upon activation with PMA and ionomycin. 154 DENDRITIC CELLS AND CHEMOKINE RECEPTOR SWITCH BY T CELLS

FIGURE 1. In vitro differentiation of naive T cells with dendritic cells FIGURE 2. Kinetics of chemokine receptor modulation on T cells un- changes chemokine receptor expression on T cells. Naive CD45RAϩ hu- dergoing differentiation from naive to memory/effector T cells in response man peripheral blood T cells (stained with CFSE) were cultured with ma- to dendritic cells. Naive T cells were cultured with mature dendritic cells ture dendritic cells at a ratio of 10:1 for 4 days followed by examination of at a ratio of 10:1 for 2Ð8 days followed by examination of chemokine chemokine receptors on T cells. Data are representative of four indepen- receptors on T cells. Data are representative of three independent Downloaded from dent experiments. experiments.

Schaerli et al. have reported that CXCR5 is transiently expressed were Th1 cells, while few were Th2 cells (Fig. 4A). When IL-4 on superantigen-activated T cells (30). There were notable differ- was added, more Th2 cells and fewer Th1 cells were formed (Fig.

ences in frequencies of T cells expressing each chemokine recep- 4A). In the presence of exogenous IL-12, more Th1 cells and al- http://www.jimmunol.org/ tor. Among the chemokine receptors examined, the frequency of most no Th2 cells were generated. Next, we examined the T cell CXCR3ϩ T cells reached over 90% after 8 days in this culture expression of chemokine receptors in these different cytokine sit- system. The frequency of CCR4ϩ T cells was often very high uations. IL-4 added to the culture resulted in generation of more (ϳ50%) as well. Frequencies of the T cells expressing CCR1, CCR4ϩ and CRTH2ϩ T cells, but decreased the frequencies of CCR2, CCR5, CCR6, CCR9, CXCR6, and CRTH2 remained low CXCR3 and CCR5 expression (Fig. 4B). In contrast, IL-12 re- (2Ð15%, Fig. 2A). duced CCR4 and CRTH2, and enhanced CCR5 expression on T When naive T cells interact with allogeneic dendritic cells in cells. IL-12 and IL-4 not only decrease the numbers of T cells culture, not all the naive T cells undergo activation at the same expressing CCR4 and CXCR3, but also the expression levels of time. Therefore, we tracked the changes in chemokine receptor CCR4 and CXCR3 respectively (Fig. 4C). Interestingly, more T by guest on September 25, 2021 expression on activated T cells that underwent different numbers of cells retained, or expressed, CXCR5 and CCR7 after culture with cell division (undivided, 3Ð14 times, and Ͼ15 times based upon exogenous IL-4. CCR3, expressed on eosinophils (reviewed in the intensity of CFSE fluorescence; Fig. 3A). CXCR3 and CXCR5 Ref. 21), has been a controversial chemokine receptor regarding its appeared on the majority of the T cells that divided 3Ð14 times. association with Th2 cells in general (31, 32). In this regard, IL-4 CXCR3 expression stayed on, but CXCR5 expression rapidly went (or IL-12) did not enhance CCR3 expression on activated T cells down on the divided T cells after day 4. Loss of CCR7 was evident in our study (not shown). Together, these results suggest the reg- on 10Ð20% of the T cells that divided Ͼ15 times. Similarly, ulation of many chemokine receptors during dendritic cell-medi- CXCR4 down-regulation was observed most evidently on this ated T cell proliferation can be dramatically altered by IL-4 and highly divided T cell population. At day 4, small numbers of the T IL-12, without requiring neutralizing Abs or extreme Th1/2 polar- cells that divided at least three times started to express CCR4. This izing conditions. Naive T cells were also cultured with IL-4 or is followed by a number of other memory/effector T cell chemo- IL-12 in the absence of dendritic cells for up to 8 days. After the kine receptors such as CCR2, CCR3, CCR5, CCR6, CXCR6, and culture, IL-4 or IL-12 did not change the chemokine receptor CRTH2. These late-appearing chemokine receptors were ex- (CCR2Ð7, CXCR3Ð6, and CRTH2) expression profile of naive T pressed more frequently by the T cells that divided Ͼ15 times, cells (data not shown), suggesting that IL-4 and IL-12 are effective while early appearing chemokine receptors (CXCR3 and CXCR5) in changing T cell chemokine receptor expression only when T were expressed more frequently by those that divided fewer (3Ð14) cells undergo activation. times. It is notable that undivided T cells retain the chemokine receptor profile of naive T cells. Most CXCR5ϩ T cells, generated Comparison of in vitro and in vivo chemokine receptor switches after 8 days in this culture system, were also positive for CCR7 As summarized in Table I, different T cell subsets at distinct de- expression reflecting the chemokine receptor profile of circulating velopmental stages in vivo express different combinations of che- ϩ CXCR5 T cells in vivo (Fig. 3B). mokine receptors, suggesting that T cells undergo changes in expression of chemokine receptors during peripheral differentiation Cytokines in determining the fate of chemokine receptor switch processes. One of the key changes in chemokine receptor expres- Dendritic cells secrete IL-4 and IL-12, which regulate T cell po- sion during naive T cell differentiation to effector T cells in vivo is larization into Th1 and Th2 cells. We examined whether these two the appearance of CCR7Ϫ, CCR4ϩ(expressed by ϳ90% of Th2), cytokines had any effect on the switch of T cell chemokine recep- and CXCR3ϩ(expressed by ϳ95% of Th1 cells) T cells (Table I). tors by dendritic cells. Naive CD4 T cells were cultured with den- Many CXCR5ϩ T cells (ϳ25%) are found among total memory T dritic cells for 5 days in the presence of IL-4 or IL-12 followed by cell pool, but their frequencies are reduced in Th1 and Th2 pop- further expansion in IL-2 for 3Ð5 days. In the absence of any ulations (ϳ10%), suggesting either a transient appearance of this exogenous cytokines, ϳ40% T cells differentiated in the culture type of cells before they become Th1/2 cells or that the expression The Journal of Immunology 155

FIGURE 3. Effects of cell division on chemokine receptor expression proﬁles. A, CFSE-labeled naive T cells were cultured Downloaded from with mature dendritic cells at a ratio of 10:1 for 2Ð8 days followed by examination of chemokine receptors on T cells. T cells that divided Ͼ15 times, 3Ð14 times, and undivided based upon CFSE ﬂuorescence intensity were separately analyzed for their

changes in chemokine receptor expression. T http://www.jimmunol.org/ cells that divided Ͼ3 times and Ͼ15 times were analyzed from day 4 and day 6, respectively, when these populations were reliably detected in all experiments. B, Most CXCR5ϩ T cells generated in the culture co- express CCR7. Activated T cells on day 8 were double-stained with Abs to CCR7 and CXCR5. Data are representative of three independent experiments. by guest on September 25, 2021

of CXCR5 is limited to non-polarized T cells. Overall, the den- shared by many non-polarized cells as well) or CCR7ϩ/Ϫ dritic cell:T cell coculture system faithfully reproduces many as- CXCR5ϪCXCR3ϩ (Th1 and non-polarized cells-associated). pects of the in vivo switches in chemokine/chemoattractant recep- These findings from the T cells differentiated in vitro in response tor expression. to dendritic cells and the data from in vivo generated peripheral blood T cells are summarized in Fig. 5. This provides insight into Discussion how differentiating T cells, in response to dendritic cell signals, Our in vitro dendritic cell:T cell coculture system suggests that the modulate their migration machinery in a stage-specific manner. development of specific chemokine receptor profiles can occur in se- Naive T cells home to lymph nodes through high endothelial quential phases during T cell activation by dendritic cells: switch 1) venules and undergo activation processes in the T cell areas in CCR7ϩCXCR5ϪCCR4ϪCXCR3Ϫ (naive) to CCR7ϩCXCR5ϩ/Ϫ response to dendritic cells when the B cells are still localized in the CCR4ϩ/ϪCXCR3ϩ/Ϫ (early memory); and switch 2) CCR7ϩ B cell-rich follicles. Both populations then migrate to the edges of CXCR5ϩ/ϪCCR4ϩ/ϪCXCR3ϩ/Ϫ (early memory) to CCR7ϩ/Ϫ the follicles and interact there, resulting in B cell proliferation and CXCR5ϪCCR4ϩ (a Th2 effector memory associated phenotype, germinal center formation (33). The first switch of chemokine 156 DENDRITIC CELLS AND CHEMOKINE RECEPTOR SWITCH BY T CELLS Downloaded from

FIGURE 5. Chemokine receptor switches during T cell differentiation from naive to effector cells. Naive T cells undergo roughly two distin- guishable changes in chemokine receptor expression during activation and differentiation to memory and effector T cells. The ﬁrst switch, which rap-

idly up-regulates CXCR3 and CXCR5, and later CCR4, occurs in vitro http://www.jimmunol.org/ within 4Ð5 days. The second switch occurs afterward, down-regulating CCR7 and CXCR5, and up-regulating CCR5, CXCR6 and CRTH2 on some T cells. In polarizing conditions in the presence of IL-4 or IL-12, two different chemokine receptor expression patterns (type 1 and 2) appear during the second switch. The typical type 1 expression pattern is CXCR3ϩCCR4ϪCCR5ϩ/ϪCXCR6ϩ/ϪCCR7ϩ/Ϫ and the typical type 2 pattern is CCR4ϩ CXCR3ϪCRTH2ϩ/ϪCCR7ϩ/Ϫ

critical for retention of the T cells in lymph nodes (35). Activated by guest on September 25, 2021 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. FIGURE 4. IL-4 and IL-12 control the terminal T cell chemokine switch induced by dendritic cells. Naive T cells were cultured with mature Dendritic cells exist in many different forms with different func- dendritic cells at a ratio of 10:1 for 5 days followed by further expansion tions. Depending on their phenotype, origin, and types and doses in IL-2 for 3Ð5 days. A, Phenotype of T cell generated in cultures with of Ags, dendritic cells are heterogeneous in production of cyto- dendritic cells in the absence and presence of IL-4 or IL-12. B, Changes in kines and in their effects on T cell polarization (23, 24, 39, 40). We T cell expression of chemokine receptors after cultures with indicated cy- have simplified these variable factors of dendritic cells in T cell tokines. After the culture (8Ð10 days), most T cells were activated and polarization by adding exogenous IL-4 or IL-12 to the culture sys- Sig- tem. Our data suggest that IL-4 and IL-12 are critical factors that ,ء .divided. Data are representative of three independent experiments nificant differences from controls (no cytokine) in three independent ex- govern the second chemokine receptor switch to type 1 vs 2. IL-4 periments. C, Changes in expression levels (mean fluorescence intensity) boosts the generation of CCR4ϩ and cells expressing a Th2 asso- of CCR4 and CXCR3 after dendritic cell: T cell cultures in IL-4 or IL-12. ciated G-protein-coupled receptor CRTH2 (41), while it sup- Data shown are relative ratio to controls (expression levels from IL-4/IL-12 ϩ ϩ cultures divided by those from the control cultures without the cytokines). presses the generation of CXCR3 (Fig. 4) and CXCR6 T cells -Significant differences from control cultures. as reported previously (20). Conversely, IL-12 significantly re ,ء duces the levels of CCR4 expression during the secondary switch. Additional levels of in vivo control undoubtedly exist as well, receptors occurring in some T cells may be required for this early potentially including tissue-specific dendritic cell effects on hom- stage of T and B cell interaction; the CXCR5 up-regulation on ing chemokine receptor expression, which can be induced, or se- some T cells is required for T cell migration toward follicles, lected, within two cell divisions during the naive to memory tran- where BLC/CXCR13 is expressed (34). The rapid up-regulation of sition in lymphoid tissues (28). CXCR3 may be important for reinforcing the T cell interaction Primary immune responses usually take 7Ð12 days to generate with dendritic cells because a CXCR3 ligand (IFN-␥-inducible memory and effector T cells. Some memory T cells retain their protein 10/CXCL10) is produced by mature dendritic cells, and is homing property to lymphoid tissues by retaining the expression of The Journal of Immunology 157

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